Preparation method of probiotic combination segment solid-state fermentation postbiotic and application thereof in regulating macrophage activity

Abstract

The present application relates to a kind of preparation method of probiotic combination segment solid-state fermentation post-biogenic substance and its application in regulating macrophage activity, by the synergistic effect of lactic acid bacteria and bifidobacterium, fermenting lactobacillus mucus IOB802, paracasei IOB413 can rapidly multiply under aerobic conditions, quickly consume oxygen and produce anaerobic environment, create good growth environment for animal bifidobacterium lactis IOB-LO7, decompose dietary fiber, resistant starch and other indigestible substances in cicer arietinum during fermentation process, produce beneficial short-chain fatty acids, enzymes and other metabolites, improve the utilization of cicer arietinum, while can improve the metabolic function of intestinal flora, enhance immunity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a preparation method of probiotic combination segment solid-state fermentation post-bion and application thereof in regulating macrophage activity. BACKGROUND

[0002] Immunity refers to the function of the body's immune system to recognize self and foreign substances and to exclude antigenic foreign matter through immune response to maintain the body's physiological balance, and is a defense line to protect the body's health. If the immune system is out of balance, the level of immunity will be affected, leading to immune dysfunction and disease. However, in modern society, people's work and life pace is accelerating, and phenomena such as stress, lack of exercise, etc. are common, and at the same time, in order to pursue convenience, fast food, take-out, etc. become the best choice. Unhealthy diet, sedentary, lack of sleep, etc. lead to malnutrition in individuals, often manifested as listlessness, fatigue, decreased appetite, sleep disorders, etc., and long-term sub-health state leads to decreased immunity. At present, immunity is adjusted through daily diet. Although there are many types of food for enhancing immunity, these products generally have the defects of complex components and non-obvious effect, and need to be taken for a long time to achieve the effect of sustained and systematic enhancement of immunity.

[0003] In order to solve these problems, researchers have been studying how to improve the health of the body by regulating the intestinal flora, among which the preparation method and application of complex probiotics have become a research hotspot. Through research and exploration, it is found that the use of probiotics such as lactic acid bacteria and bifidobacterium can produce more beneficial substances and better maintain intestinal health through synergistic effect. Different ecological relationships exist between different microorganisms, and different strains can make up for their own metabolic characteristics short board by utilizing metabolic products to achieve mutual symbiosis.

[0004] There are many limitations to solid-state fermentation, such as: ① Limited selection of strains: Solid-state fermentation is mainly suitable for low-water activity-tolerant microorganisms, which limits the selection of strains. ② Slow fermentation speed and long cycle: Due to the limitations of mass and heat transfer in solid-state fermentation, the fermentation speed is slow, resulting in a long production cycle. ③ Difficult to detect and control process parameters: Solid-state fermentation is a heterogeneous reaction process, and it is difficult to measure and control parameters. ④ More labor-intensive operation: Due to the difficulty in accurately controlling process parameters, more labor and time are required during operation. ⑤ Limited by mass and heat transfer: In solid-state fermentation, the transfer of oxygen and nutrients required for microbial growth is limited, resulting in unstable fermentation effects. ⑥ Product quality and yield affected by raw materials: Natural raw materials have complex and variable compositions, affecting the quality and yield of fermentation products. These shortcomings limit the efficiency and stability of solid-state fermentation in some applications, which need to be paid attention to and improved in actual operation.

[0005] In summary, at present, almost all composite bacteria fermentation adopts liquid fermentation, and after liquid fermentation is completed, centrifugal separation or other solid-liquid separation methods are used to add protective agents to prepare composite bacterial agents. In the solid-liquid separation process, organic acids, polypeptides and other substances with bacteriostatic effects produced by probiotics during liquid fermentation are removed. No similar literature reports have been found.

[0006] Therefore, the preparation method and application of the probiotic combination segmented solid-state fermentation post-biogenic agent have important significance. It not only provides an effective method to improve intestinal health, enhance immune function, etc., but also provides a new research direction and application prospect for the field of medicine, health care products, etc. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art, provide a preparation method of probiotic combination segmented solid-state fermentation post-biogenic agent and its application in regulating macrophage activity, enrich the types of post-biogenic agents, improve the content of active metabolites, and not be limited by gastric acid and bile salts, which can play a probiotic function in the intestinal tract.

[0008] The technical problem of the present application is solved by adopting the following technical scheme:

[0009] A preparation method of a probiotic combination segmented solid-state fermentation post-biogenic agent, comprising the following steps:

[0010] S1, strain activation: respectively activate and culture the fermentation Lactobacillus mui IOB802, Paracasei IOB413 and Bifidobacterium animalis lactis IOB-LO7 to obtain the fermentation Lactobacillus mui IOB802 secondary seed liquid, Paracasei IOB413 secondary seed liquid and Bifidobacterium animalis lactis IOB-LO7 secondary seed liquid;

[0011] S2, aerobic fermentation: inoculating the activated and cultured secondary seed liquid of Paracasei IOB413 into the sterilized solid-state fermentation substrate at an inoculation amount of 1:1.5-1:1.75 of material to liquid ratio, then performing aerobic fermentation, and then low-temperature drying treatment to obtain the Paracasei IOB413 postbiotic;

[0012] S3, facultative anaerobic fermentation: inoculating the activated and cultured secondary seed liquid of Limosilactobacillus IOB802 into the Paracasei IOB413 postbiotic at an inoculation amount of 1:1.5-1:1.75 of material to liquid ratio, then performing facultative anaerobic fermentation, and then low-temperature drying treatment to obtain the Paracasei IOB413 and Limosilactobacillus IOB802 compound postbiotic;

[0013] S4, strict anaerobic fermentation: inoculating the activated and cultured secondary seed liquid of Bifidobacterium animalis ssp. lactis IOB-LO7 into the Paracasei IOB413 and Limosilactobacillus IOB802 compound postbiotic at an inoculation amount of 1:1.5-1:1.75 of material to liquid ratio, then performing strict anaerobic fermentation, and then high-temperature inactivation, drying, and crushing treatment to obtain the probiotic combination segmented solid-state fermentation postbiotic.

[0014] Moreover, the Paracasei IOB413 has a strain name of IOB413, a classification name of Lacticasseibacillus paracasei, a preservation number of CGMCC No. 16022, a preservation date of June 29, 2018, a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing;

[0015] The Limosilactobacillus IOB802 has a strain name of IOB802, a classification name of Limosilactobacillus fermentum, a preservation number of CGMCC No. 23120, a preservation date of August 5, 2021, a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing;

[0016] The animal bifidobacterium lactis IOB-LO7 has a strain name of IOB-LO7, a classification name of Bifidobacterium animalis subsp. lactis, a preservation number of CGMCC No. 24185, a preservation date of Dec. 23, 2021, and a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0017] Moreover, in step S1, the Paracasei IOB413 strain activation method is as follows: the Paracasei IOB413 is taken out from a strain cryopreservation tube, inoculated in an activation medium at a volume ratio of 1:20, incubated at 37±2°C for 22±2h, to obtain a first-stage seed liquid, and the first-stage seed liquid is inoculated in an activation medium at a volume ratio of 1:25, incubated at 37±2°C for 22±2h, to obtain a second-stage seed liquid.

[0018] The activation medium of the Paracasei IOB413 is MRS medium, and the components include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO·7H2O, 5.0g of CH3COONa·3H2O, 2.0g of C6H5O7(NH4)3, 0.2g of MgSO 4` 7H2O, 0.05g of MnSO4·4H2O, 15.0g of agar, and the volume is adjusted to 1L, the pH is adjusted to 6.2±0.2, and sterilized at 121°C for 15min.

[0019] The fermentation of the L. mesenteroides IOB802 strain activation method is as follows: the L. mesenteroides IOB802 is taken out from a strain cryopreservation tube, inoculated in an activation medium at a volume ratio of 1:20, incubated at 37±2°C for 22±2h, to obtain a first-stage seed liquid, and the first-stage seed liquid is inoculated in an activation medium at a volume ratio of 1:25, incubated at 37±2°C for 22±2h, to obtain a second-stage seed liquid.

[0020] The activation medium of the L. mesenteroides IOB802 is MRS medium, and the components include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO·7H2O, 5.0g of CH3COONa·3H2O, 2.0g of C6H5O7(NH4)3, 0.2g of MgSO 4`7H2O 0.2g, MnSO4·4H2O 0.05g, agar 15.0g, constant volume to 1L, adjust pH=6.2±0.2, sterilize at 121℃ for 15min;

[0021] The animal bifidobacterium lactis IOB-LO7 strain activation method is as follows: the animal bifidobacterium lactis IOB-LO7 is taken out from a strain cryopreservation tube, inoculated in an activation culture medium at a volume ratio of 1:20, activated in a closed anaerobic environment at 37±2℃, and the first-stage seed liquid is obtained after 20±2h of activation; the first-stage seed liquid is inoculated in an activation culture medium at a volume ratio of 1:25, activated in a closed anaerobic environment at 37±2℃, and the second-stage seed liquid is obtained after 24±2h of activation.

[0022] The activation culture medium of the animal bifidobacterium lactis IOB-LO7 is a modified TPY liquid culture medium, and the components include: hydrolyzed casein 10.0g, plant peptone 5.0g, yeast powder 2.0g, glucose 5.0g, L-cysteine 1.0g, dipotassium hydrogen phosphate 2.0g, constant volume to 1L, adjust pH 6.5±0.1, sterilize at 121℃ for 15min.

[0023] Moreover, in step S2, the solid-state fermentation substrate is chickpea.

[0024] Moreover, in step S2, the aerobic fermentation condition is that the culture temperature is 37±2℃, the ventilation amount is 0.2m³ / min, and the standing culture is 12±2h, and the Lactobacillus paracasei IOB413 fermentation product is obtained.

[0025] Moreover, in step S3, the facultative anaerobic fermentation condition is that the culture temperature is 37±2℃, the standing culture is 12±2h, and the Lactobacillus paracasei IOB413 and the Lactobacillus mucosus IOB802 compound fermentation product is obtained.

[0026] Moreover, in step S4, the strict anaerobic fermentation condition is that the culture temperature is 37±1℃, the nitrogen ventilation amount is 0.1m³ / min, the culture is in a closed anaerobic environment, and the standing culture is 12±2h, and the probiotic combination segmented solid-state fermentation product is obtained.

[0027] Moreover, in step S4, the high-temperature inactivation temperature is 75±2℃, and the drying temperature is 55±2℃.

[0028] A probiotic combination segmented solid-state fermentation postbiotic is prepared by the above method.

[0029] The probiotic combination segmented solid-state fermentation postbiotic is used for preparing an immunoregulation preparation.

[0030] The advantages and positive effects of the present application are as follows:

[0031] 1. This invention utilizes the synergistic effect of lactic acid bacteria and bifidobacteria to ferment Lactobacillus mucinus IOB802 and Lactobacillus paracasei IOB413, enabling them to rapidly multiply under aerobic conditions and quickly consume oxygen to create an anaerobic environment. This provides a favorable growth environment for Bifidobacterium animalis subsp. lactis IOB-LO7. During fermentation, it decomposes indigestible substances such as dietary fiber and resistant starch in chickpeas, producing beneficial short-chain fatty acids, enzymes, and other metabolites, thereby improving the utilization rate of chickpeas. At the same time, it can improve the metabolic function of intestinal flora and enhance the body's immunity.

[0032] 2. This invention utilizes a three-stage fermentation process—aerobic, facultative, and strictly anaerobic—to produce extracellular polysaccharides, organic acids, short-chain fatty acids, and small-molecule peptides, while retaining nutrients such as soluble fiber, amino acids, and flavonoids from the fermentation substrate. These nutrients efficiently promote the proliferation of fermenting *Lactobacillus mucinus* IOB802, *Lactobacillus paracasei* IOB413, and *Bifidobacterium animalis* subsp. *lactobacter* IOB-LO7, while effectively protecting the activity of the strains and increasing the content of intracellular active substances in these strains. This enriches the types of metabolites, increases the content of active metabolites, and allows them to exert probiotic functions in the intestines, unaffected by gastric acid and bile salts.

[0033] 3. This invention obtains a variety of probiotic postbiotics through a segmented fermentation preparation method. Cell experiments and animal experiments have verified that the postbiotics can activate macrophages with appropriate concentrations of Bifidobacterium, thereby increasing the release of IL-6 and TNF-α from macrophages, restoring the spleen coefficient reduction and inhibiting the proliferation of splenic lymphocytes caused by CTX, and effectively enhancing the immunity of immunocompromised mice.

[0034] 4. This invention uses chickpeas as a solid-state fermentation substrate. The resistant starch and dietary fiber in chickpeas are progressively broken down by probiotics during staged fermentation, generating highly active metabolites such as short-chain fatty acids and extracellular polysaccharides. Simultaneously, the soluble fiber, flavonoids, and amino acids in the substrate are fully preserved. Through a three-stage fermentation process—aerobic-facultative anaerobic-strict anaerobic—different bacterial strains (Lactobacillus paracasei IOB413, Lactobacillus fermentatus IOB802, and Bifidobacterium animalis subsp. lactis IOB-LO7) sequentially utilize the complex carbohydrates in chickpeas, forming a synergistic system of metabolites and substrate nutrients. Compared to liquid fermentation, solid-state fermentation is closer to the intestinal microenvironment, promoting symbiotic interactions among bacterial strains. This results in a 30% increase in short-chain fatty acid content in the metabolites, a flavonoid retention rate of over 90%, and significantly enhanced stability. It can regulate intestinal flora and enhance immunity without relying on live bacteria.

[0035] 5、The present application first adds postbiotics in solid-state culture medium, postbiotics refers to the metabolites or cell components produced by microbial fermentation, which has biological activity and can improve the various deficiencies of existing solid-state fermentation culture, the present application first finds that postbiotics can change the fermentation characteristics: from aerobic fermentation, facultative anaerobic fermentation to strict anaerobic fermentation, simulate the process of food digestion in the intestine in vitro, make full use of the characteristics of different strains, improve the efficiency of the fermentation process, to ensure the full accumulation of metabolites, meet the growth needs of various probiotics. After the addition of postbiotics, the characteristics of fermentation products change greatly: various metabolites (such as short-chain fatty acids, enzymes, polypeptides, etc.) and cell components (such as cell wall, cytoplasm, etc.) have high stability after processing, which is convenient for storage and application. Improve the function of existing postbiotics, further enhance the health functions of postbiotics such as regulating intestinal flora, enhancing immunity and anti-inflammatory; after strict screening and evaluation, the safety is higher.

[0036] 6、The present application first finds that postbiotics contains various bioactive components such as short-chain fatty acids, antibacterial peptides, cell wall components, etc., which can be used as functional substrates or signal molecules in the fermentation process, further promote the growth and metabolism of target microorganisms, thereby enhancing the biological activity of fermentation products, at the same time, activate specific metabolic pathways of microorganisms, produce more metabolites with immunomodulatory function, oligosaccharides, organic acids and other components in postbiotics can significantly promote the growth and metabolism of bifidobacteria. Polypeptides and vitamins can enhance the fermentation capacity of lactic acid bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The figure is the content of crude polysaccharide in different groups of the present application.

[0038] Figure 2 The figure is the content of polypeptide in different groups of the present application.

[0039] Figure 3 The figure is the content of organic acid in different groups of the present application.

[0040] Figure 4 The figure is the content of acetic acid in different groups of the present application.

[0041] Figure 5 The figure is the content of propionic acid in different groups of the present application.

[0042] Figure 6 The figure is the content of butyric acid in different groups of the present application.

[0043] Figure 7 The figure is the content of valeric acid in different groups of the present application.

[0044] Figure 8 The figure is the relative activity of RAW264.7 cells in the present application.

[0045] Figure 9Figure for the amount of NO released by RAW264.7 cells in the present application.

[0046] Figure 10 Figure for the amount of IL-6 released by RAW264.7 cells in the present application.

[0047] Figure 11 Figure for the amount of TNF-α released by RAW264.7 cells in the present application.

[0048] Figure 12 Figure for the spleen coefficient of mice in different groups in the present application.

[0049] Figure 13 Figure for the spleen lymphocyte proliferation of mice in different groups in the present application.

[0050] Figure 14 Figure for the flow cytometry results of spleen lymphocytes of mice in different groups in the present application. DETAILED DESCRIPTION

[0051] The present application will be further described below through specific examples, which are only descriptive and not limiting, and cannot limit the protection scope of the present application.

[0052] The probiotic combination used in the present application includes Lacticasseibacillus paracasei IOB413, Fermented Mucilaginous Lactobacillus IOB802, and Animal Bifidobacterium lactis Subspecies IOB-LO7, which are obtained by independent screening.

[0053] Lacticasseibacillus paracasei IOB413 is screened from natural fermented sourdough in Tianjin residents' homes. The strain has been preserved and the physicochemical indicators have been detected. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 29, 2018, with the preservation number of CGMCC No. 16022 and the address of 1st North Chenxi Road, 3rd Courtyard, Chaoyang District, Beijing. The strain has been preserved and the physicochemical indicators have been detected. Bacterial identification detection was carried out in China Food Fermentation Industry Research Institute Co., Ltd. on March 19, 2021. The colony is white, round, surface wet, opaque, and the edge is neat.

[0054] Limosilactobacillus fermentum IOB802 is screened from fermented pickles, and the strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 5, 2021, with a preservation number of CGMCC No. 23120 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing. The strain has been preserved and detected for physical and chemical indicators, and the bacterial identification detection was carried out in China Food Fermentation Research Institute Co., Ltd. on April 3, 2023. The colony is white, round, surface wet, opaque, and the edge is neat.

[0055] Bifidobacterium animalis subsp. lactis IOB-LO7 is screened from the feces of healthy infants, and the strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 23, 2021, with a preservation number of CGMCC No. 24185. The preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0056] The Lacticaseibacillus paracasei IOB413 has a strain name of IOB413, a classification name of Lacticaseibacillus paracasei, a preservation number of CGMCC No. 16022, a preservation date of June 29, 2018, a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing;

[0057] The Limosilactobacillus fermentum IOB802 has a strain name of IOB802, a classification name of Limosilactobacillus fermentum, a preservation number of CGMCC No. 23120, a preservation date of August 5, 2021, a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing;

[0058] The Bifidobacterium animalis subsp. lactis IOB-LO7 has a strain name of IOB-LO7, a classification name of Bifidobacterium animalis subsp. lactis, a preservation number of CGMCC No. 24185, a preservation date of December 23, 2021, a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0059] The fermentation substrate is: the pretreatment of Cicer arietinum Linn. solid-state fermentation substrate, whole beans are soaked for 6-8 hours, sterilized, and inoculated. The present application can also use chickpea powder, and the particle size of the powder is 60-80 mesh.

[0060] Example 1

[0061] A preparation method of a probiotic combination segmented solid-state fermentation bio-preparation, and the present embodiment is a preparation method of Paracasei IOB413 bio-preparation, which comprises the following steps:

[0062] S1, strain activation: Paracasei IOB413 is taken out from the strain frozen tube, inoculated in the activation medium at 1:20, 37℃, and incubated for 22h, to obtain the first level seed liquid, and the first level seed is inoculated in the activation medium at 1:25, incubated at 37℃ for 22h, to obtain the second level seed liquid.

[0063] The activation medium is MRS medium, and the components thereof include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO·7H2O, 5.0g of CH3COONa·3H2O, 2.0g of C6H5O7(NH4)3, 0.2g of MgSO 4` 4H2O, 0.05g of MnSO4·4H2O, 15.0g of agar, constant volume to 1L, pH=6.2, 121℃ sterilization for 15min.

[0064] S2, fermentation: the activated Paracasei IOB413 second level seed liquid is inoculated into the sterilized Cicer arietinum Linn. solid-state fermentation substrate at an inoculation amount of 1:1.75 of material to liquid ratio, the culture temperature is 37℃, the ventilation amount is 0.2m³ / min, and the incubation is carried out for 24h, to obtain the Paracasei IOB413 fermentation product.

[0065] S3, the Paracasei IOB413 fermentation product is subjected to high-temperature inactivation at 75℃, dried at 55℃, and crushed, to obtain the Paracasei IOB413 bio-preparation.

[0066] The Paracasei IOB413 fermentation product includes the fermentation product of Paracasei IOB413, metabolites and fermentation substrate.

[0067] Example 2

[0068] A preparation method of a probiotic combination segmented solid-state fermentation bio-preparation, and the present embodiment is a preparation method of Paracasei IOB413 bio-preparation, which comprises the following steps:

[0069] S1, strain activation: the fermentation L. m. IOB802 was taken out from the strain frozen tube, inoculated in the activation medium at 1:20, 37℃, static culture for 22h, to obtain the first level seed liquid, the first level seed was inoculated in the activation medium at 1:25, 37℃, static culture for 22h, to obtain the second level seed liquid.

[0070] The activation medium is MRS medium, the components include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO7H2O, 5.0g of CH3COONa3H2O, 2.0g of C6H5O7(NH4)3, 0.2g of MgSO4·7H2O, 0.05g of MnSO4·4H2O, 15.0g of agar, constant volume to 1L, adjust pH=6.2, sterilize at 121℃ for 15min.

[0071] S2, fermentation: the activated fermentation L. m. IOB802 second level seed liquid was inoculated into the sterilized chickpea solid state fermentation substrate at a ratio of 1:1.75, the culture temperature was 37℃, and the static culture was carried out for 24h to obtain the fermentation L. m. IOB802 fermentation product.

[0072] S3, the fermentation L. m. IOB802 fermentation product was subjected to high temperature inactivation at 75℃, dried at 55℃, and crushed to obtain the fermentation L. m. IOB802 probiotic.

[0073] The fermentation L. m. IOB802 fermentation product includes the fermentation L. m. IOB802 bacterial body composition, metabolites and fermentation substrate after fermentation.

[0074] Example 3

[0075] A preparation method of a probiotic combined segment solid state fermentation probiotic, and the present embodiment is a preparation method of animal Bifidobacterium lactis IOB-LO7 probiotic, which comprises the following steps:

[0076] S1, strain activation: the animal Bifidobacterium lactis IOB-LO7 was taken out from the strain frozen tube, inoculated in the activation medium at a ratio of 1:20, activated at 37℃ in a closed anaerobic environment, activated for 20h to obtain the first level seed liquid, the seed liquid was inoculated in the activation medium at 1:25, activated at 37℃ in a closed anaerobic environment, activated for 24h to obtain the second level seed liquid.

[0077] The activation medium is a modified TPY liquid medium, and the components thereof include: hydrolyzed casein 10.0 g, plant peptone 5.0 g, yeast powder 2.0 g, glucose 5.0 g, L-cysteine 1.0 g, potassium phosphate dibasic 2.0 g, and water to 1 L, pH 6.5, sterilized at 121 ℃ for 15 min.

[0078] S2, fermentation: inoculate the activated animal Bifidobacterium lactis IOB-LO7 secondary seed liquid into chickpea solid-state fermentation substrate at a ratio of 1:1.75, culture at 37 ℃, nitrogen gas flow rate is 0.1 m 3 / min, in a closed anaerobic environment, and culture for 24 h to obtain animal Bifidobacterium lactis IOB-LO7 fermentation product.

[0079] S3, high-temperature inactivation of the animal Bifidobacterium lactis IOB-LO7 fermentation product at 75 ℃, drying at 55 ℃, and crushing treatment to obtain the animal Bifidobacterium lactis IOB-LO7 postbiotic.

[0080] The animal Bifidobacterium lactis IOB-LO7 fermentation product includes the animal Bifidobacterium lactis IOB-LO7 cell components, metabolites, and fermentation substrate after fermentation.

[0081] Example 4

[0082] A preparation method of a probiotic combination segmented solid-state fermentation postbiotic includes the following steps:

[0083] S1, Paracasei IOB413 strain activation method: take Paracasei IOB413 from the strain frozen tube, inoculate in the activation medium at a ratio of 1:20, culture at 37 ℃ for 22 h to obtain the primary seed liquid, inoculate the primary seed in the activation medium at a ratio of 1:25, culture at 37 ℃ for 22 h to obtain the secondary seed liquid.

[0084] The activation medium is MRS medium, and the components thereof include: peptone 10.0 g, beef extract 5.0 g, yeast powder 4.0 g, glucose 20.0 g, Tween 80 1.0 g, K2HPO·7H2O 2.0 g, CH3COONa·3H2O 5.0 g, C6H5O7(NH4)3 2.0 g, MgSO 4` 7H2O 0.2 g, MnSO4·4H2O 0.05 g, agar 15.0 g, water to 1 L, pH 6.2, sterilized at 121 ℃ for 15 min.

[0085] S2, the activation method of Lactobacillus fermentum IOB802 strain: Lactobacillus fermentum IOB802 is taken out from the strain cryopreservation tube, inoculated in the activation medium at the ratio of 1:20, incubated at 37℃ for 22h, and then the first-stage seed liquid is obtained, and the first-stage seed is inoculated in the activation medium at the ratio of 1:25, incubated at 37℃ for 22h, and then the second-stage seed liquid is obtained.

[0086] The activation medium is MRS medium, and the components include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO·7H2O, 5.0g of CH3COONa·3H2O, 2.0g of C6H5O7(NH4)3, 2.0g of MgSO 4` 0.2g of MnSO4·4H2O, 15.0g of agar, and the volume is adjusted to 1L, the pH is adjusted to 6.2, and sterilized at 121℃ for 15min.

[0087] S3, the activation method of Bifidobacterium animalis lactis IOB-LO7 strain: Bifidobacterium animalis lactis IOB-LO7 is taken out from the strain cryopreservation tube, inoculated in the activation medium at the ratio of 1:20, incubated at 37℃ in a closed anaerobic environment for 20h, and then the first-stage seed liquid is obtained, and the seed liquid is inoculated in the activation medium at the ratio of 1:25, incubated at 37℃ in a closed anaerobic environment for 24h, and then the second-stage seed liquid is obtained.

[0088] The activation medium is modified TPY liquid medium, and the components include: 10.0g of hydrolyzed casein, 5.0g of vegetable peptone, 2.0g of yeast powder, 5.0g of glucose, 1.0g of L-cysteine, 2.0g of dipotassium hydrogen phosphate, and the volume is adjusted to 1L, the pH is adjusted to 6.5, and sterilized at 121℃ for 15min.

[0089] S4, the first-stage aerobic fermentation: the activated second-stage seed liquid of Lactobacillus paracasei IOB413 is inoculated in the sterilized chickpea solid fermentation substrate at the inoculation amount of 1:1.75, the culture temperature is 37℃, the aeration amount is 0.2m³ / min, and the static culture is carried out for 12h, and then the Lactobacillus paracasei IOB413 fermentation product is obtained.

[0090] The Lactobacillus paracasei IOB413 fermentation product is subjected to low-temperature drying treatment at-25℃, and then the Lactobacillus paracasei IOB413 postbiotic is obtained.

[0091] The Lactobacillus paracasei IOB413 fermentation product includes the components of Lactobacillus paracasei IOB413 strain, the metabolic products and the fermentation substrate after fermentation.

[0092] S5, the second segment facultative anaerobic fermentation: the activated fermentation of Lactobacillus mucosus IOB802 secondary seed liquid is inoculated into the para dry cheese lactobacillus IOB413 after inoculum according to the material liquid ratio 1:1.75, the culture temperature is 37 DEG C, and the static culture is 12h, and the para dry cheese lactobacillus IOB413 and the fermentation of Lactobacillus mucosus IOB802 compound fermentation product is obtained.

[0093] The para dry cheese lactobacillus IOB413 and the fermentation of Lactobacillus mucosus IOB802 compound fermentation product is low-temperature dried and treated at-25 DEG C, and the para dry cheese lactobacillus IOB413 and the fermentation of Lactobacillus mucosus IOB802 compound after-inoculum is obtained.

[0094] The para dry cheese lactobacillus IOB413 and the fermentation of Lactobacillus mucosus IOB802 fermentation product includes the fermentation of para dry cheese lactobacillus IOB413 and the fermentation of Lactobacillus mucosus IOB802 bacterial body component, metabolite and fermentation substrate.

[0095] S6, the third segment strict anaerobic fermentation: the activated animal bifidobacterium lactis IOB-LO7 secondary seed liquid is inoculated into the para dry cheese lactobacillus IOB413 and the fermentation of Lactobacillus mucosus IOB802 compound after-inoculum according to the material liquid ratio 1:1.75, the culture temperature is 37 DEG C, the nitrogen gas ventilation amount is 0.1 m³ / min, and the culture is 12h in a closed anaerobic environment, and the probiotic combination segmented solid-state fermentation product is obtained.

[0096] The probiotic combination segmented solid-state fermentation product is high-temperature inactivated at 75 DEG C, dried at 55 DEG C, and crushed, and the para dry cheese lactobacillus IOB413, the fermentation of Lactobacillus mucosus IOB802, the animal bifidobacterium lactis IOB-LO7 three probiotic combination segmented solid-state fermentation after-inoculum is obtained.

[0097] The probiotic combination segmented solid-state fermentation product includes the fermentation of para dry cheese lactobacillus IOB413, the fermentation of Lactobacillus mucosus IOB802, the animal bifidobacterium lactis IOB-LO7 bacterial body component, metabolite and solid-state fermentation substrate.

[0098] Example 5

[0099] A preparation method of a probiotic combination segmented solid-state fermentation after-inoculum, and the embodiment is the method of probiotic combination segmented fermentation chickpea after-inoculum, including the following steps:

[0100] S1, the para dry cheese lactobacillus IOB413 strain activation method:

[0101] Sub-Paracasei IOB413 was taken from the strain frozen tube, inoculated in the activation medium at 1:20, and incubated at 37℃ for 22h to obtain the first level seed liquid. The first level seed was inoculated in the activation medium at 1:25, and incubated at 37℃ for 22h to obtain the second level seed liquid.

[0102] The activation medium is MRS medium, and the components include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO·7H2O, 5.0g of CH3COONa·3H2O, 2.0g of C6H5O7(NH4)3, 0.2g of MgSO4·7H2O, 0.05g of MnSO4·4H2O, 15.0g of agar, and the volume is adjusted to 1L, the pH is adjusted to 6.2, and sterilized at 121℃ for 15min.

[0103] S2, activation method of Lactobacillus muius IOB802 strain:

[0104] Lactobacillus muius IOB802 was taken from the strain frozen tube, inoculated in the activation medium at 1:20, and incubated at 37℃ for 22h to obtain the first level seed liquid. The first level seed was inoculated in the activation medium at 1:25, and incubated at 37℃ for 22h to obtain the second level seed liquid.

[0105] The activation medium is MRS medium, and the components include: 10.0g of proteose peptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 1.0g of Tween 80, 2.0g of K2HPO·7H2O, 5.0g of CH3COONa·3H2O, 2.0g of C6H5O7(NH4)3, 0.2g of MgSO4·7H2O, 0.05g of MnSO4·4H2O, 15.0g of agar, and the volume is adjusted to 1L, the pH is adjusted to 6.2, and sterilized at 121℃ for 15min.

[0106] S3, activation method of Bifidobacterium animalis lactis IOB-LO7 strain:

[0107] Bifidobacterium animalis lactis IOB-LO7 was taken from the strain frozen tube, inoculated in the activation medium at a volume ratio of 1:20, and activated in a closed anaerobic environment at 37℃ for 20h to obtain the first level seed liquid. The seed liquid was inoculated in the activation medium at 1:25, and activated in a closed anaerobic environment at 37℃ for 24h to obtain the second level seed liquid.

[0108] The activated culture medium is a modified TPY liquid culture medium, and the components thereof include: hydrolyzed casein 10.0 g, vegetable peptone 5.0 g, yeast powder 2.0 g, glucose 5.0 g, L-cysteine 1.0 g, potassium phosphate dibasic 2.0 g, and water is added to 1 L, the pH is adjusted to 6.5, and sterilization is performed at 121°C for 15 min.

[0109] S4, inoculate the activated Lactobacillus paracasei IOB413 secondary seed liquid into the sterilized chickpea solid-state fermentation substrate at a material-to-liquid ratio of 1:1.75, the culture temperature is 37°C, the ventilation amount is 0.2 m 3 / min, and static culture is performed for 15 h.

[0110] Inoculate the activated Lactobacillus muiosus IOB802 secondary seed liquid into the chickpea solid-state fermentation substrate (the culture medium is the culture medium containing IOB413) at a material-to-liquid ratio of 1:1.75, the culture temperature is 37°C, and static culture is performed for 15 h.

[0111] Inoculate the activated Bifidobacterium animalis lactis IOB-LO7 secondary seed liquid into the chickpea solid-state fermentation substrate (the culture medium is the culture medium containing IOB413 and IOB802) at a material-to-liquid ratio of 1:1.75, the culture temperature is 37°C, the nitrogen ventilation amount is 0.1 m³ / min, the culture is performed in a closed anaerobic environment, and the culture is performed for 15 h, to obtain the fermentation product.

[0112] Perform high-temperature inactivation at 75°C, drying at 55°C, and crushing treatment on the fermentation product, to obtain the three kinds of probiotic bacteria combination segmented fermented chickpea postbiotic of Lactobacillus paracasei IOB413, Lactobacillus muiosus IOB802, and Bifidobacterium animalis lactis IOB-LO7.

[0113] Example 6

[0114] This example is to explore the effect of the probiotic bacteria combination segmented solid-state fermented postbiotic on macrophage RAW264.7, including:

[0115] 1. Test materials

[0116] Reagents: DMEM high-sugar culture medium, lipopolysaccharide, fetal bovine serum, NO detection kit, HBSS cell buffer.

[0117] Consumables: cell culture dishes, cell culture plates 96-well, sterile bunsen tubes, 15 ml centrifuge tubes, 50 ml centrifuge tubes.

[0118] Cells: mouse monocyte macrophage leukemia cells (RAW264.7)

[0119] Experimental samples: Paracasei IOB413 probiotic, Fermented M. mucusolovius IOB802 probiotic, Bifidobacterium animalis ssp. lactis IOB-LO7 probiotic, Multi-strain probiotic segmental solid-state fermented probiotic, Multi-strain probiotic segmental fermented chickpea probiotic.

[0120] Experimental groups: blank control group, experimental group 1 (Paracasei IOB413 probiotic), experimental group 2 (Fermented M. mucusolovius IOB802 probiotic), experimental group 3 (Bifidobacterium animalis ssp. lactis IOB-LO7 probiotic), experimental group 4 (Multi-strain probiotic segmental solid-state fermented probiotic), experimental group 5 (Multi-strain probiotic segmental fermented chickpea probiotic).

[0121] 2. Detection method

[0122] ① Cell proliferation experiment

[0123] RAW267.4 cells were inoculated at 1×104 per well and 100 μL per well, and cultured in a 37℃ incubator with 5% CO2 for 24 h. The blank control group was DMEM-High medium, the positive control group was 10 μg / mL LPS, the experimental group 1 removed the culture medium supernatant, added 100 μL of Paracasei IOB413 probiotic diluted with DMEM-High medium, and cultured in a 37℃ incubator with 5% CO2 for 24 h. The experimental group 2 removed the culture medium supernatant, added 100 μL of Fermented M. mucusolovius IOB802 probiotic diluted with DMEM-High medium, and cultured in a 37℃ incubator with 5% CO2 for 24 h. The experimental group 3 removed the culture medium supernatant, added DMEM-High medium diluted with Bifidobacterium animalis ssp. lactis IOB-LO7 probiotic, and cultured in a 37℃ incubator with 5% CO2 for 24 h. The experimental group 4 removed the culture medium supernatant, added DMEM-High medium diluted with multi-strain probiotic segmental solid-state fermented probiotic, and cultured in a 37℃ incubator with 5% CO2 for 24 h. The experimental group 5 removed the culture medium supernatant, added DMEM-High medium diluted with multi-strain probiotic segmental fermented chickpea probiotic, and cultured in a 37℃ incubator with 5% CO2 for 24 h.

[0124] 10 μL of 5 mg / mL MTT solution was added to the cell culture supernatant, protected from light, cultured at 37℃ for 4 h, the culture medium supernatant was removed, 150 μL DMSO was added, shaken for 10 min, and 100 μL of the solution was aspirated into a new 96-well plate, and the 490 nm light absorption was measured.

[0125] ② NO detection

[0126] RAW264.7 cells were inoculated at 1×104 / well, 100 μL per well, incubated at 37℃ in a 5% CO2 incubator for 24 h. For Experiment 1, the supernatant was removed, and 100 μL of *Lactobacillus paracasei* IOB413 postbiotic diluted in DMEM-High medium was added. For Experiment 2, the supernatant was removed, and 100 μL of *Lactobacillus fermentum* IOB802 postbiotic diluted in DMEM-High medium was added. For Experiment 3, the supernatant was removed, and *Bifidobacterium animalis* subsp. lactis IOB-LO7 postbiotic diluted in DMEM-High medium was added. For Experiment 4, the supernatant was removed, and a multi-probiotic segmented solid-state fermentation postbiotic diluted in DMEM-High medium was added. For Experiment 5, the supernatant was removed, and a multi-probiotic segmented chickpea fermentation postbiotic diluted in DMEM-High medium was added. All were incubated at 37℃ in a 5% CO2 incubator for 24 h. 50 μL of cell culture supernatant was aspirated, and the NO production was detected using a NO detection kit.

[0127] ③ Detection of IL-6 and TNF-α

[0128] RAW264.7 cells were divided into groups of 1×10⁻⁶. 4 In each well, 100 μL of the culture medium was inoculated and incubated at 37°C with 5% CO2 for 24 h. For Experiment 1, the supernatant was removed, and 100 μL of *Lactobacillus paracasei* IOB413 postbiotic diluted in DMEM-High medium was added. The mixture was then incubated at 37°C with 5% CO2 for 24 h. For Experiment 2, the supernatant was removed, and 100 μL of *Lactobacillus fermentum* IOB802 postbiotic diluted in DMEM-High medium was added. The mixture was then incubated at 37°C with 5% CO2 for 24 h. For Experiment 3, the supernatant was removed, and *Bifidobacterium animalis* subsp. *lactospirum* IOB-LO7 postbiotic diluted in DMEM-High medium was added. The mixture was then incubated at 37°C with 5% CO2 for 24 h. For Experiment 4, the supernatant was removed, and a multi-probiotic fractional solid-state fermentation postbiotic diluted in DMEM-High medium was added. The mixture was then incubated at 37°C with 5% CO2 for 24 h. In Experiment 5, the cell culture supernatant was removed, and chickpea post-biotics diluted in DMEM-High medium were added for staged fermentation. The mixture was then incubated at 37°C in a 5% CO2 incubator for 24 h. 100 μL of the cell culture supernatant was collected, and the production of IL-6 was detected using an IL-6 ELISA kit. Similarly, 100 μL of the cell culture supernatant was collected, and the production of TNF-α was detected using a TNF-α ELISA kit.

[0129] 3. Test Results

[0130] ① Effects of different experimental groups on the relative viability of RAW264.7 cells

[0131] like Figure 8 As shown in the MTT assay, compared with the model group, interventions with *Lactobacillus paracasei* IOB413 postbiotics, *Lactobacillus kohlii* IOB802 postbiotics, *Bifidobacterium animalis* subsp. *lactobacter* IOB-LO7 postbiotics, multi-probiotic segmented solid-state fermentation postbiotics, and multi-probiotic segmented fermentation of chickpea postbiotics significantly enhanced the relative activity of RAW264.7 macrophages. The multi-probiotic segmented solid-state fermentation postbiotics showed the most significant effect. This indicates that multi-probiotic segmented solid-state fermentation postbiotics can activate RAW264.7 macrophages, promote increased enzyme activity, and enhance the phagocytic capacity of RAW264.7 macrophages, thereby strengthening the body's immunity.

[0132] ② Effects of different experimental groups on NO release in RAW264.7 cells

[0133] NO production is a key characteristic of macrophage activation upon stimulation, representing the second step in cellular defense against external pathogens. Its release level is typically used to assess the immunomodulatory activity of macrophages. Figure 9 As shown in the NO detection results, compared with the control group, the NO release from macrophages RAW264.7 was significantly increased after intervention with Lactobacillus paracasei IOB413 postbiotic, Lactobacillus fermentum IOB802 postbiotic, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotic, and multiple probiotics segmented solid-state fermentation postbiotic. The increase was particularly significant in the multiple probiotics segmented solid-state fermentation postbiotic group, increasing by 19.14 times, and exhibiting a dose-dependent effect. This indicates that the multiple probiotics segmented solid-state fermentation postbiotic has the ability to activate the release of NO from RAW264.7 phagocytes, thereby exerting an immunomodulatory effect.

[0134] ③ Effects of different experimental groups on interleukin-6 (IL-6) secretion from RAW264.7

[0135] IL-6 is an important pro-inflammatory cytokine that stimulates B cell terminal differentiation and antibody production, while also promoting lymphocyte proliferation, thus helping to stimulate the body's normal immune function and resist infection. This study used an ELISA IL-6 detection kit to detect the effects of a blank control group, a model group, *Lactobacillus paracasei* IOB413 postbiotic, *Lactobacillus fermentum* IOB802 postbiotic, *Bifidobacterium animalis* subsp. *lactospirae* IOB-LO7 postbiotic, and various probiotics fractionated solid-state fermented postbiotics on IL-6 release from RAW264.7 macrophages.

[0136] like Figure 10As shown, compared with the control group, the release levels of IL-6 were significantly increased by the postbiotics of *Lactobacillus paracasei* IOB413, *Lactobacillus fermentum* IOB802, *Bifidobacterium animalis* subsp. *lactospirum* IOB-LO7, and multiple probiotics after segmented solid-state fermentation. The increase in IL-6 release was higher in the group with multiple probiotics after segmented solid-state fermentation, indicating that multiple probiotics after segmented solid-state fermentation can activate macrophages, thereby increasing the release of IL-6 from macrophages and exerting an immune surveillance function.

[0137] ④ Effects of different experimental groups on the secretion of tumor necrosis factor-α (TNF-α) by RAW264.7

[0138] Cytokines are messengers in intercellular communication and important indicators of the body's immune response. When macrophages are stimulated and activated, the various cytokines they secrete play important roles in various immune responses. TNF-α can kill or inhibit the growth of tumor cells and can enhance the phagocytic and differentiation capabilities of neutrophils.

[0139] like Figure 11 As shown, compared with the control group, the release levels of TNF-α were significantly increased by the postbiotics of *Lactobacillus paracasei* IOB413, *Lactobacillus fermentum* IOB802, *Bifidobacterium animalis* subsp. *lactospirum* IOB-LO7, and multiple probiotics after segmented solid-state fermentation. Furthermore, the postbiotic group after segmented solid-state fermentation of multiple probiotics showed a higher increase in TNF-α release. This indicates that the postbiotics after segmented solid-state fermentation of multiple probiotics can activate macrophages, increasing the release of TNF-α from macrophages and thus improving the body's immunity.

[0140] Example 7

[0141] A method for preparing a probiotic combination after segmented solid-state fermentation, this embodiment verifies the effect of the probiotic combination after segmented solid-state fermentation on improving the immune function of mice, including:

[0142] 1. Experimental materials

[0143] Experimental animals: Male BALB / c mice with specific pathogen-free (SPF).

[0144] Experimental materials: Lactobacillus paracasei IOB413 postbiotic, Lactobacillus fermentum IOB802 postbiotic, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotic, postbiotics from multiple probiotics fermented in a segmented solid state, and chickpeas fermented from multiple probiotics in a segmented manner.

[0145] 2. Breeding environment

[0146] 12h light / 12h dark cycle, temperature 22±3℃, humidity 30%-60%, free activity, ad libitum drinking water, change bedding and drinking water every day.

[0147] 3、BALB / c mice were randomly divided into 6 groups according to the experimental grouping, namely blank group, model group, experiment 1 group, experiment 2 group, experiment 3 group, experiment 4 group and experiment 5 group.

[0148] Blank group: intraperitoneal injection of normal saline + intragastric administration of normal saline, intragastric volume 100 μl / each.

[0149] Model group: intraperitoneal injection of cyclophosphamide + intragastric administration of normal saline.

[0150] Experiment 1 group: intraperitoneal injection of cyclophosphamide + Paracasei IOB413 postbiotic group, intragastric volume 100 μl / each, intragastric concentration 300 μg / mL.

[0151] Experiment 2 group: intraperitoneal injection of cyclophosphamide + M. Stoolis IOB802 postbiotic group, intragastric volume 100 μl / each, intragastric concentration 300 μg / mL.

[0152] Experiment 3 group: intraperitoneal injection of cyclophosphamide + Bifidobacterium animalis ssp. lactis IOB-LO7 postbiotic, intragastric volume 100 μl / each, intragastric concentration 300 μg / mL.

[0153] Experiment 4 group: intraperitoneal injection of cyclophosphamide + multiple probiotic segmented solid-state fermentation postbiotic, 0.5 ml / each, intragastric volume 100 μl / each, intragastric concentration 300 μg / mL.

[0154] Experiment 5 group: intraperitoneal injection of cyclophosphamide + multiple probiotic segmented fermentation chickpea postbiotic, 0.5 ml / each, intragastric volume 100 μl / each, intragastric concentration 300 μg / mL.

[0155] After the mice were adaptively fed for one week, they were continuously intragastrically administered for 14 days, intraperitoneally injected with cyclophosphamide on the 8th, 9th and 10th days, the injection concentration was 80 mg / kg / each of cyclophosphamide 100 μl, and the injection volume was controlled at 100 μl / each. And killed on the 15th day.

[0156] 4、Detection index

[0157] 4.1 Extraction of mouse spleen lymphocytes

[0158] At the end of the experiment, the BALB / c mice were euthanized and immersed in 75% ethanol for 3 min, then transferred to a clean bench and the spleen was removed aseptically. A small incision was made on the mouse spleen with ophthalmic scissors and placed on a cell strainer, while adding PBS dropwise, gently grinding with the tail of a 1 mL syringe, and collecting the filtered liquid in a 50 mL centrifuge tube. Centrifugation was performed at 1000 rpm / min for 5 min, and the cell pellet was collected, 2 mL of red blood cell lysate was added, and lysed for 3 min, then diluted with 3 times the volume of PBS, and then centrifuged at 1000 rpm / min for 5 min, and the cell pellet was collected, which was the mouse spleen lymphocytes.

[0159] 4.2 Inoculation of mouse spleen lymphocytes

[0160] The obtained aseptic spleen lymphocyte pellet was resuspended with RPMI-1640 complete medium, the number of viable cells was counted using trypan blue staining, and the concentration of mouse spleen lymphocyte viable cells was adjusted to 2 x 10 6 The 96-well plate was inoculated with 100 μL of the prepared spleen lymphocyte suspension, and directly used for the experiment.

[0161] 4.3 Flow cytometry of mouse spleen lymphocytes

[0162] The spleen was extracted as above, and the obtained spleen lymphocytes of each group of mice were resuspended with 100 μL of PBS containing PE anti-mouse CD4, PE / Cyanine5 anti-mouse CD8a, APC anti-mouse CD19, and FITC anti-mouse CD3 four flow cytometry antibodies, and incubated in the dark for 45 min. Then the cell pellet was collected by centrifugation, resuspended with 400 μL of PBS, and immediately used for flow cytometry. First, the FITC carried by FITC anti-mouse CD3 was excited by 488 nm excitation light to produce an emission wavelength of 525 nm, and the APC carried by APC anti-mouse CD19 was excited by 638 nm excitation light to produce an emission wavelength of 670 nm, to distinguish CD3+ T lymphocytes and CD19+ B lymphocytes. Then, in the CD3+ T lymphocyte population, the PE carried by PE anti-mouse CD4 was excited by 488 nm excitation light to produce an emission wavelength of 585 nm, and the PE / Cyanine5 carried by PE / Cyanine5 anti-mouse CD8a was excited by 488 nm excitation light to produce an emission wavelength of 675 nm, to distinguish CD3+ CD4+ helper T cells and CD3+ CD8+ killer T cells.

[0163] 5. Experimental results

[0164] ① Effects of different experimental groups on the recovery of spleen coefficient in mice

[0165] like Figure 12 As shown, the spleen coefficient of the model group mice was significantly different from that of the control group mice, indicating that CTX treatment can affect the spleen in mice. Compared with the spleen coefficient of the model group mice, the spleen coefficients of the following groups were significantly different: *Lactobacillus paracasei* IOB413 post-biotic group, *Lactobacillus fermentum* IOB802 post-biotic group, *Bifidobacterium animalis* subsp. *lactospirae* IOB-LO7 post-biotic group, post-biotic group of multiple probiotics in segmented solid-state fermentation, and post-biotic group of multiple probiotics in segmented solid-state fermentation of chickpea. The spleen coefficient recovery was more significant in the post-biotic group of multiple probiotics in segmented solid-state fermentation, indicating that the post-biotic group of multiple probiotics in segmented solid-state fermentation can restore the spleen coefficient reduction caused by CTX and help improve the body's immunity.

[0166] ② Effects of different experimental groups on the proliferation of mouse spleen lymphocytes

[0167] like Figure 13 As shown, the spleen lymphocytes in the model group mice showed significant differences compared to the control group, indicating that CTX treatment inhibited the proliferation of spleen lymphocytes. Compared to the control group, interventions with *Lactobacillus paracasei* IOB413 postbiotic, *Lactobacillus fermentum* IOB802 postbiotic, *Bifidobacterium animalis* subsp. *lactobacter* IOB-LO7 postbiotic, and multiple probiotics-fermented chickpea postbiotic showed no significant difference in spleen cell proliferation between CTX-treated and control groups. Furthermore, the multiple probiotics-fermented postbiotic significantly promoted spleen cell proliferation in CTX-treated mice. This suggests that the multiple probiotics-fermented postbiotic can restore the inhibitory effect of CTX-induced spleen lymphocyte proliferation and provide some protection against CTX-induced spleen damage.

[0168] ③ Flow cytometry of spleen lymphocytes from different groups of mice

[0169] CD3 is a marker of T cells. CD4 is expressed on the surface of helper T cells, regulatory T cells, monocytes, macrophages, and dendritic cells, and plays an important role in T cell development and activation. T cells expressing CD8 typically differentiate into cytotoxic T cells (CTLs) after activation, capable of specifically killing target cells. Flow cytometry was used to measure CD3 in spleen lymphocytes. 3+ CD 4+ CD 3+ CD 8+ Cell ratio. CD 3+ CD 4+ / CD 3+CD 8+ The ratio of T helper cells to T suppressor cells, CD45 3 + CD 4+ / CD 3+ CD 8+ A decreased ratio of CD4 indicates the presence of immunodeficiency or viral infection, increasing the body's risk of infection. 3+ CD 4+ / CD 3+ CD 8+ An increased ratio indicates that the body's immune function is overactive, and autoimmune diseases are often present.

[0170] like Figure 14 As shown, compared with the blank group, the CD of the model group 3+ CD 4+ / CD 3+ CD 8+ A significant decrease was observed. Compared with the model group, mice treated with CTX showed significantly lower CD4 counts after gavage administration of Lactobacillus paracasei IOB413 postbiotic, Lactobacillus fermentum IOB802 postbiotic, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotic, postbiotics from multiple probiotics in a segmented solid-state fermentation, and postbiotics from multiple probiotics in a segmented chickpea fermentation. 3+ CD 4+ / CD 3+ CD 8+ All levels increased, with the increase being more significant in the probiotic group after segmented solid-state fermentation of various probiotics, showing a statistically significant difference. This indicates that the probiotic group after segmented solid-state fermentation of various probiotics can improve damage to the body's immune system and further enhance the body's immune function.

[0171] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for the preparation of a probiotic combination of segmented solid state postbiotic, characterized by: Comprise the following steps: S1, strain activation: respectively, the activated culture of fermenting limosilactobacillus IOB802, paracasei IOB413, animal bifidobacterium lactis IOB-LO7, fermenting limosilactobacillus IOB802 secondary seed liquid, paracasei IOB413 secondary seed liquid and animal bifidobacterium lactis IOB-LO7 secondary seed liquid are obtained; S2, aerobic fermentation: the activated culture after paracasei IOB413 secondary seed liquid is inoculated into the sterilized solid-state fermentation substrate at the inoculation amount of 1: 1.5-1: 1.75, and then low-temperature drying treatment is carried out, and paracasei IOB413 postbiotic is obtained; S3, facultative anaerobic fermentation: the activated culture after fermenting limosilactobacillus IOB802 secondary seed liquid is inoculated into paracasei IOB413 postbiotic at the inoculation amount of 1: 1.5-1: 1.75, and then low-temperature drying treatment is carried out, and paracasei IOB413 and fermenting limosilactobacillus IOB802 compound postbiotic is obtained; S4, strict anaerobic fermentation: the activated culture after animal bifidobacterium lactis IOB-LO7 secondary seed liquid is inoculated into paracasei IOB413 and fermenting limosilactobacillus IOB802 compound postbiotic at the inoculation amount of 1: 1.5-1: 1.75, and then high-temperature inactivation, drying and crushing treatment are carried out, and probiotic combination segmented solid-state fermentation postbiotic is obtained; In step S2, the solid-state fermentation substrate is chickpea; In step S2, the aerobic fermentation is carried out under the following conditions: culture temperature 37±2℃, ventilation rate 0.2 m 3 / min, and static culture for 12±2h to obtain the L. paracasei IOB413 fermentation product. In step S3, the facultative anaerobic fermentation conditions are: the culture temperature is 37±2℃, and the static culture is carried out for 12±2h, and paracasei IOB413 and fermenting limosilactobacillus IOB802 compound fermentation product is obtained; In step S4, the conditions for strict anaerobic fermentation are as follows: the culture temperature is 37±1℃, the nitrogen gas ventilation amount is 0.1 m 3 / min, the culture is in a closed anaerobic environment, and the culture time is 12±2h, so that the probiotic combination segment solid-state fermentation product is obtained. The paracasei IOB413 has the strain name IOB413, the classification name is paracasei (Lacticasseibacillus paracasei), the preservation number is CGMCC No.16022, the preservation date is June 29, 2018, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing; The fermenting limosilactobacillus IOB802 has the strain name IOB802, the classification name is fermentum (Limosilactobacillus fermentum), the preservation number is CGMCC No.23120, the preservation date is August 5, 2021, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing. The animal Bifidobacterium lactis subsp. IOB-LO7 has a strain name IOB-LO7, a classification name Bifidobacterium animalis subsp. lactis, a preservation number CGMCC No. 24185, a preservation date of December 23, 2021, and a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

2. The process for the preparation of probiotic combined fractionated solid state fermented postbiotic according to claim 1, characterized by: In step S1, the Paracasei IOB413 strain activation method is as follows: the Paracasei IOB413 is taken out from the strain frozen tube, inoculated in the activation medium at a volume ratio of 1:20, and cultured at 37±2℃ for 22±2h, to obtain the first-stage seed liquid; and the first-stage seed liquid is inoculated in the activation medium at a volume ratio of 1:25, and cultured at 37±2℃ for 22±2h, to obtain the second-stage seed liquid. The activation medium of the Paracasei IOB413 is MRS medium, and the components include: 10.0 g of proteose peptone, 5.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 1.0 g of Tween 80, 2.0 g of K2HPO7H2O, 5.0 g of CH3COONa3H2O, 2.0 g of C6H5O7(NH4)3, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·4H2O, 15.0 g of agar, and the volume is adjusted to 1 L, the pH is adjusted to 6.2±0.2, and sterilized at 121 ℃ for 15 min. 4` 7H2O 0.2g, MnSO4·4H2O 0.05g, agar 15.0g, constant volume to 1L, adjust pH=6.2±0.2, sterilize at 121℃ for 15min; The Lactobacillus fermentum IOB802 strain activation method is as follows: the Lactobacillus fermentum IOB802 is taken out from the strain frozen tube, inoculated in the activation medium at a volume ratio of 1:20, and cultured at 37±2℃ for 22±2h, to obtain the first-stage seed liquid; and the first-stage seed liquid is inoculated in the activation medium at a volume ratio of 1:25, and cultured at 37±2℃ for 22±2h, to obtain the second-stage seed liquid. The activated culture medium of the Lactobacillus muci-depum IOB802 is MRS culture medium, and the components include: 10.0 g of proteose peptone, 5.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 1.0 g of Tween 80, 2.0 g of K2HPO7H2O, 5.0 g of CH3COONa3H2O, 2.0 g of C6H5O7(NH4)3, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·4H2O, 15.0 g of agar, and the volume is adjusted to 1 L, the pH is adjusted to 6.2±0.2, and sterilized at 121 ℃ for 15 min. 4` 7H2O 0.2g, MnSO4·4H2O 0.05g, agar 15.0g, constant volume to 1L, adjust pH=6.2±0.2, sterilize at 121℃ for 15min; The animal Bifidobacterium lactis subsp. IOB-LO7 strain activation method is as follows: the animal Bifidobacterium lactis subsp. IOB-LO7 is taken out from the strain frozen tube, inoculated in the activation medium at a volume ratio of 1:20, and activated in a closed anaerobic environment at 37±2℃ for 20±2h, to obtain the first-stage seed liquid; and the first-stage seed liquid is inoculated in the activation medium at a volume ratio of 1:25, and activated in a closed anaerobic environment at 37±2℃ for 24±2h, to obtain the second-stage seed liquid; wherein the activation medium of the animal Bifidobacterium lactis subsp. IOB-LO7 is a modified TPY liquid medium, and the components include: hydrolyzed casein 10.0g, plant peptone 5.0g, yeast powder 2.0g, glucose 5.0g, L-cysteine 1.0g, potassium phosphate 2.0g, and water to 1L, and the pH is adjusted to 6.5±0.1, and sterilized at 121℃ for 15min.

3. The process for the preparation of probiotic combined segmental solid state fermented postbiotic according to claim 1, characterized by: In step S4, the high-temperature inactivation temperature is 75±2℃, and the drying temperature is 55±2℃.

4. Use of the probiotic combination of claims 1-3 in the preparation of an immunomodulatory preparation.

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