A kind of Lactobacillus paracasei microcapsule and its preparation method and application

Abstract

The invention relates to the field of functional microorganism screening and application technology, and specifically provides a Lactobacillus paracasei microcapsule and a preparation method and application thereof. The Lactobacillus paracasei microcapsule contains a novel Lactobacillus paracasei VHProbi F22 strain with a preservation number of CCTCC No: M2019941, which not only has a high amount of live bacteria, but also has good tolerance to artificial gastric juice and artificial intestinal juice, can reduce the level of pro-inflammatory cytokines, and alleviate the symptoms of intestinal immune disorders, and can be widely used in the fields of food, health care products, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic application, and particularly relates to a Lactobacillus paracasei microcapsule and a preparation method and application thereof. Background Art

[0002] In the past few decades, influenced by the Western lifestyle or industrialized lifestyle, human allergies have shown a sharp increase. Diseases caused by allergic reactions include rhinitis, asthma, food allergies, atopic dermatitis and contact dermatitis. There is increasing evidence that intestinal flora is an important environmental factor affecting immune tolerance.

[0003] When the order of establishing the structure of the intestinal flora community changes, it will lead to changes in the differentiation of helper T cells, and the imbalance of type I helper T cells (Th1 cells) and type II helper T cells (Th2 cells) in the body's immune system will be imbalanced, especially the balance tends to Th2 cells. For example, in infants and young children, the use of broad-spectrum antibiotics will lead to changes in intestinal flora and intestinal ecological imbalance, and increase susceptibility to allergies. In fact, epidemiological surveys and studies in recent years have shown that environmental factors that affect the colonization of intestinal flora are associated with the risk of allergies.

[0004] At present, allergic diseases are mainly treated with anti-allergic drugs and antihistamines, but they cannot fundamentally solve the problem for patients with weak immunity, such as lactose intolerance caused by imperfect intestinal development in infants and young children; in addition, histamine can cause adverse reactions such as reduced attention and slow reaction in the human body. Therefore, it is of great significance to find a more reasonable method to treat allergies. In recent years, the treatment of allergic diseases with probiotics has received increasing attention. Clinical studies have shown that probiotics can induce the body to produce immune responses and immune tolerance, and can improve immune regulation ability by presenting antigens through the gastrointestinal mucosa. Probiotics can increase IFN-γ, IL-2 and IL-12, promote T cell differentiation to Th1, and reduce IL-4 to reduce T cell differentiation to Th2, regulate the immune balance of Th1 and Th2, and thus achieve anti-allergic effects.

[0005] There are some related studies on probiotics regulating allergic reactions in the prior art. For example, the Chinese invention patent with publication number CN111560330 A discloses a strain of Lactobacillus casei, which can regulate immunity, regulate the balance of M1 / M2 macrophages, increase the number of Treg cells, improve the symptoms of colitis in mice and resist cervical cancer; the Chinese invention patent with publication number CN102373173 B discloses a strain of Lactobacillus casei, which can induce macrophages to produce IL-12, IL-6, TNF-α, and has an enhanced immunomodulatory effect; the Chinese invention patent with publication number CN 101139557 B discloses a strain of Lactobacillus casei, which can affect the peripheral blood T lymphocyte subsets of mice and their serum IgG levels and intestinal mucosal SigA levels, and enhance the cellular immunity, humoral immunity and intestinal mucosal local immune function of mice. Screening probiotics with immune regulation function and relieving the symptoms of intestinal immune disorders has a very important application value. Summary of the invention

[0006] The present invention provides a Lactobacillus paracasei microcapsule and a preparation method and application thereof to solve the problems of the prior art. The provided Lactobacillus paracasei microcapsule not only has a high amount of viable bacteria, but also has good tolerance to artificial gastric juice and artificial intestinal juice, can reduce the level of pro-inflammatory cytokines, alleviate the symptoms of intestinal immune disorders, and can be widely used in the fields of food, health care products, etc.

[0007] The invention firstly provides a Lactobacillus paracasei microcapsule, wherein the core material of the microcapsule contains Lactobacillus paracasei.

[0008] The Lactobacillus paracasei, named Lactobacillus paracasei VHProbi F22 (Lacticaseibacillus paracasei VHProbi F22), was deposited in the China Type Culture Collection of Wuhan University, China on November 18, 2019, and its deposit number is CCTCC No: M2019941.

[0009] The core material of the microcapsule also contains starch.

[0010] The embedding material used in the outer layer of the microcapsule comprises sodium alginate, gelatin and dextrin.

[0011] The microcapsule has a preparation method comprising: mixing 2-5% (W / W) porous starch with a suspension of Lactobacillus paracasei, adjusting the pH value to 6.0, and shaking at 25-35°C for 30 minutes; after sufficient adsorption, sequentially adding the following outer layer embedding materials according to weight percentage: 1-3% sodium alginate, 1-5% gelatin, and 1-5% dextrin, and stirring evenly; dripping 2-6% (W / W) calcium chloride solution until the mixed solution solidifies; and freeze drying to obtain Lactobacillus paracasei microcapsules.

[0012] Further preferably, the microcapsules are prepared by the following method: 3% porous starch is mixed with a suspension of Lactobacillus paracasei, the pH value is adjusted to 6.0, and the mixture is oscillated at 25-35° C. for 30 minutes; after sufficient adsorption, the following outer layer embedding materials are added in order according to weight percentage: 1.5% sodium alginate, 2.5% gelatin, and 3.5% dextrin, and stirred evenly; 6% (W / W) calcium chloride solution is dripped until the mixture solidifies; and freeze-drying is performed to obtain Lactobacillus paracasei microcapsules.

[0013] The invention also provides application of the Lactobacillus paracasei microcapsule in regulating intestinal immune disorder.

[0014] The invention also provides application of the Lactobacillus paracasei microcapsules in preparing functional food with the effect of regulating intestinal immune disorders.

[0015] The invention also provides a functional food comprising the Lactobacillus paracasei microcapsules.

[0016] 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 biosafety; and can tolerate higher salinity, with a maximum tolerance salt concentration of 6%.

[0017] The strain has strong antioxidant capacity and can effectively degrade cholesterol. In addition, the cell surface hydrophobicity of the strain is more than 70%, and it has a certain adhesion ability.

[0018] Lactobacillus paracasei VHProbi F22 has good efficacy in regulating intestinal immune disorders. The Lactobacillus paracasei VHProbi F22 provided by the invention can effectively alleviate the symptoms of intestinal immune disorders.

[0019] 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 mice with intestinal immune disorder model, and the effect of the probiotic pretreatment group is better than that of the post-treatment group.

[0020] According to the HE staining results of mouse jejunal tissue, treatment with Lactobacillus paracasei VHProbi F22 can reduce the swelling of jejunal mucosal villi, reduce the shedding of epithelial cells, and reduce the atrophy of the mucosal lamina propria. The jejunal mucosal villi have good integrity, and the effect of the probiotic pretreatment group is better than that of the post-treatment group.

[0021] The use of Lactobacillus paracasei VHProbi F22 can reduce the Verrucomicrobia in the intestines and increase the Bacteroidetes. Most probiotics belong to the Bacteroidetes. The intestinal flora structure of the allergic mouse model is closer to that of the normal intestine, and the diversity of the intestinal flora also increases.

[0022] The 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 with the function of alleviating intestinal immune disorders, and has broad application prospects.

[0023] The Lactobacillus paracasei microcapsules provided by the invention have high live bacteria quantity, strong tolerance to artificial gastric juice and intestinal juice, simple preparation process, cheap embedding material, low industrialization cost, can effectively improve the stability of Lactobacillus paracasei VHProbi F22 during storage, processing and transportation, greatly improve the actual use effect of Lactobacillus paracasei VHProbi F22, and can be widely used in the fields of food, health care products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For cultured colonies and light microscopic photos;

[0025] Figure 2 It is the protein fingerprint;

[0026] Figure 3 It is the Riboprinter fingerprint image;

[0027] Figure 4 It is the RAPD fingerprint;

[0028] Figure 5 is the rep-PCR fingerprint;

[0029] Figure 6 The intestinal immune disorder symptom score and wet stool frequency chart, where * is compared with the intestinal immune disorder group, P < 0.05;

[0030] Figure 7 The results of HE staining of jejunum of mice in each group;

[0031] Figure 8 Figure 2 shows the cytokine levels in each group, where *P<0.05 compared with the intestinal immune disorder group;

[0032] Fig. 9 It is a species diversity dilution curve graph;

[0033] Fig.10 This is the LAD value diagram of species abundance with statistical differences between the O1 and O2 groups;

[0034] Fig.11 This is a bar graph showing the relative abundance of intestinal flora in each group of mice. DETAILED DESCRIPTION

[0035] The screening method of the present invention is not limited to the examples described in the embodiments, and any known method that can achieve the screening purpose can be used. The screening description of the embodiments is only an explanation of the present invention, and is not a limitation on the protection scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0036] The present invention is further described below in conjunction with specific embodiments.

[0037] Example 1 Isolation and screening of Lactobacillus paracasei VHProbi F22

[0038] 1. Initial screening of Lactobacillus

[0039] Prepare MRS (Man Rogosa Sharpe) agar medium: 1000 mL of purified water, 10 g of peptone, 10 g of beef extract, 5.0 g of yeast extract, 5 g of sodium acetate, 5 g of glucose, 2 g of potassium dihydrogen phosphate, 1.0 mL of Tween 80, 2.0 g of diammonium citrate, 20 g of calcium carbonate, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate heptahydrate, 15 g of agar, adjust pH to 6.2-6.5, and sterilize at 121°C for 15 min.

[0040] Take 1g of cheese fermented by herders from the grasslands of Xilin Gol League, Inner Mongolia, dilute it with sterile saline, put it into a sterile sample bag, and beat it with a homogenizer to mix it; take 100μL of the mixed solution for gradient dilution, spread it on MRS agar medium, and culture it at 37℃ for 48h, and wait for single colonies to grow on the plate for microscopic examination. According to the results of microscopic examination, the applicant screened out a total of 11 potential lactobacilli, named NL-1, NL-2, NL-3, NL-4, NL-5, NL-6, NL-7, NL-8, NL-9, NL-10, and NL-11.

[0041] 2. Lactobacillus re-screening

[0042] Prepare 1L of MRS liquid culture medium and sterilize it under high pressure at 115℃ for 30min. After the culture medium has cooled, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to prepare an acid-resistant culture medium.

[0043] 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 above-mentioned acid-resistant culture medium at an inoculation rate of 6%, cultured at 37°C for 48 hours, and the fermentation broth was taken for bacterial count.

[0044] The results showed that the logarithmic values ​​of the viable bacteria in the fermentation broth of the 11 lactobacilli were 7.19, 8.22, 7.16, 7.52, 5.83, 5.61, 7.85, 6.74, 7.32, 7.08, and 8.58 Log CFU / mL, respectively. The NL-11 strain had the largest amount of viable bacteria after rescreening with an acid-resistant medium, with a logarithmic value of up to 8.58 Log CFU / mL. This shows that the NL-11 strain screened by the present invention has the highest acid resistance.

[0045] Example 2 Strain Identification

[0046] 2.1 Colony morphology identification

[0047] The NL-11 strain was inoculated on MRS agar medium and cultured at 37°C for 48 hours. The NL-11 single colony was rough and grayish white. Under the microscope, it was long or short rod-shaped with both ends flat. The NL-11 single colony and the photos under the optical microscope were shown in Figure 1 .

[0048] 2.2 Identification of physiological and biochemical characteristics

[0049] The inoculation solution in this embodiment was prepared as follows: Under sterile conditions, an appropriate amount of fresh NL-11 bacterial solution was taken, centrifuged at 5000 rpm / min for 5 min, washed twice with PBS buffer, and then resuspended with the same volume of PBS buffer and diluted 50 times to serve as the inoculation solution.

[0050] 1. Salinity tolerance test

[0051] 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 a 96-well plate. Do 3 parallels for each salt concentration, 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 the culture process. Incubate at 37°C and observe whether the culture medium becomes turbid.

[0052] The results showed that the NL-11 strain grew at a salt concentration of 1% to 6%, did not grow at a salt concentration of 7% to 8%, and its maximum tolerance salt concentration was 6%.

[0053] 2. Catalase test

[0054] Take a fresh bacterial liquid and drop it on a clean slide, 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.

[0055] 3. Carbon source metabolism test

[0056] The basal medium formula used in this embodiment is as follows:

[0057] Peptone 1.5g; yeast extract 0.6g; Tween 80 0.1g; saline solution 0.5mL; phenol red 18mg; distilled water 100mL; pH 7.4±0.2. Saline solution ingredients: MgSO4·7H2O 11.5g, MnSO4·4H2O 2.8g, distilled water 100mL.

[0058] Prepare 10g / 100mL sugar, alcohol and glycoside carbohydrate solutions and filter with a 0.22μm sterile filter. Under sterile conditions, add 20μL of sterilized carbohydrate solution to a 96-well plate, 4 parallels for each carbohydrate, then add 170μL of sterilized basal medium containing phenol red, and then add 10μL of inoculation solution. The uninoculated reaction wells are used as controls. Add 50μL of liquid paraffin to each well to prevent water evaporation during the culture process. Anaerobic culture was performed at 37℃, and phenol red was used as an indicator to observe the color change of the culture medium. The specific results are shown in Table 1.

[0059] Table 1 Carbon source metabolism results of NL-11 strain

[0060] Cellobiose Melibiose Raffinose Mannitol Amygdalin sucrose Galactose + - - + + + + lactose maltose Mannose Salicin Trehalose Arabinose Sodium Gluconate + + + - + - + Melezitose Ribose Sorbitol Xylose Rhamnose / / + + + - - / /

[0061] Note: “+” means positive reaction; “-” means negative reaction.

[0062] 2.3 MALDI-TOF-MS detection of full protein expression of strains

[0063] Inoculate fresh bacterial liquid in MRS liquid culture medium at a 0.1% inoculation rate, culture at 37°C, 150rpm for 48h, collect the bacteria, wash with sterile water 4 times, and dry the surface moisture. Then take a small amount of fresh bacteria and evenly apply it on the target plate in the form of a film, add 1μL of lysis solution to cover the sample, dry it, add 1μL of matrix solution to cover the sample, and after drying, put the sample target into the mass spectrometer for identification. Use laser to irradiate the co-crystallized film formed by the sample and the matrix to ionize the protein in the sample. The ions are accelerated to fly through the flight pipe under the action of 10-20KV electric field, and the molecular weight of the protein is detected according to the different flight time to the detector. The protein fingerprint was obtained using Autofms 1000 analysis software Autof Analyzer v1.0. The main ion peaks of the NL-11 strain were: m / z 9397.500, 7482.800, 6867.117, 5893.371, 5303.893, 4452.130, and 4699.426. The results are as follows Figure 2 shown.

[0064] 2.4 Molecular Biological Identification

[0065] 2.4.1 16S rDNA gene sequence analysis

[0066] 1. Genomic DNA Extraction

[0067] The operation was performed according to the Tiangen Bacteria Genomic DNA Extraction Kit (Catalog Number: DP302).

[0068] 2. 16s rDNA gene amplification

[0069] 1) Primer sequences:

[0070] 27F:AGAGTTTGATCCTGGCTCA;

[0071] 1492R: GGTTACCTTGTTACGACTT.

[0072] 2) Reaction system (50 μL)

[0073] Table 2: 16s rDNA PCR amplification system

[0074]

[0075] 3) Electrophoresis verification of PCR product nucleic acid electrophoresis results of about 1500bp meets the requirements.

[0076] 4) PCR product sequencing

[0077] The 16s rDNA sequence SEQ ID NO: 1 of the NL-11 strain was obtained by sequencing, and the sequence was compared in the NCBI database, and the NL-11 strain was preliminarily determined to be Lactobacillus paracasei.

[0078]

[0079] 2.4.2 Riboprinter fingerprint

[0080] A single colony was picked from the agar plate with a bacterial stick and placed in a sample tube with buffer. The colony was stirred with a hand-held stirrer to suspend it in the buffer. The sample rack was then placed in a heater to inactivate the colony and placed in the Riboprinter system. After DNA preparation, membrane transfer, imaging detection and data processing, the Riboprinter fingerprint of strain NL-11 was obtained ( Figure 3 ).

[0081] 2.4.3 RAPD and rep-PCR fingerprint identification

[0082] 1. RAPD fingerprint identification

[0083] 1) Primer sequence: M13 (5'-GAGGGTGGCGGTTCT-3');

[0084] 2) RAPD reaction system

[0085] Table 3 RAPD reaction system

[0086]

[0087] 3) Electrophoresis

[0088] Prepare a 1.5% agarose gel plate, use DL2000 DNA Marker as the result control, and run the electrophoresis at 100V for 80 minutes. Finally, use the gel imaging system to detect the electrophoresis pattern. Figure 4 shown.

[0089] 2. rep-PCR fingerprint

[0090] 1) rep-PCR primers

[0091] CTACGGCAAGGCGACGCTGACG.

[0092] 2) Rep-PCR reaction system

[0093] Table 4 Reaction system of rep-PCR

[0094]

[0095] 3) Electrophoresis

[0096] DL2000 DNA Marker was used as a control. The voltage was 100V and the electrophoresis time was 80min to detect the amplification results. The rep-PCR fingerprint of NL-11 strain is shown in Figure 5 shown.

[0097] 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 compared with the results published in the literature De Clerck E, et al. Systematic and applied microbiology, 2004, 27 (1) 50. Based on the molecular biological identification results, it can be concluded that the NL-11 strain is a new strain of Lactobacillus paracasei, which is named Lactobacillus paracasei VHProbi F22.

[0098] Example 3 Tolerance test of Lactobacillus paracasei VHProbi F22 to artificial gastric juice and artificial intestinal juice

[0099] 3.1. Preparation of artificial gastric juice

[0100] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl respectively, add 1000mL of distilled water, adjust pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Then add 3.2g of porcine mucosal pepsin before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0101] 3.2 Preparation of artificial intestinal fluid

[0102] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4 and 3.0g of ox bile salt respectively, add 77mL of 0.2mol / L NaOH solution, dilute to 1000mL, adjust pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, sterilize at 115℃ for 20min. Then add 1g of pancreatic enzyme before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0103] 3.2 Test methods

[0104] Take 2mL of fresh bacterial solution, centrifuge at 5000rpm / min for 5min to collect the bacteria, wash the bacteria 3 times with saline, and resuspend with 2mL of saline as the inoculum. Take 1mL of the inoculum, add it to 24mL of artificial intestinal fluid, place it in a 37℃ water bath shaker (200rpm / min) for 3h, take 1mL of the sample, and detect the amount of live bacteria.

[0105] The live bacteria count method was carried out in accordance with the national standard GB4789.35-2016-Food Microbiology Inspection Lactic Acid Bacteria Inspection to determine the bacterial count. The live bacteria count (Log CFU / mL) of the strain after digestion with artificial intestinal fluid is shown in Table 5.

[0106] Table 5 The amount of viable bacteria after digestion with artificial gastric juice and artificial intestinal juice

[0107]

[0108] As shown in Table 5, after the Lactobacillus VHProbi F22 screened by the present invention was digested by artificial gastric juice and artificial intestinal juice, the amount of live bacteria decreased by only about 1.2 Log CFU / mL, which indicates that the strain has strong tolerance to artificial gastric juice and artificial intestinal juice.

[0109] Example 4 Antibiotic tolerance experiment of Lactobacillus paracasei VHProbi F22

[0110] 1) Antibiotic preparation

[0111] Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were prepared into 2048 μg / mL stock solutions and stored at -20°C for later use. When used, the stock solution was diluted 2-fold with BSM liquid medium to form a working solution, with a total of 11 gradients of gradient dilution concentrations ranging from 1 to 1024 μg / mL.

[0112] 2) Preparation of inoculum

[0113] Take an appropriate amount of fresh bacterial liquid (cultured for 24 hours at 37°C), centrifuge at 5000 rpm for 5 minutes, wash once with sterile saline, resuspend the bacteria with the same volume of saline and dilute 50 times as the inoculum.

[0114] 3) Determination of the minimum inhibitory concentration (MIC) of antibiotics against Lactobacillus paracasei VHProbi F22 by broth microdilution method

[0115] a. Add MRS liquid culture medium without antibiotics to the first column of the 96-well plate as a negative control, and add 190 μL of MRS liquid culture medium containing different concentrations of antibiotics to the 2nd to 12th columns, and then inoculate 10 μL of the above inoculation solution to make 3 parallel wells, and use one well without bacterial solution as a blank.

[0116] b. Add 50 μL paraffin oil to cover to prevent water evaporation.

[0117] c. Incubate the 96-well plate at 37°C for 24 hours and then take it out to measure OD 600 The MIC values ​​of antibiotics against the strains were calculated using the results of 24 h. The specific results are shown in Table 6.

[0118] Table 6 Antibiotic MIC values ​​of Lactobacillus paracasei VHProbi F22

[0119]

[0120] MIC unit: μg / mL

[0121] It can be seen from the results in Table 6 that the Lactobacillus paracasei VHProbi F22 provided by the present invention is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biosafety.

[0122] Example 5 Determination of the antioxidant function of Lactobacillus paracasei VHProbi F22

[0123] 1. Determination of the ability of strains to scavenge DPPH free radicals

[0124] 1) Preparation of PBS bacterial suspension

[0125] Inoculate a single colony with good growth status into 3mL of MRS liquid culture medium, culture at 37℃ for 24h, use this culture solution as inoculum, inoculate into 50mL of MRS liquid culture medium at a 2% inoculum amount, and culture for 24h to obtain the culture solution of the strain. Collect the bacteria by absorbing 1mL of bacterial solution, wash the bacteria twice with 1mL of PBS buffer, and then add 2mL of PBS solution to resuspend the bacteria for later use.

[0126] 2) Determination of the ability of strains to scavenge DPPH free radicals

[0127] Take 1mL of the PBS bacterial suspension of the strain to be tested, add 1mL of 0.4mM DPPH free radical solution, mix well and then place it at room temperature for 30 minutes in the dark, then measure the absorbance of the sample at a wavelength of 517nm A sample, measure 3 times in parallel. The control group sample is equal to the volume of PBS solution and DPPH·ethanol mixture, and the blank is zeroed with an equal volume of PBS bacterial suspension and ethanol mixture. The clearance rate is calculated according to the following formula: Clearance rate % = [1-(A 样品 -A 空白 ) / A 对照 ]×100%.

[0128] The Lactobacillus paracasei IMC-4 strain was used as a positive control. The results are shown in Table 7.

[0129] Table 7 DPPH free radical scavenging rate

[0130]

[0131] 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 that of the Lactobacillus paracasei IMC-4 strain.

[0132] 2. Identification of strains against lipid peroxidation

[0133] 1) Cultivation of lactic acid bacteria and preparation of fermentation supernatant, bacterial cells, and intracellular extracts: Lactic acid bacteria were cultured in MRS liquid medium at 37°C for 24 h. After three generations, the culture was centrifuged at 6000 rpm / min and 4°C for 10 min, and the supernatant was collected as the fermentation supernatant. The collected bacterial cells were centrifuged at 6000 r / min for 10 min with PBS buffer (pH 7.4) and washed three times. The bacterial cells were resuspended in PBS buffer and the bacterial cell concentration was adjusted to 1.0×10 9 cells / mL to obtain bacterial suspension.

[0134] 2) Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0135] 3) Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample, and place in a 37°C water bath for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture, and place in a 100°C water bath for 30 min. Cool rapidly, centrifuge at 4000 rpm / min for 15 min, collect the supernatant, and measure the absorbance at 532 nm to obtain A. The control group uses 0.5 mL of distilled water instead of sample to obtain A0. Inhibition rate / % = (A0-A) / A0×100%

[0136] Note: A is the absorbance of the sample group; A0 is the absorbance of the control group. Lactobacillus paracasei IMC-4 was used as the positive control. The results are shown in Table 8.

[0137] Table 8 Anti-lipid peroxidation inhibition rate

[0138]

[0139] It can be seen from the data in Table 8 that the supernatant anti-lipid peroxidation inhibition rate of Lactobacillus paracasei VHProbi F22 provided by the present invention is 50.64%, which is lower than that of Lactobacillus paracasei IMC-4 strain; and the anti-lipid peroxidation inhibition rate of the bacterial body is 51.66%, which is higher than that of Lactobacillus paracasei IMC-4 strain.

[0140] Example 6 In vitro cholesterol degradation experiment of Lactobacillus paracasei VHProbi F22

[0141] 1. Preparation of cholesterol micelle solution: Accurately weigh 1 g of cholesterol, dissolve it in anhydrous ethanol, and make up to 100 mL. Filter and sterilize it with a 0.22 μm microporous filter membrane under sterile conditions.

[0142] 2. Weigh 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, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt, and 1000mL of distilled water, adjust the pH value to 7.3, sterilize at 115℃ for 30min, and then add cholesterol solution to make the final cholesterol concentration 0.1%.

[0143] Inoculate fresh bacterial liquid at 0.1% inoculation volume, culture at 37℃ for 48h, then take 0.2mL of bacterial liquid, add 1.8mL of anhydrous ethanol, mix, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and take the supernatant for determination of cholesterol content. Cholesterol determination method is in accordance with GB / T 5009.128-2003 <Determination of cholesterol in food>.

[0144] The results showed that the degradation rate of cholesterol by Lactobacillus paracasei VHProbi F22 provided by the present invention reached 34.55%.

[0145] Example 7 Test of hydrophobicity of Lactobacillus paracasei VHProbi F22 cell surface

[0146] 1. Preparation of test solution: 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 24-48h. Then inoculate it into MRS liquid medium at a 1% (V / V) inoculation amount and continue to culture it at 37°C for 24-48h, then centrifuge it at 6000×g for 10min, collect the bacteria and rinse it twice with sterile saline, and then resuspend the bacteria with 1mL sterilized 0.1M KNO3 solution as the test solution.

[0147] 2. Surface hydrophobicity determination: Pipette 50 μL of the above bacterial suspension, add 2450 μL of 0.1 M KNO3 and record the OD 600 Take 1.5 ml of bacterial suspension and 500 μL of xylene, mix well and let stand at room temperature for 10 minutes (a two-phase system is formed at this time). Vortex the two-phase system for 2 minutes and then let stand for 20 minutes to re-form the aqueous phase and the organic phase. Carefully aspirate the aqueous phase (do not aspirate the organic phase) and measure the absorbance A1 at 600 nm. The cell hydrophobicity is calculated according to the formula Hydrophobicity% = (A0-A1) / A1×%, and the average value is taken after three experiments.

[0148] The results showed that the hydrophobicity of the cell surface of Lactobacillus paracasei VHProbi F22 provided by the present invention was 70.61%.

[0149] Example 8 Application of Lactobacillus paracasei VHProbi F22 in Alleviating Symptoms of Intestinal Immune Disorders in Mice

[0150] 1.1 Experimental Materials

[0151] 1.1.1 Experimental animals

[0152] BALB / c mice SPF grade, 30 females, 4-6 weeks old, weighing 19-25 g. Provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., production license number SCXK(Lu)20140007.

[0153] Animal quarantine and identification: After all animals arrive, they will be adapted for at least one week and then quarantined and observed. The animals' activities, diet, and other behaviors will be observed. Animals must pass the inspection before the experiment, and qualified animals can be used for the experiment. After the animal quarantine passes, each animal will be assigned a single animal number and marked at the tail of the animal. During the quarantine observation period, the cage card will indicate the topic number, animal number, cage number, gender, animal receipt date, and topic person in charge; after grouping, the cage card will indicate the topic number, animal number, cage number, gender, group, experiment start and end dates, and topic person in charge.

[0154] The environmental conditions for the feeding and management of experimental animals were as follows: room temperature 20-26°C, daily temperature difference ≤4°C, relative humidity 40-70%, and light-dark alternation time 12 / 12h. Animals were kept in standard mouse cages, with 6 mice in each cage.

[0155] Animal feed and drinking water: free access to food and water. Feed is SPF-grade growth and breeding feed for mice and rats, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Batch No.: 20190905). Drinking water is city tap water that has been sterilized at high temperature.

[0156] 1.1.2 Reagents and consumables

[0157] Ovalbumin (OVA) (Batch No.: S12016): Shanghai Yuanye Biotechnology Co., Ltd.;

[0158] D-biotin (lot number: S13004): Shanghai Yuanye Biotechnology Co., Ltd.;

[0159] Vitamin B2 (batch number: S13020): Shanghai Yuanye Biotechnology Co., Ltd.;

[0160] Aluminum adjuvant (Batch No.: UL292268): Thermo Fisher Scientific (China) Co., Ltd.;

[0161] IL-5 (Batch No.: E20200605-20187B) kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0162] IL-6 (Batch No.: E20200605-20188B) kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0163] IL-10 (Batch No.: E20200601-20162B) kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0164] IFN-γ (Batch No.: E20200602-20140B) kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0165] TNF-α cytokine (lot number: E20200607-20852B) kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0166] OVA-specific IgE (lot number: E20200604-20508B) kit: Shanghai ELISA Biotechnology Co., Ltd.;

[0167] Eosin (Batch No.: 20181204): Beijing Solebow Technology Co., Ltd.;

[0168] Hematoxylin (Batch No.: 20181204): Beijing Solebow Technology Co., Ltd.

[0169] 1.2 Experimental methods

[0170] 1.2.1 Preparation of bacterial solution

[0171] Streak a single colony on an MRS plate and culture it anaerobically at 37°C for 24-48 hours. Pick a single colony and expand it in MRS broth medium for 16 hours. Collect the bacterial solution and adjust its concentration to 10 9 CFU / mL bacterial suspension.

[0172] 1.2.2 Grouping

[0173] After 3 days of adaptive feeding, SPF female BALB / c mice aged 4-6 weeks were randomly divided into a blank control group, an intestinal immune disorder group, a probiotic pretreatment group, and a probiotic post-treatment group, with 6 mice in each group. The probiotic pretreatment group and the post-treatment group were given probiotic solution by oral gavage at 0.2 mL / 10 g.

[0174] 1.2.3 Modeling and probiotic intervention program

[0175] The mice in the probiotic pretreatment group were given probiotics by gavage before modeling, and the treatment lasted for 10 days, while the other groups were not treated. Modeling began 10 days later, and 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, with each mouse 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); from day 28, the blank control group was gavaged with 200 μL PBS solution every three days, and the intestinal immune disorder group and the two probiotic groups were gavaged with 50 mg OVA every three days, for a total of 6 times of sensitization; probiotic pretreatment was given bacterial solution from 3 days after adaptive feeding to the day before sacrifice, and the probiotic posttreatment group was given bacterial solution by gavage from the beginning of basic sensitization on day 0 to the day before sacrifice.

[0176] 1.3 Indicator Observation

[0177] 1.3.1 Symptoms of intestinal immune disorders in mice

[0178] One hour after each oral administration of OVA, the intestinal immune disorder symptoms of the mice were observed and scored. The scoring details are shown in Table 9.

[0179] Table 9 Scoring table for intestinal immune disorders in mice

[0180]

[0181] 1.3.2 Detection of IL-5, IL-6, IL-10, IFN-γ, TNF-α and OVA-specific IgE

[0182] 24 hours after the last administration of the mice, blood was collected from the eyeballs to prepare serum, which was stored at -80°C. The IL-5, IL-6, IL-10, IFN-γ, TNF-α cytokine levels and OVA-specific IgE levels in the serum of each group of mice were detected by ELISA.

[0183] 1.3.3 Histopathological examination

[0184] After the mice were killed, the jejunal tissues were obtained, fixed with paraformaldehyde, sampled, dehydrated, embedded in paraffin, sliced, and HE stained to observe the pathological changes of the tissues.

[0185] 1.4 Data Statistical Processing Methods

[0186] All experimental data were expressed as mean ± standard deviation "± SD" and were statistically analyzed and plotted using Microsoft EXCEL. The t-test was used to compare the data between the two groups, and P < 0.05 was considered to be significantly different.

[0187] 1.5 Experimental Results

[0188] 1.5.1 Symptoms of intestinal immune disorders in mice

[0189] Compared with the blank control group, the immune disorder symptoms and wet stool frequency of mice in the intestinal immune disorder group increased significantly, and with the increase in the number of sensitization times, the immune disorder and diarrhea symptoms increased; at the end of the trial period, the intestinal immune disorder group had the highest allergy symptom score, and the mice had severe diarrhea and restlessness, while the allergy symptom scores of the probiotic pretreatment and post-treatment groups were reduced, the diarrhea symptoms were milder, and the intestinal immune disorder symptoms were improved. Symptom scores and wet stool frequency of each group are shown in Figure 6 .

[0190] 1.5.2 Histopathological examination

[0191] Observation under an optical microscope showed that compared with the intestinal immune disorder group, probiotic treatment could reduce the swelling of jejunal mucosal villi, reduce epithelial cell shedding, improve integrity, and reduce the atrophy of the mucosal lamina propria. The effect of the pretreatment group was better than that of the posttreatment group. Figure 7 .

[0192] 1.5.3 Detection of IL-5, IL-6, IL-10, IFN-γ, TNF-α and OVA-specific IgE

[0193] Elisa method was used to detect the IL-5, IL-6, IL-10, IFN-γ, TNF-α cytokine content and OVA-specific IgE level 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 the mice in the intestinal immune disorder group were significantly increased, indicating that the OVA-induced intestinal immune disorder model was successfully established. Compared with the intestinal immune disorder group, the IL-5, IL-6, IFN-γ, and TNF-α cytoinflammatory factors in the serum of mice in the probiotic post-treatment group were reduced, the IL-10 cytoinflammatory factor was increased, and the OVA-specific IgE was reduced, and there were significant differences (P<0.05); the IL-5, IL-6, IFN-γ, and TNF-α cytoinflammatory factors in the serum of mice in the probiotic pre-treatment group were reduced, the IL-10 cytoinflammatory factor was increased, and the OVA-specific IgE was reduced, and there were significant differences (P<0.05). Comparison of serum cytokine contents of L-5, IL-6, IL-10, IFN-γ, TNF-α and OVA-specific IgE in each group of mice Figure 8 shown.

[0194] From the above indicators, it can be seen that compared with the intestinal immune disorder group, the 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 posttreatment group. Compared with the mice in the intestinal immune disorder group, probiotic treatment can reduce the degree of axial atrophy of the jejunal mucosal villi, reduce the gap between the axial connective tissue and the surface epithelium, reduce the shedding of epithelial cells, and alleviate the degree of lesions.

[0195] Example 9 Application of Lactobacillus paracasei VHProbi F22 in regulating intestinal flora of mice

[0196] 1.1 Experimental methods

[0197] The same animal experiment scheme as in Example 1 was used. The feces of mice in the intestinal immune disorder group and the probiotic pretreatment group were collected before OVA sensitization and after the experiment, and the intestinal flora structure was analyzed. The collected fecal samples were sent to Novogene for sequencing, and the community diversity and abundance changes of the flora microorganisms, including the richness index and diversity index, were analyzed using amplicon sequencing technology.

[0198] 1.2 Experimental Results

[0199] On the basis of obtaining 97% similarity OTU, the Alpha diversity of all samples was analyzed and the corresponding dilution curve was constructed, such as Fig. 9 As shown in the figure, when the curve tends to be flat, it means that the number of sequencing is reasonable, most of the microorganisms can be detected, and the sequencing depth can basically cover all species in the sample. It can also be indirectly seen that the species abundance of the sample after the probiotic pretreatment group (Y2) is higher than that before sensitization of the intestinal immune disorder group (O1) and before sensitization of the probiotic pretreatment group (Y1), and the species abundance of the sample before sensitization of the intestinal immune disorder group (O1) is higher than that after the end of the intestinal immune disorder group (O2), indicating that the species abundance of the intestinal flora decreases after OVA sensitization causes symptoms of intestinal immune disorder. Intervention with Lactobacillus paracasei VHProbiF22 can increase the species abundance of the intestinal flora of mice with intestinal immune disorder and restore the intestinal flora.

[0200] The LDA value distribution bar chart shows species with LDA scores greater than the set value (the default setting is 4), that is, species with statistical differences between groups, and species with significant abundance differences between different groups. The length of the bar chart represents the impact size of the different species (that is, the LDA score). The results are shown in the attached Fig.10As shown, in O1, Bacillus, Lactobacillus, Lactobacillus intestinalis, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus delbrueckii, etc. were all significantly highly abundant; in O2, Verrucomicrobia, Eckermannia, Erysipelotrichia and Turicibacter were all significantly highly abundant, indicating that intestinal immune disorders would cause a decrease in the abundance of Lactobacillus in the intestinal flora.

[0201] Comparison of the data of the two groups O1 / O2 showed that after the experiment, the relative abundance of Firmicutes in the intestinal immune disorder group decreased, while the relative abundance of Bacteroidetes and Verrucomicrobia increased; comparison of the data of the two groups O1 / Y1 showed that the pretreatment with probiotics in the early stage reduced the relative abundance of Firmicutes and increased the relative abundance of Bacteroidetes in the mouse intestine; comparison of the data of the two groups O2 / Y2 showed that after the experiment, the relative abundance of Verrucomicrobia in the probiotic group was reduced compared with the intestinal immune disorder group, while the relative abundance of Bacteroidetes increased; the results showed that after the intestinal immune disorder of mice, the intestinal flora structure of mice was more similar to that of normal mice after intervention with Lactobacillus paracasei VHProbi F22, and the relative abundance of species in each group was shown. Fig.11 .

[0202] After OVA sensitization caused symptoms of intestinal immune disorder, the species abundance of intestinal flora decreased. Intervention with Lactobacillus paracasei VHProbi F22 can increase the species abundance of intestinal flora in mice with intestinal immune disorder, restore the intestinal flora, increase the abundance of Lactobacillus in the intestinal flora, and make the intestinal flora structure more similar to that of healthy mice.

[0203] In summary, the lactobacillus paracasei VHProbi F22 provided by the present invention has a strong tolerance to simulated artificial gastrointestinal fluid, which lays the foundation for the probiotic strain to smoothly colonize in the colon through the gastrointestinal tract and play a probiotic function. Hemolytic test confirms that lactobacillus paracasei VHProbi F22 does not produce hemolysin, can not dissolve blood cells, and has good biosafety. Meanwhile, lactobacillus paracasei VHProbi F22 can remove DPPH free radicals, inhibit lipid peroxidation, has certain antioxidant function activity, can degrade cholesterol, and has the probiotic properties of reducing serum cholesterol. Animal experiments confirm that lactobacillus paracasei VHProbi F22 can improve the inflammatory response of intestinal immune disorder model mice, can suppress TH2 type immune response while strengthening TH1 type cellular immune response, reduce the inflammatory state of body, enhance immunity, which shows that lactobacillus paracasei VHProbi F22 has potential application value in alleviating intestinal immune disorder inflammation.

[0204] Example 10 Lactobacillus paracasei microcapsules and preparation method thereof

[0205] 1. Preparation of Lactobacillus paracasei fermentation liquid

[0206] Under aseptic conditions, the seed liquid of Lactobacillus paracasei VHProbi F22 is inoculated into a fermentation medium at a volume ratio of 3% to 10%, wherein the components and their contents in the fermentation medium are respectively: 50 to 70 g / L of sucrose, 20 to 30 g / L of yeast powder, 5 to 25 g / L of soy peptone, 1.5 to 2 g / L of magnesium sulfate and 0.08 to 0.12 g / L of manganese sulfate; the culture is carried out at 37°C for 18 to 24 hours, the rotation speed is 50 to 100 rpm, the ventilation volume is 0.3 to 1 L / min, and the tank pressure is 0.05 to 0.08 MPa. After the fermentation is completed, the fermentation liquid of Lactobacillus paracasei VHProbi F22 is obtained, wherein the viable bacteria amount of Lactobacillus paracasei is 10 9 -10 10 CFU / mL.

[0207] 2. Preparation of Lactobacillus paracasei microcapsules

[0208] The fermentation broth of Lactobacillus paracasei VHProbi F22 was centrifuged at 4°C and 5000 r / min for 10 min, the bacteria were collected, washed twice with sterile saline, and resuspended to prepare a bacterial suspension.

[0209] 5% (W / W) porous starch was mixed with the bacterial suspension, the pH value was adjusted to 6.0, and the temperature was controlled at 25-35°C for 30 minutes; after sufficient adsorption, the following outer layer embedding materials were added in order according to weight percentage: 3% sodium alginate, 5% gelatin, and 5% dextrin, and stirred evenly; 2% (W / W) calcium chloride solution was dripped until the mixture solidified; freeze-dried to obtain Lactobacillus paracasei microcapsules. The live bacterial count of the Lactobacillus paracasei VHProbi F22 microcapsules was 1.5×10 9 CFU / mL.

[0210] Example 11 Lactobacillus paracasei microcapsules and preparation method thereof

[0211] 1. Preparation of Lactobacillus paracasei fermentation liquid

[0212] Under aseptic conditions, the seed liquid of Lactobacillus paracasei VHProbi F22 is inoculated into a fermentation medium at a volume ratio of 3% to 10%, wherein the components and their contents in the fermentation medium are respectively: 50 to 70 g / L of sucrose, 20 to 30 g / L of yeast powder, 5 to 25 g / L of soy peptone, 1.5 to 2 g / L of magnesium sulfate and 0.08 to 0.12 g / L of manganese sulfate; the culture is carried out at 37°C for 18 to 24 hours, the rotation speed is 50 to 100 rpm, the ventilation volume is 0.3 to 1 L / min, and the tank pressure is 0.05 to 0.08 MPa. After the fermentation is completed, the fermentation liquid of Lactobacillus paracasei VHProbi F22 is obtained, wherein the viable bacteria amount of Lactobacillus paracasei is 109 -10 10 CFU / mL.

[0213] 2. Preparation of Lactobacillus paracasei microcapsules

[0214] The fermentation broth of Lactobacillus paracasei VHProbi F22 was centrifuged at 4°C and 5000 r / min for 10 min, the bacteria were collected, washed twice with sterile saline, and resuspended to prepare a bacterial suspension.

[0215] 2% (W / W) porous starch was mixed with the bacterial suspension, the pH value was adjusted to 6.0, and the temperature was controlled at 25-35°C for 30 minutes; after sufficient adsorption, the following outer layer embedding materials were added in order according to weight percentage: 1% sodium alginate, 1% gelatin, and 1% dextrin, and stirred evenly; 4% (W / W) calcium chloride solution was dripped until the mixture solidified; freeze-dried to obtain Lactobacillus paracasei microcapsules. The live bacterial count of the Lactobacillus paracasei VHProbi F22 microcapsules was 4×10 9 CFU / mL.

[0216] Example 12 Lactobacillus paracasei microcapsules and preparation method thereof

[0217] 1. Preparation of Lactobacillus paracasei fermentation liquid

[0218] Under aseptic conditions, the seed liquid of Lactobacillus paracasei VHProbi F22 is inoculated into a fermentation medium at a volume ratio of 3% to 10%, wherein the components and their contents in the fermentation medium are respectively: 50 to 70 g / L of sucrose, 20 to 30 g / L of yeast powder, 5 to 25 g / L of soy peptone, 1.5 to 2 g / L of magnesium sulfate and 0.08 to 0.12 g / L of manganese sulfate; the culture is carried out at 37°C for 18 to 24 hours, the rotation speed is 50 to 100 rpm, the ventilation volume is 0.3 to 1 L / min, and the tank pressure is 0.05 to 0.08 MPa. After the fermentation is completed, the fermentation liquid of Lactobacillus paracasei VHProbi F22 is obtained, wherein the viable bacteria amount of Lactobacillus paracasei is 10 9 -10 10 CFU / mL.

[0219] 2. Preparation of Lactobacillus paracasei microcapsules

[0220] The fermentation broth of Lactobacillus paracasei VHProbi F22 was centrifuged at 4°C and 5000 r / min for 10 min, the bacteria were collected, washed twice with sterile saline, and resuspended to prepare a bacterial suspension.

[0221] 3% porous starch was mixed with bacterial suspension, pH value was adjusted to 6.0, and the temperature was controlled at 25-35°C for 30 minutes; after sufficient adsorption, the following outer layer embedding materials were added in order according to weight percentage: 1.5% sodium alginate, 2.5% gelatin, 3.5% dextrin, and stirred evenly; 6% (W / W) calcium chloride solution was dripped until the mixture solidified; freeze-dried to obtain Lactobacillus paracasei microcapsules. The live bacterial count of Lactobacillus paracasei VHProbi F22 microcapsules was 1×10 10 CFU / mL;

[0222] Example 13 Performance test of Lactobacillus paracasei microcapsules

[0223] 1. Stress resistance test

[0224] 1.1 Preparation of artificial gastric juice

[0225] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl respectively, add 1000mL of distilled water, adjust pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Then add 3.2g of porcine mucosal pepsin before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0226] 1.2 Preparation of artificial intestinal fluid

[0227] Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4 and 3.0g of ox bile salt respectively, add 77mL of 0.2mol / L NaOH solution, dilute to 1000mL, adjust pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, sterilize at 115℃ for 20min. Then add 1g of pancreatic enzyme before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0228] 1.3 Test methods

[0229] Take 2g of Lactobacillus paracasei microcapsules prepared in Examples 10 to 12, resuspend them with 2mL of physiological saline, and use them as inoculation solution. Take 1mL of inoculation solution, add it to 9mL of artificial gastric juice that has been warmed for 1h in advance, place it in a 37℃ water bath shaker and oscillate at 200rpm / min for 2h, sample 1mL at 0h and 2h, and detect the amount of viable bacteria. Then take 1mL of artificial gastric juice after digestion for 2h, add it to 24mL of artificial intestinal juice, place it in a 37℃ water bath shaker (200rpm / min) for 3h, sample 1mL, and detect the amount of viable bacteria. The viable bacteria counting method is determined according to the national standard "GB4789.35-2016-Food Microbiology Inspection Lactic Acid Bacteria Inspection". The amount of viable bacteria (Log CFU / mL) of the microcapsules prepared in Examples 10 to 12 after digestion by artificial gastric juice and artificial intestinal juice is shown in Table 10.

[0230] Table 10 Tolerance effect of Lactobacillus paracasei microcapsules on artificial gastric juice and artificial intestinal juice

[0231]

[0232]

[0233] It can be seen from the results in Table 10 that the Lactobacillus paracasei microcapsules provided by the present invention can still maintain a relatively high amount of viable bacteria after being digested by artificial gastric juice and artificial intestinal juice, which indicates that the Lactobacillus paracasei VHProbi F22 microcapsules have strong gastric acid and choline resistance.

[0234] The applicant fed the Lactobacillus paracasei microcapsules provided by the present invention to 4-6 week old female BALB / c mice in advance for 10 days, and then conducted an intestinal immune disorder modeling test on the mice to observe the intestinal immune disorder and other indicators of the mice. The results showed that compared with the negative group mice, the levels of cellular inflammatory factors and OVA-specific antibodies in the mice gavaged with Lactobacillus paracasei microcapsules in advance were significantly reduced, and the degree of jejunal pathology in the mice was alleviated.

[0235] The Lactobacillus paracasei microcapsules provided by the present invention were fed to female BALB / c mice aged 4-6 weeks in advance for 10 days, and then the mice were subjected to an intestinal immune disorder modeling test to detect changes in their intestinal flora. The results showed that compared with the negative group mice, the abundance of Lactobacillus species in the intestinal flora of mice gavaged with Lactobacillus paracasei microcapsules increased, and the intestinal flora structure of mice with intestinal immune disorder was more similar to that of healthy mice.

[0236] The preparation process of the Lactobacillus paracasei microcapsules provided by the invention is simple, the embedding material is cheap, the industrialization cost is low, the stability of the Lactobacillus paracasei VHProbi F22 during storage, processing and transportation can be effectively improved, the actual use effect of the Lactobacillus paracasei VHProbi F22 is greatly improved, and the microcapsules can be widely used in the fields of food, health care products, etc. Sequence Listing <110> Qingdao Blue Biotechnology Co., Ltd. Qingdao Blue Biotechnology Group Co., Ltd. <120> A kind of Lactobacillus paracasei microcapsule and its preparation method and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1468 <212> DNA <213> Lacticaseibacillus paracasei <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 360 420 ctctgttgtt ggaagaat ggtcggcaga gtaactgttg tcggcgtgac ggtatccaac 480 cagaaagcca cggctacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540 tccggatttta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagccc 600 660 gaactccatg tgtagcggtg aaatgcgtag atatatgaa 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 acggggcccc 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

1. A Lactobacillus paracasei microcapsule, characterized in that The core material of the Lactobacillus paracasei microcapsule contains Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CCTCC No: M2019941.

2. The Lactobacillus paracasei microcapsule according to claim 1, wherein The core material of the microcapsule contains starch.

3. The Lactobacillus paracasei microcapsule as claimed in claim 1, characterized in that The embedding material used in the outer layer of the microcapsule comprises sodium alginate, gelatin and dextrin.

4. Use of the Lactobacillus paracasei microcapsules according to any one of claims 1 to 3 in preparing functional foods having the effect of regulating intestinal immune disorders.

5. A functional food, characterized in that: The functional food contains the Lactobacillus paracasei microcapsules according to any one of claims 1 to 3.

Images