Preparation method of Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis and its application in immunity

Abstract

The present invention relates to a preparation method of Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis and its application in the immune direction, comprising activating Lactobacillus paracasei IOB413 strain; inoculating the activated strain into an optimized culture medium for culturing to obtain a secondary seed liquid; inoculating the secondary seed liquid into a sterilized fermentation broth medium for fermentation; centrifuging the fermentation broth medium, collecting bacterial mud, freeze-drying the bacterial mud, and preparing Lactobacillus paracasei IOB413 bacterial powder. The present invention can significantly increase the amount of butyric acid synthesized by Lactobacillus paracasei IOB413 in the intestine by optimizing the culture medium, and further elaborates in detail that Lactobacillus paracasei IOB413 affects intestinal function by promoting butyric acid synthesis, and finally exhibits the effects of enhancing immunity, repairing damaged immune organs, improving intestinal damage, regulating intestinal microecology, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, in particular to a method for preparing Lactobacillus paracasei IOB413 bacterial powder capable of regulating butyric acid synthesis and an application thereof in the immune direction. Background Art

[0002] The intestinal microbiota plays an important role in human intestinal health. The carbohydrates ingested by the human body are anaerobically fermented in the cecum and colon, and the final product is short-chain fatty acids (SCFAs), which can provide energy for the growth of intestinal microorganisms and maintain intestinal cell function. It has a significant impact on multiple organs and metabolism of the human body and is crucial to human health.

[0003] SCFAs are mainly composed of acetic acid, propionic acid and butyric acid, and the content of the three accounts for 90% to 95% of the total intestinal SCFAs. Studies have found that butyrate-based enzyme activity and its genes exist in some butyrate-producing strains. In the human intestine, the CoAT pathway may be the main pathway for butyrate production. Studies have found that butyrate also has the function of inhibiting tumor cell proliferation and differentiation, enhancing the body's immune function and preventing colitis. Similarly, SCFAs also have the potential to repair the immune system.

[0004] Immunomodulation refers to the interaction between immune cells in the human immune system and other systems to maintain the stability of the immune response. Immune disorders can cause many diseases, including allergic reactions (allergies, immune complexes, delayed immune diseases, cytotoxic immune diseases), immunodeficiency (AIDS, etc.), and impaired immune system.

[0005] Black beans are rich in nutrients, containing protein, fat, vitamins, trace elements and other nutrients. They also have a variety of biologically active substances, such as black bean pigments, black bean polysaccharides and isoflavones. Black bean crude polysaccharides can promote It can inhibit the proliferation of pathogens, prevent constipation and abdominal distension, promote digestion, and regulate gastrointestinal function.

[0006] Sodium alginate is an edible macromolecular polysaccharide that is not digested by the human body. It is widely used in thickening. Its film-forming and chelating properties can be used as a freeze-drying protective agent to improve the survival rate of bacterial powder. Sodium alginate also has the effects of lowering blood pressure, lowering blood lipids, lowering cholesterol, preventing fatty liver, accelerating gastrointestinal motility, and preventing constipation.

[0007] Studies have shown that optimizing the composition of fermentation medium can affect the synthesis and composition of extracellular polysaccharides produced by lactic acid bacteria. Regulating the immune system is one of the main probiotic properties of Lactobacillus paracasei. It is speculated whether optimizing the culture medium can affect the butyrate synthesis and immune regulation of Lactobacillus paracasei in the body. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art, provide a method for preparing Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis and its application in the immune direction, and can significantly increase the amount of butyric acid synthesized by Lactobacillus paracasei IOB413 in the intestine.

[0009] The present invention solves the technical problem by adopting the following technical solutions:

[0010] A method for preparing Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis, comprising the following steps:

[0011] S1, activating the strain of Lactobacillus paracasei IOB413;

[0012] S2, inoculating the activated strain into the optimized culture medium for cultivation to obtain a secondary seed solution;

[0013] S3, inoculating the secondary seed liquid into the sterilized fermentation medium for fermentation;

[0014] S4, centrifuging the fermentation broth medium, collecting bacterial mud, and freeze-drying the bacterial mud to prepare Lactobacillus paracasei IOB413 bacterial powder;

[0015] The specific steps of preparing the secondary seed solution are as follows: inoculate the strain into the optimized culture medium at a ratio of 1:15-25, and culture at 37±2°C for 24-48 hours under anaerobism to obtain the primary seed solution; inoculate the primary seeds into the optimized culture medium at a ratio of 1:20-30, and culture at 37±2°C for 24-48 hours under anaerobism to obtain the secondary seed solution;

[0016] The optimized culture medium has the following formula: 4.5-5.5 g of peptone, 9-11 g of crude black bean polysaccharide, 4.5-5.5 g of glucose, 6.5-7.5 g of beef extract powder, and 4.5-5.5 g of sodium alginate. The culture medium is dissolved in 1000 ml of distilled water, mixed, and the pH of the solution is adjusted to 6.0-6.5, and then sterilized at 110-121° C. for 20-30 min.

[0017] Moreover, the specific steps of strain activation are: inoculating the strain in the cryopreserved tube into the slant culture medium and placing it at 37±2°C for culturing, and the activation time is 20±2h;

[0018] The slant culture medium is a modified MRS culture medium, and its formula is: peptone 4.5g-5.5g, beef extract 4.5g-5.5g, yeast powder 5.5g-6.5g, glucose 14.0g-16.0g, Tween 80 0.8mL-1.2mL, dipotassium hydrogen phosphate 1.8g-2.2g, sodium acetate 3.5g-4.5g, triammonium citrate 0.8g-1.2g, magnesium sulfate 0.18g-0.22g, manganese sulfate 0.04g-0.06g, agar 14.0g-16.0g, pH=6.2±0.2, and the amount of water added is 1L.

[0019] Moreover, the specific steps of fermentation are: inoculating the cultured secondary seed liquid into the sterilized fermentation liquid medium according to the inoculation amount of 1:40-60, stirring the fermentation tank at a speed of 20-40r / min, 37±2℃, and anaerobic culture for 6-20h to obtain the fermentation liquid;

[0020] The fermentation medium is formulated as follows: 4.5-5.5 g of peptone, 9-11 g of crude black bean polysaccharide, 4.5-5.5 g of glucose, 6.5-7.5 g of beef extract powder, and 4.5-5.5 g of sodium alginate. The components are dissolved in 1000 ml of distilled water, mixed, and the pH of the solution is adjusted to 5.1-6.2, and then sterilized at 110-130° C. for 10-20 min. The inoculation amount is 3×10 7 -7×10 7 CFU / mL.

[0021] Moreover, after the fermentation was completed, the activity of the solid fermentation product obtained by centrifugation and freeze-drying was 5×10 10 -9×10 10 CFU / g.

[0022] Moreover, the Lactobacillus paracasei IOB413, strain name: IOB413, classification name: Lactobacillus paracasei, preservation number: CGMCC No.16022, preservation date: June 29, 2018, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] The invention discloses an application of the Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis in promoting butyric acid synthesis.

[0024] The advantages and positive effects of the present invention are:

[0025] 1. The present invention can significantly increase the amount of butyrate synthesized by Lactobacillus paracasei IOB413 in the intestine by optimizing the culture medium; at the same time, it significantly promotes the synthesis of other short-chain fatty acids and restores the intestinal environment; the present invention elaborates in detail that Lactobacillus paracasei IOB413 affects intestinal function by promoting butyrate synthesis.

[0026] 2. The present invention optimizes the original culture medium formula and provides a new fermentation culture method of Lactobacillus paracasei IOB413 and a preparation process of bacterial powder. By optimizing the culture medium, the obtained bacterial powder can affect butyric acid synthesis, and Lactobacillus paracasei IOB413 ultimately shows the effects of enhancing immunity, repairing damaged immune organs, improving intestinal damage, and regulating intestinal microecology.

[0027] 3. The Lactobacillus paracasei IOB413 powder product obtained by the method provided by the present invention promotes the synthesis of butyric acid in the body and plays an important role in improving immune damage and assisting in the treatment of intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a comparison of the appearance of mice in the examples of the present invention, with the left side being the blank group and the right side being the model group.

[0029] Figure 2 Graph showing the acetic acid content in mouse feces according to an embodiment of the present invention.

[0030] Figure 3 Graph showing the propionic acid content in mouse feces according to an embodiment of the present invention.

[0031] Figure 4 Graph showing the isobutyric acid content in mouse feces according to an embodiment of the present invention.

[0032] Figure 5 Graph showing the butyric acid content in mouse feces according to an embodiment of the present invention.

[0033] Figure 6 Graph showing the valeric acid content in mouse feces according to an embodiment of the present invention.

[0034] Figure 7 Graph showing the isovaleric acid content in mouse feces according to an embodiment of the present invention.

[0035] Figure 8 This is a stimulation index diagram of mouse spleen T lymphocytes in an example of the present invention.

[0036] Fig. 9 This is a stimulation index diagram of mouse spleen B lymphocytes in an example of the present invention.

[0037] Fig.10 Graph showing the relative transcription level of T-bet in the spleen of each group of mice in the examples of the present invention.

[0038] Fig.11 This is a graph showing the relative transcription level of GATA-3 in the spleen of each group of mice in the examples of the present invention.

[0039] Strain name: IOB413, classification name: Lacticaseibacillus paracasei, deposit number: CGMCC No.16022, deposit date: June 29, 2018, deposit unit: General Microbiology Center of China Culture Collection Administration, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0041] A method for preparing Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis, comprising the following steps:

[0042] S1, activating the strain of Lactobacillus paracasei IOB413;

[0043] S2, inoculating the activated strain into the optimized culture medium for cultivation to obtain a secondary seed solution;

[0044] S3, inoculating the secondary seed liquid into the sterilized fermentation medium for fermentation;

[0045] S4, the fermentation medium is centrifuged, the bacterial sludge is collected, and the bacterial sludge is freeze-dried to prepare the bacterial powder of Lactobacillus paracasei IOB413.

[0046] The Lactobacillus paracasei IOB413 used in the present invention is isolated from naturally fermented sour dough in the homes of residents in Tianjin. This strain has been preserved and tested for physical and chemical indicators. On March 19, 2021, a bacterial identification test was carried out at China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges.

[0047] Strain name: IOB413, classification name: Lacticaseibacillus paracasei, deposit number: CGMCC No.16022, deposit date: June 29, 2018, deposit unit: General Microbiology Center of China Culture Collection Administration, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0048] Example 1

[0049] A method for preparing Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis, wherein the present embodiment is a method for preparing Lactobacillus paracasei IOB413 bacterial powder No. 1 by optimizing the fermentation medium for the first time, comprising the following steps:

[0050] S1. Strain activation: inoculate the bacteria in the cryopreservation tube into the slant culture medium and place it at 37°C for 20 hours. The activated colonies are white, round, moist, opaque, with neat edges. Microscopically, the bacteria are rod-shaped, uniform and strong. After activation, the unfermented samples of Lactobacillus paracasei IOB413 are harvested.

[0051] Among them, the slant culture medium used was a modified MRS culture medium with the following formula: 5.0 g peptone, 5.0 g beef extract, 6.0 g yeast powder, 15.0 g glucose, 1.0 mL Tween 80, 2.0 g potassium dihydrogen phosphate (7H2O), 4.0 g sodium acetate (3H2O), 1.0 g triammonium citrate, 0.2 g magnesium sulfate (7H2O), 0.05 g manganese sulfate (4H2O), 15.0 g agar, pH = 6.2, and the amount of water added was 1 L.

[0052] S2. Inoculate the strain into an optimized culture medium at a ratio of 1:20, and culture at 37°C for 36 hours under anaerobism to obtain a primary seed solution; wherein the optimized culture medium is 5 g of peptone, 10 g of crude black bean polysaccharide, 5 g of glucose, 7 g of beef extract powder, and 5 g of sodium acetate, which are dissolved in 1000 ml of distilled water, mixed, and the pH of the solution is adjusted to 6.0-6.5, and then sterilized at 121°C for 15 min.

[0053] S3, inoculate the first-level seeds into the optimized culture medium (the same as the activation culture medium formula in step S2) at a ratio of 1:25, and culture at 37°C for 36 hours under anaerobism to obtain the second-level seed solution;

[0054] S4, fermentation: according to the inoculation amount of 1:50, the cultured secondary seed liquid is inoculated into the sterilized fermentation liquid medium, the fermentation tank is stirred at a speed of 30r / min, 37°C, and anaerobic culture is carried out for 14h to obtain the fermentation liquid; wherein, the fermentation liquid medium is: 5g peptone, 10g black bean crude polysaccharide, 5g glucose, 7g beef extract powder, 5g sodium acetate, which is dissolved and prepared with 1000ml distilled water, the above components are mixed, and the pH of the solution is adjusted to 6.5, and then sterilized at 121°C for 15min.

[0055] S5. Bacterial powder: centrifuge the fermentation broth medium, collect bacterial mud, and freeze-dry the bacterial mud to obtain bacterial powder of No. 1 Lactobacillus paracasei IOB413.

[0056] Example 2

[0057] A method for preparing Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis, in this embodiment, Lactobacillus paracasei IOB413 bacterial powder No. 2 prepared by optimizing the fermentation medium for the second time, comprises the following steps:

[0058] S1. The bacterial activation step is the same as that in Example 1.

[0059] S2. Inoculate the strain into the optimized culture medium at a ratio of 1:20, and culture at 37°C under anaerobism for 36 hours to obtain the primary seed solution; wherein the optimized culture medium is 5 g of peptone, 15 g of glucose, 7 g of beef extract powder, and 5 g of sodium alginate, which are dissolved in 1000 ml of distilled water, mixed, and the pH of the solution is adjusted to 6.5, and then sterilized at 121°C for 25 minutes.

[0060] S3, inoculate the first-level seeds into the optimized culture medium at a ratio of 1:25, and culture at 37°C for 36 hours under anaerobism to obtain the second-level seed solution;

[0061] S4, fermentation: according to the inoculation amount of 1:50, the cultured secondary seed liquid is inoculated into the sterilized fermentation liquid medium, the fermentation tank is stirred at a speed of 30r / min, 37°C, and anaerobic culture is carried out for 12h to obtain the fermentation liquid; wherein, the fermentation liquid medium is: 5g peptone, 15g glucose, 7g beef extract powder, 5g sodium alginate, which is dissolved and prepared with 1000ml distilled water, the above components are mixed, and the pH of the solution is adjusted to 6.2, and then sterilized at 121°C for 15min.

[0062] S5. Bacterial powder: centrifuge the fermentation broth medium to collect bacterial mud; freeze-dry the bacterial mud to obtain bacterial powder of No. 2 Lactobacillus paracasei IOB413.

[0063] Example 3

[0064] A method for preparing Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis, wherein the present embodiment is a method for preparing Lactobacillus paracasei IOB413 bacterial powder No. 3 prepared by optimizing the fermentation medium for the third time, comprising the following steps:

[0065] S1. The bacterial activation step is the same as that in Example 1.

[0066] S2. Inoculate the strain into the optimized culture medium at a ratio of 1:20, and culture at 37°C under anaerobism for 36 hours to obtain the first-level seed solution, wherein the optimized culture medium is 5 g of peptone, 10 g of crude black bean polysaccharide, 5 g of glucose, 7 g of beef extract powder, and 5 g of sodium alginate, which are dissolved in 1000 ml of distilled water, mixed, and the pH of the solution is adjusted to 6.5, and then sterilized at 121°C for 25 minutes.

[0067] S3, inoculate the first-level seeds into the optimized culture medium at a ratio of 1:25, and culture at 37°C for 36 hours under anaerobism to obtain the second-level seed solution;

[0068] S4, fermentation: according to the inoculation amount of 1:50, the cultured secondary seed liquid is inoculated into the sterilized fermentation liquid medium, the fermentation tank is stirred at a speed of 30r / min, 37°C, and anaerobic culture is carried out for 12h to obtain the fermentation liquid; wherein, the fermentation liquid medium is: 5g peptone, 10g black bean crude polysaccharide, 5g glucose, 7g beef extract powder, 5g sodium alginate, which is dissolved and prepared with 1000ml of distilled water, the above components are mixed, and the pH of the solution is adjusted to 6.2, and then sterilized at 121°C for 15min.

[0069] S5. Bacterial powder: centrifuge the fermentation broth medium to collect bacterial mud; freeze-dry the bacterial mud to obtain bacterial powder of No. 3 Lactobacillus paracasei IOB413.

[0070] Example 4

[0071] The preparation method of Lactobacillus paracasei IOB413 bacterial powder, which is prepared from Lactobacillus paracasei IOB413 bacterial powder using an unoptimized fermentation medium, comprises the following steps:

[0072] S1. The bacterial activation step is the same as that in Example 1.

[0073] S2. Inoculate the strain into the optimized culture medium at a ratio of 1:20, and culture at 37°C under anaerobism for 36 hours to obtain the first-level seed solution. The unoptimized culture medium formula is 5 g of peptone, 15 g of glucose, 7 g of beef extract powder, and 5 g of sodium acetate. Use 1000 ml of distilled water to dissolve and prepare. Mix the above components, adjust the solution pH to 6.5, and then sterilize at 121°C for 25 minutes.

[0074] S3, inoculate the first-level seeds into the unoptimized culture medium at a ratio of 1:25, and culture at 37°C for 36 hours under anaerobism to obtain the second-level seed solution;

[0075] S4, fermentation: according to the inoculation amount of 1:50, the cultured secondary seed liquid is inoculated into the sterilized fermentation liquid medium, the fermentation tank is stirred at a speed of 30r / min, 37°C, and anaerobic culture is carried out for 12h to obtain the fermentation liquid; wherein, the fermentation liquid medium is: 5g peptone, 15g glucose, 7g beef extract powder, 5g sodium acetate, which is dissolved and prepared with 1000ml distilled water, the above components are mixed, and the pH of the solution is adjusted to 6.2, and then sterilized at 121°C for 15min.

[0076] S5. Bacterial powder: centrifuge the fermentation broth medium to collect bacterial mud; freeze-dry the bacterial mud to obtain bacterial powder of No. 4 Lactobacillus paracasei IOB413.

[0077] Example 5

[0078] Study on the effect of Lactobacillus paracasei IOB413 powder obtained by different preparation methods on improving immunosuppression damage in mice

[0079] 1. Experimental Materials

[0080] 1.1 Experimental animals

[0081] 7-week-old SPF BALB / c male mice (weighing about 22 g) were purchased from Sbefor (Beijing, China) with a license number of SCXK (Beijing) 2019-0010. The animals were kept in the barrier animal room of Tianjin University of Science and Technology with a temperature of (23±2)°C, humidity of (55±5%), and a light exposure time of 12 h in 24 h. The mice were required to adapt to the environment for one week before the formal experiment. During the animal experiment, the mice were kept free to eat, and all experimental procedures were carried out in accordance with the provisions of the Guide for the Care and Use of Laboratory Animals.

[0082] 1.2 Reagents and consumables

[0083] The main biological and chemical reagents used in the experiment are shown in Table 1:

[0084] Table 1 Main biochemical reagents in the experiment

[0085]

[0086] 2. Experimental Methods

[0087] 2.1 Preparation of bacterial culture

[0088] 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.

[0089] 2.2 Preparation of main solutions

[0090] Con A storage solution: Weigh about 1 mg of concanavalin (Con A) powder and dissolve it in about 1 mL of PBS to prepare 1 mg / mL Con A storage solution. Filter it with a 0.22 μm water filter membrane and place it in a 4°C refrigerator for use.

[0091] LPS stock solution: Dissolve 1 mg of LPS powder in 1 mL of PBS solution, filter through a 0.22 μm water filter membrane to prepare a 1 mg / mL LPS stock solution, and store in a refrigerator at 4°C for later use.

[0092] 2.3 Grouping

[0093] Forty-eight 7-week-old SPF mice were divided into 6 groups, namely, a blank group, a model group, a group treated with the powder of Lactobacillus paracasei 10B413 of Example 3 (experimental group 1), a group treated with the powder of Lactobacillus paracasei 10B413 of Example 1 (experimental group 2), a group treated with the powder of Lactobacillus paracasei 10B413 of Example 2 (experimental group 3), and a group treated with the powder of Lactobacillus paracasei 10B413 of Example 4 (experimental group 4), with 8 mice in each group.

[0094] 2.4 Modeling and intervention plan

[0095] The immunosuppressive mouse model was constructed by intraperitoneal injection of cyclophosphamide (CTX). After a 7-day adaptation period, cyclophosphamide (80 mg / kg) was injected intraperitoneally on the 1st, 2nd, 3rd, 18th, and 22nd days of the experiment to establish an immunosuppressive mouse model. The blank group was given an equal amount of normal saline. From the 4th to the 28th day, the mice were gavaged daily. Experimental group 1 was given the powder of Lactobacillus paracasei IOB413 (1×10 9 CFU / mL) 0.2mL / day; Experimental group 2 was given Lactobacillus paracasei IOB413 powder (1×10 9 CFU / mL) 0.2 mL / day, and the experimental group 3 was given the powder of Lactobacillus paracasei IOB413 of Example 2 (1×10 9 CFU / mL) 0.2 mL / day, and the experimental group 4 was given the powder of Lactobacillus paracasei IOB413 of Example 4 (1×10 9 CFU / mL), and the gavage volume was also 0.2 mL. The normal group and the model group were gavaged with an equal amount of normal saline (0.2 mL) every day.

[0096] 3.Indicator observation

[0097] 3.1 Observe the changes in the body shape, hair and other appearance of the mice and weigh the mice on the 1st, 4th and 28th days of the experiment to observe the effect of Lactobacillus paracasei IOB413 powder on the weight of the mice before and after the optimization of the culture medium composition.

[0098] 3.2 Effects on SCFAs content in the intestine of mice

[0099] Mouse feces were collected for SCFAs detection.

[0100] 3.3 Effects on mouse immune organ indexes

[0101] At the end of the experiment, the complete spleen and the fat tissue attached to the spleen were removed, and the other organs of the mouse were removed. The blood stains were washed with saline and then dried with filter paper. The weights were weighed and recorded. The mouse immune organ index was calculated according to the following formula:

[0102] Immune organ coefficient = organ weight (mg) / body weight (g)

[0103] 3.4 Effect on proliferation of mouse spleen lymphocytes

[0104] Immediately place the mouse spleen that was removed in the clean bench in the previous step in pre-cooled PBS. Subsequently, place it on a sterilized 200-mesh cell sieve and grind it through the sieve. Rinse the sieve with PBS during the grinding process and collect the rinse solution into a 10 mL centrifuge tube. Subsequently, centrifuge at 1000 rpm for 5 min and discard the supernatant. Add red blood cell lysis solution to the precipitate, mix well and centrifuge at 1000 rpm for 5 min. Repeat the above steps 2-3 times. Subsequently, suspend the precipitate in RPMI-1640 cell culture medium to obtain a spleen cell suspension. The cell suspension was counted by trypan blue staining. After determining that the number of live cells was >95%, the cell concentration was adjusted to 1×10 6 cells / mL.

[0105] ConA stock solution and LPS stock solution were added to RPMI-1640 cell culture medium, respectively, to prepare RPMI-1640 cell culture medium containing 5 μg / mL ConA and 10 μg / mL LPS, respectively.

[0106] Take 100 μL of spleen lymphocyte suspension and add it to a 96-well plate. Add 100 μL of RPMI-1640 complete medium to the control well, and add 100 μL of RPMI-1640 cell culture medium containing ConA and LPS (concentrations are 5 μg / mL and 10 μg / mL, respectively) to the test well. Set up 3 parallels for each well. Place the cell culture plate in a 37°C, 5% CO2 cell culture incubator for 44 h, and remove the supernatant with a pipette. Add 20 μL of MTT reagent (5 mg / mL) and 180 μL of RPMI-1640 cell culture medium again and culture for another 4 h. Discard the supernatant, add 150 μL of DMSO and shake well. Subsequently, use an enzyme-labeled instrument to detect the absorbance value of each well at 490 nm. Calculate the absorbance values ​​of the test wells and control wells according to the following formula:

[0107] Irritation Index (SI) = A 试验组 / A 对照组

[0108] 3.5 Effects on Th cell differentiation in mouse spleen

[0109] qPCR was used to analyze the relative transcription levels of T-bet (gene ID: 57765), GATA-3 (gene ID: 14462), and β-actin (gene ID: 11461) in the spleen of mice and the changes in the spleen tissue of mice.

[0110] RNA was extracted from spleen tissues of mice in each group using Trizol reagent. After the concentration of the extracted RNA was tested, cDNA was synthesized using HiFi-Script cDNA Kit reverse transcription kit.

[0111] The desired mouse spleen immune factor target gene sequence was searched in the NCBI database, and the primers were designed as shown in Table 2.

[0112] Table 2 Primer sequences

[0113]

[0114] Quantitative PCR (qPCR) was performed using the fluorescent dye SYBR-Green to analyze the transcription level of each transcription factor. The qPCR reaction system is shown in Table 3, and ddH2O was used instead of cDNA as a template as a blank control.

[0115] Table 3 RT-qPCR reaction system

[0116]

[0117] The qPCR program was as follows: 95°C for 2 min, 95°C for 15 s, 60°C for 15 s, and 55°C for 30 s. 40 cycles were performed under this parameter setting. -△△Ct The relative transcription level of the target gene mRNA after the reaction was calculated by this method.

[0118] 4. Data statistical processing methods

[0119] All data were expressed as mean ± standard deviation and plotted using GraphPad Prism 8 and Origin 2021 software.

[0120] 5. Experimental Results

[0121] 5.1 Changes in appearance and weight of mice

[0122] After three consecutive days of CTX injection (80 mg / kg BW), the status of mice was as follows Figure 1 As shown in the figure, the mice in the blank group had a balanced body shape, smooth and shiny hair, and were full of energy, while the mice in the immunosuppressed model group had unkempt and dry hair and were listless. There was a clear difference between the two groups of mice.

[0123] The mice were weighed on the 1st, 4th and 28th days of the experiment to observe the effect of bacterial powder obtained by different preparation methods on the weight of mice. The results are shown in Table 4.

[0124] Table 4. Changes in mouse body weight

[0125]

[0126] After mice were injected with CTX for 3 consecutive days, the body weight of the blank group, model group, experimental group 1, experimental group 2, experimental group 3 and experimental group 4 decreased significantly. On the 28th day, only the body weight of mice in the model group was still significantly lower than that in the normal group, while the body weight of mice in the four experimental groups increased to varying degrees.

[0127] This shows that the intervention of four groups of Lactobacillus paracasei IOB413 powder had a good recovery effect on the weight loss of mice caused by CTX-induced immunosuppression.

[0128] 5.2 Effects on SCFAs and butyrate content in the intestine of mice

[0129] Table 5 Synthesis of short-chain fatty acids in the intestine of mice

[0130]

[0131] From the data in the above table, it can be seen that the fermentation of Lactobacillus paracasei IOB413 powder with the addition of crude black bean polysaccharide and sodium alginate significantly increased the synthesis of butyric acid in the mouse intestine. Compared with the unoptimized fermentation, the butyric acid synthesis increased by 77.51%, compared with the powder fermented with crude black bean polysaccharide alone, the butyric acid synthesis increased by 78.97%, and compared with the powder fermented with sodium alginate alone, the butyric acid synthesis increased by 76.15%. This shows that the fermentation culture of Lactobacillus paracasei IOB413 after component optimization significantly increased the synthesis of butyric acid and other SCFAs in the mouse intestine.

[0132] Effects of high-viable Lactobacillus paracasei IOB413 powder obtained by different preparation methods on the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in feces of immunosuppressed mice Figure 2-Figure 7 There was no significant difference in the content of valeric acid and isovaleric acid in the feces of mice in different groups; there were extremely significant differences in the levels of acetic acid, propionic acid, isobutyric acid and butyric acid in the intestine of mice in experimental group 1 compared with those in the model group; there were also significant differences in acetic acid, propionic acid, isobutyric acid and butyric acid between experimental group 1 and experimental groups 2, 3 and 4, among which the difference in butyric acid synthesis was the largest, indicating that the fermented Lactobacillus paracasei IOB413 after component optimization can significantly increase the synthesis of butyric acid in the intestine of mice, and at the same time significantly increase the synthesis of SCFAs in the intestine of mice.

[0133] 5.3 Effects on mouse immune organ indices

[0134] Under the influence of CTX, the immune system of mice was destroyed and the spleen became abnormally enlarged. However, the two groups of Lactobacillus paracasei IOB413 powder showed different improvement effects.

[0135] Table 6 Effect on mouse spleen index

[0136]

[0137] In each intervention group, the thymus index of mice in the three experimental groups was not significantly different from that in the model group. Combined with the experimental results, under the influence of CTX, the immune system of mice was destroyed, the thymus atrophied, and the improvement effect of experimental group 1 on the thymus of mice was slightly better than that of experimental groups 2, 3, and 4.

[0138] Table 7 Effects on mouse thymus index

[0139]

[0140] The above results indicate that the Lactobacillus paracasei IOB413 powder in Experimental Group 1 significantly increased the amount of butyrate synthesis and exhibited a better immunoregulatory effect.

[0141] 5.4 Effects on the proliferation of mouse spleen lymphocytes

[0142] The stimulation index of mouse spleen T lymphocytes is as follows Figure 8 As shown in the figure, the T lymphocyte stimulation index of the model group was significantly lower than that of the normal group, indicating that under the influence of CTX, the proliferation of T lymphocytes in mice was inhibited. Compared with the model group, the stimulation index of experimental group 1 and experimental group 2 increased to varying degrees. Among them, the experimental group 2 was significantly improved compared with the model group. Compared with the model group, the T lymphocyte stimulation index of experimental group 1 was significantly improved.

[0143] Stimulation index of mouse spleen B lymphocytes Fig. 9 As shown. Compared with the normal group, the stimulation index of B lymphocytes in the model group was significantly reduced, reflecting the strong inhibitory effect of CTX on the proliferation of B lymphocytes in mice. The stimulation index of each intervention group was increased to varying degrees compared with the model group. There were significant differences between experimental groups 2 and 3 and the model group, and there was a very significant difference between experimental group 1 and the model group.

[0144] The results of the mouse splenic lymphocyte proliferation test showed that Lactobacillus paracasei IOB413 powder could effectively promote the proliferation of splenic lymphocytes in immunosuppressive mice, and the effect of experimental group 1 was significantly stronger than that of experimental groups 2, 3, and 4.

[0145] 5.5 Effects on differentiation of Th cells in mouse spleen

[0146] The relative transcription levels of T-bet in the spleen of each group of mice are shown in Fig.10As shown. Compared with the blank group, the relative transcription level of T-bet mRNA in the spleen of mice in the model group decreased by 56.50%, with a very significant difference; compared with the model group, the relative transcription level of T-bet mRNA in the spleen of mice in experimental groups 1 and 2 increased to varying degrees. And the relative transcription level of T-bet mRNA was significantly increased in experimental group 1.

[0147] The relative transcription level of GATA-3 in the spleen of each group of mice is shown in Fig.11 Compared with the blank group, the relative transcription level of GATA-3 in the spleen of mice in the model group decreased by 56.75%, which was a very significant difference; the two groups of bacterial powder showed significant changes compared with the model group, and the relative transcription level of GATA-3 mRNA in the spleen of mice in the experimental group 1 increased significantly.

[0148] In summary, it can be seen that under the influence of CTX, the levels of T-bet and GATA-3 in the spleen of mice decreased, which hindered the differentiation of T cells into Th1 cells and Th2 cells, and reduced the immunity of mice. Lactobacillus paracasei IOB413 can affect intestinal function by increasing butyrate synthesis and binding to SCFAs receptors, activating Th1 and Th2 cells in the spleen of immunosuppressive mice and improving the immunity of mice.

[0149] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a Lactobacillus paracasei IOB413 bacterial powder for regulating butyric acid synthesis, characterized in that: Here are the steps: S1, activating the strain of Lactobacillus paracasei IOB413; S2, inoculating the activated strain into the optimized culture medium for cultivation to obtain a secondary seed solution; S3, inoculating the secondary seed liquid into the sterilized fermentation medium for fermentation; S4, centrifuging the fermentation broth medium, collecting bacterial mud, and freeze-drying the bacterial mud to prepare Lactobacillus paracasei IOB413 bacterial powder; The specific steps of preparing the secondary seed solution are as follows: inoculate the strain into the optimized culture medium at a ratio of 1:15-25, and culture at 37±2°C for 24-48 hours under anaerobism to obtain the primary seed solution; inoculate the primary seeds into the optimized culture medium at a ratio of 1:20-30, and culture at 37±2°C for 24-48 hours under anaerobism to obtain the secondary seed solution; The optimized culture medium has the following formula: 4.5-5.5 g of peptone, 9-11 g of crude black bean polysaccharide, 4.5-5.5 g of glucose, 6.5-7.5 g of beef extract powder, and 4.5-5.5 g of sodium alginate. The mixture is dissolved in 1000 ml of distilled water, and the pH of the solution is adjusted to 6.0-6.5, and then sterilized at 110-121° C. for 20-30 min. The specific steps of fermentation are: inoculating the cultured secondary seed liquid into the sterilized fermentation liquid medium according to the inoculation amount of 1:40-60, stirring the fermentation tank at a speed of 20-40r / min, 37±2℃, and anaerobic culture for 6-20h to obtain the fermentation liquid; The fermentation medium is formulated as follows: 4.5-5.5 g of peptone, 9-11 g of crude black bean polysaccharide, 4.5-5.5 g of glucose, 6.5-7.5 g of beef extract powder, and 4.5-5.5 g of sodium alginate. The components are dissolved in 1000 ml of distilled water, mixed, and the pH of the solution is adjusted to 5.1-6.2, and then sterilized at 110-130° C. for 10-20 min. The inoculation amount is 3×10 7 -7×10 7 CFU / mL; The Lactobacillus paracasei IOB413, strain name: IOB413, classification name: Lactobacillus paracasei ( Lacticaseibacillus paracasei ), deposit number: CGMCC No.16022, deposit date: June 29, 2018, deposit unit: General Microbiology Center of China Culture Collection Administration, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. the preparation method of the synthetic Lactobacillus paracasei IOB413 bacteria powder of regulation and control butyric acid according to claim 1, is characterized in that: The specific steps of strain activation are as follows: inoculate the strain in the cryopreserved tube into the slant culture medium and place it at 37±2℃ for incubation, and the activation time is 20±2h; The slant culture medium is a modified MRS culture medium, and its formula is: peptone 4.5g-5.5g, beef extract 4.5g-5.5g, yeast powder 5.5g-6.5g, glucose 14.0g-16.0g, Tween 80 0.8mL-1.2mL, dipotassium hydrogen phosphate 1.8g-2.2g, sodium acetate 3.5g-4.5g, triammonium citrate 0.8g-1.2g, magnesium sulfate 0.18g-0.22g, manganese sulfate 0.04g-0.06g, agar 14.0g-16.0g, pH=6.2±0.2, and the amount of water added is 1L.

3. the preparation method of the synthetic Lactobacillus paracasei IOB413 bacteria powder of regulation and control butyric acid according to claim 1, is characterized in that: After the fermentation, the solid fermentation product was centrifuged and freeze-dried, and the activity of the solid fermentation product was 5×10 10 -9×10 10 CFU / g.

4. A Lactobacillus paracasei IOB413 powder for regulating butyric acid synthesis, characterized in that: Prepared by the method of claim 1.

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