Preparation method of probiotics combined segmented solid-state fermentation prebiotics and application of probiotics combined segmented solid-state fermentation prebiotics in regulating activity of macrophages

Abstract

The invention relates to a preparation method of probiotics combined segmented solid-state fermentation prebiotics and an application of the prebiotics in regulation of macrophage viability, through a synergistic effect of lactic acid bacteria and bifidobacteria, fermented lactobacillus mucus IOB802 and casei paracasei IOB413 can rapidly breed under an aerobic condition, oxygen is rapidly consumed to generate an anaerobic environment, and the effect of regulating the activity of macrophages is achieved. A good growth environment is created for the bifidobacterium animalis subsp. Lactis IOB-LO7, dietary fibers, resistant starch and other difficult-to-digest substances in the chickpeas are decomposed in the fermentation process, beneficial short-chain fatty acids, enzymes and other metabolites are generated, the utilization rate of the chickpeas is increased, meanwhile, the metabolic function of intestinal flora can be improved, and the immunity of the organism can be enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular to a method for preparing probiotics by segmented solid-state fermentation of a probiotic combination and an application thereof in regulating macrophage activity. Background Art

[0002] Immunity refers to the function of the body's immune system to identify itself and foreign substances, and to eliminate antigenic foreign substances through immune response to maintain the body's physiological balance. It is a line of defense to protect the body's health. If the immune system is out of balance, it will affect the level of immunity, leading to immune dysfunction and the occurrence of diseases. However, in modern society, people's work and life pace is accelerating, and phenomena such as high pressure, staying up late, and lack of exercise are common. At the same time, in pursuit of convenience, low-nutrition, high-calorie foods such as fast food and takeout have become the best choice. Poor diet, long-term sitting, lack of sleep, etc. lead to malnutrition in individuals, which are often manifested as mental depression, fatigue, decreased appetite, sleep disorders, etc., and are in a sub-healthy state for a long time, resulting in a decrease in the body's immunity. At present, immunity is regulated through daily diet. Although there are many kinds of foods used to enhance immunity, these products generally have the defects of complex ingredients and unobvious effects. They need to be taken for a long time to achieve a continuous and systematic effect of enhancing 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 them, the preparation method and application of compound probiotics have become a hot topic of research. Through research and exploration, it is found that the mixed use of probiotics such as lactic acid bacteria and bifidobacteria can play a synergistic role, produce more beneficial substances, and better maintain intestinal health. Different microorganisms have different ecological relationships. Different strains can achieve mutual symbiosis by using each other's metabolites to make up for their own metabolic characteristics.

[0004] There are many limitations of solid-state fermentation, such as: ① Limited strain selectivity: Solid-state fermentation is mainly suitable for microorganisms that tolerate low water activity, which makes the selectivity of strains relatively limited. ② Slow fermentation speed and long cycle: Due to the limitations of mass transfer and heat transfer in the solid-state fermentation process, 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 the determination and control of parameters are relatively difficult. ④ The operation consumes a lot of labor: Since the process parameters are difficult to accurately control, more labor and time are required during the operation. ⑤ Limited by mass transfer 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 are affected by raw materials: The composition of natural raw materials is complex and may change, affecting the quality and yield of fermentation products. These shortcomings limit the efficiency and stability of solid-state fermentation in certain applications, and need to be paid attention to and improved in actual operations.

[0005] In summary, currently, almost all composite bacterial fermentation adopts liquid fermentation. After the liquid fermentation is completed, the composite bacterial agent is prepared by centrifugal separation or other solid-liquid separation methods. In the solid-liquid separation process, the organic acids, polypeptides and other substances with antibacterial effects produced by the probiotics in the liquid fermentation process are removed. No literature reports similar to the present invention have been found.

[0006] Therefore, the preparation method and application of probiotic combination segmented solid-state fermentation postbiotics proposed in the present invention are of great significance. It can not only provide people with an effective method to improve intestinal health, enhance body immunity, etc., but also provide new research directions and application prospects in the fields of medicine and health products. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art, provide a method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination and its application in regulating macrophage activity, enrich the types of postbiotics, increase the content of active metabolites, are not restricted by gastric acid and bile salts, and can play a probiotic function in the intestine.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] A method for preparing probiotics by segmented solid-state fermentation, comprising the following steps:

[0010] S1, strain activation: fermented Lactobacillus mucilaginosus IOB802, Lactobacillus paracasei IOB413, and Bifidobacterium animalis subspecies lactis IOB-LO7 were activated and cultured to obtain fermented Lactobacillus mucilaginosus IOB802 secondary seed liquid, Lactobacillus paracasei IOB413 secondary seed liquid, and Bifidobacterium animalis subspecies lactis IOB-LO7 secondary seed liquid;

[0011] S2, aerobic fermentation: the activated cultured Lactobacillus paracasei IOB413 secondary seed liquid is inoculated into the sterilized solid fermentation substrate at a material-liquid ratio of 1:1.5-1:1.75 for aerobic fermentation, followed by low-temperature drying to obtain Lactobacillus paracasei IOB413 postbiotics;

[0012] S3, facultative anaerobic fermentation: the activated cultured fermented Lactobacillus mucilaginosus IOB802 secondary seed liquid is inoculated into the Lactobacillus paracasei IOB413 postbiotic at a material-liquid ratio of 1:1.5-1:1.75 for facultative anaerobic fermentation, followed by low-temperature drying to obtain a composite postbiotic of Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802;

[0013] S4. Strict anaerobic fermentation: The activated cultured animal Bifidobacterium lactis subsp. IOB-LO7 secondary seed liquid is inoculated into the composite postbiotics of Lactobacillus paracasei IOB413 and Lactobacillus mucilaginosus IOB802 at a solid-liquid ratio of 1:1.5-1:1.75 for strict anaerobic fermentation, followed by high-temperature inactivation, drying, and pulverization to obtain the probiotic combination segmented solid-state fermentation postbiotics.

[0014] Moreover, the Lactobacillus 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 the General Microbiology Center of the China Microbiological Culture Collection Administration, and a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0015] The fermented mucus lactobacillus IOB802, strain name: IOB802, classification name: fermented mucus lactobacillus (Limosilactobacillus fermentum), preservation number: CGMCC No.23120, preservation date: August 5, 2021, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing;

[0016] The animal Bifidobacterium lactis subsp. lactis IOB-LO7, the strain name is: IOB-LO7, the classification name is: Bifidobacterium animalis subsp. lactis, the preservation number is: CGMCC No. 24185, the preservation date is: December 23, 2021, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0017] Moreover, in step S1, the activation method of the Lactobacillus paracasei IOB413 strain is as follows: taking out the Lactobacillus paracasei IOB413 from the strain freezing tube, inoculating it into an activation medium at a volume ratio of 1:20, culturing it at 37±2°C for 22h±2h to obtain a primary seed solution, inoculating the primary seed solution into an activation medium at a volume ratio of 1:25, culturing it at 37±2°C for 22±2h to obtain a secondary seed solution;

[0018] The activation medium of Lactobacillus paracasei IOB413 is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2 .0g, CH 3 COONa·3H 2 O 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4` 7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2 ± 0.2, sterilize at 121℃ for 15min;

[0019] The activation method of the fermented mucus lactobacillus IOB802 strain is as follows: taking the fermented mucus lactobacillus IOB802 from a strain freezing tube, inoculating it into an activation medium at a volume ratio of 1:20, culturing it at 37±2°C for 22±2h, to obtain a first-level seed liquid, inoculating the first-level seed liquid into an activation medium at a volume ratio of 1:25, culturing it at 37±2°C for 22±2h, to obtain a second-level seed liquid;

[0020] The activation medium for fermenting Lactobacillus mucilaginosus IOB802 is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2 .0g, CH 3 COONa·3H 2 O 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4` 7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2 ± 0.2, sterilize at 121℃ for 15min;

[0021] The method for activating the animal Bifidobacterium lactis subspecies IOB-LO7 strain is as follows: taking out the animal Bifidobacterium lactis subspecies IOB-LO7 from a strain freezing tube, inoculating it into an activation medium at a volume ratio of 1:20, activating it in a closed anaerobic environment at 37±2°C for 20±2h to obtain a first-level seed liquid, inoculating the first-level seed liquid into an activation medium at a volume ratio of 1:25, activating it in a closed anaerobic environment at 37°C±2°C for 24±2h to obtain a second-level seed liquid;

[0022] Among them, the activation culture medium of animal Bifidobacterium lactis subspecies IOB-LO7 is a modified TPY liquid culture medium, the ingredients of which include: 10.0g hydrolyzed casein, 5.0g plant peptone, 2.0g yeast powder, 5.0g glucose, 1.0g L-cysteine, 2.0g dipotassium hydrogen phosphate, fixed volume to 1L, adjusted pH 6.5±0.1, and sterilized at 121°C for 15min.

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

[0024] Moreover, in step S2, the conditions for aerobic fermentation are: culture temperature of 37±2°C, ventilation volume of 0.2m³ / min, static culture for 12±2h, and obtaining the fermentation product of Lactobacillus paracasei IOB413.

[0025] Moreover, in step S3, the conditions of facultative anaerobic fermentation are: the culture temperature is 37±2° C., and the culture is statically cultured for 12±2 hours to obtain a composite fermentation product of Lactobacillus paracasei IOB413 and Lactobacillus mucilaginosus IOB802.

[0026] Moreover, in step S4, the conditions for strict anaerobic fermentation are: culture temperature of 37±1°C, nitrogen ventilation of 0.1m³ / min, closed anaerobic environment, culture for 12±2h, and obtaining the probiotic combination segmented solid fermentation product.

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

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

[0029] Application of a probiotic combination segmented solid-state fermentation postbiotic in the preparation of immunomodulatory preparations.

[0030] The advantages and positive effects of the present invention are:

[0031] 1. The present invention uses the synergistic effect of lactic acid bacteria and bifidobacteria to ferment Lactobacillus mucilaginosus IOB802 and Lactobacillus paracasei IOB413, which can reproduce rapidly under aerobic conditions, quickly consume oxygen to produce an anaerobic environment, and create a good growth environment for animal Bifidobacterium lactis subspecies IOB-LO7. During the fermentation process, the indigestible substances such as dietary fiber and resistant starch in chickpeas are decomposed to produce beneficial short-chain fatty acids, enzymes and other metabolites, thereby improving the utilization rate of chickpeas, and at the same time improving the metabolic function of intestinal flora and enhancing the body's immunity.

[0032] 2. The present invention produces metabolites such as extracellular polysaccharides, organic acids, short-chain fatty acids, and small-molecule peptides through aerobic, facultative, and strictly anaerobic three-stage fermentation processes, while retaining nutrients such as soluble fiber, amino acids, and flavonoids in the fermentation substrate. These nutrients can efficiently promote the proliferation of fermented mucus lactobacillus IOB802, Lactobacillus paracasei IOB413, and animal bifidobacterium lactis subspecies IOB-LO7, while effectively protecting the activity of the strain, improving the intracellular active substance content of fermented mucus lactobacillus IOB802, Lactobacillus paracasei IOB413, and animal bifidobacterium lactis subspecies IOB-LO7, thereby enriching the types of postbiotics, improving the content of active metabolites, not being restricted by gastric acid and bile salts, and can play a prebiotic function in the intestinal tract.

[0033] 3. The present invention obtains a variety of probiotic postbiotics through a staged fermentation preparation method. Cell experiments and animal experiments have verified that the postbiotics can activate macrophages with an appropriate concentration of bifidobacteria, increase the release of IL-6 and TNF-α of macrophages, restore the decrease in spleen coefficient caused by CTX, inhibit the proliferation of spleen lymphocytes, and effectively enhance the immunity of immunocompromised mice.

[0034] 4. The present invention uses chickpeas as solid-state fermentation substrates, and the resistant starch and dietary fiber rich in chickpeas are decomposed step by step by probiotics in segmented fermentation to generate highly active metabolites such as short-chain fatty acids and extracellular polysaccharides, while completely retaining the nutrients such as soluble fiber, flavonoids and amino acids in the substrate. Through aerobic-facultative anaerobic-strict anaerobic three-stage fermentation, different strains (Lactobacillus paracasei IOB413, fermented Lactobacillus mucus IOB802, and animal Bifidobacterium lactis subspecies IOB-LO7) sequentially utilize the complex carbohydrates in chickpeas to form a synergistic system of metabolites and substrate nutrition. Compared with liquid fermentation, solid-state fermentation is closer to the intestinal microenvironment, promotes the mutual symbiosis of strains, increases the content of short-chain fatty acids in postbiotics by 30%, and the retention rate of flavonoids reaches more than 90%, and the stability is significantly enhanced, and the function of regulating intestinal flora and enhancing immunity can be exerted without relying on live bacteria.

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

[0036] 6. The present invention first discovered that postbiotics contain a variety of bioactive ingredients, such as short-chain fatty acids, antimicrobial peptides, cell wall components, etc., which can be used as functional substrates or signal molecules in the fermentation process to further promote the growth and metabolism of target microorganisms, thereby enhancing the biological activity of fermentation products, while activating specific metabolic pathways of microorganisms to produce more metabolites with immunomodulatory functions. Oligosaccharides, organic acids and other ingredients in postbiotics can significantly promote the growth and metabolism of bifidobacteria. Peptides and vitamins can enhance the fermentation ability of lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing the crude polysaccharide content of different groups of the present invention.

[0038] Figure 2 This is a diagram showing the content of polypeptides in different groups of the present invention.

[0039] Figure 3This is a graph showing the organic acid content of different groups of the present invention.

[0040] Figure 4 This is a graph showing the acetic acid content of different groups of the present invention.

[0041] Figure 5 This is a diagram of propionic acid content in different groups of the present invention.

[0042] Figure 6 This is a diagram of butyric acid content in different groups of the present invention.

[0043] Figure 7 This is a graph showing the valeric acid content in different groups of the present invention.

[0044] Figure 8 This is a graph showing the relative viability of RAW264.7 cells of the present invention.

[0045] Fig. 9 This is a graph showing the NO release from RAW264.7 cells of the present invention.

[0046] Fig.10 This is a graph showing the release of IL-6 from RAW264.7 cells of the present invention.

[0047] Fig.11 This is a graph showing the release of TNF-α in RAW264.7 cells of the present invention.

[0048] Fig.12 This is a graph of spleen coefficients of mice in different groups according to the present invention.

[0049] Fig.13 This is a diagram of the proliferation of spleen lymphocytes in different groups of mice in the present invention.

[0050] Fig.14 The figure is a flow cytometry result of spleen lymphocytes of different groups of mice in the present invention. DETAILED DESCRIPTION

[0051] 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.

[0052] The probiotic combination used in the present invention includes Lactobacillus paracasei IOB413, Lactobacillus mucosa fermentation IOB802, and Bifidobacterium animalis subspecies lactis IOB-LO7, all of which are obtained through independent screening.

[0053] Lacticasseibacillus paracasei IOB413 was screened from naturally fermented sourdough in the homes of residents in Tianjin. This strain has been preserved and tested for physical and chemical indicators. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) on June 29, 2018. Its preservation number is CGMCC No. 16022, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. This strain has been preserved and tested for physical and chemical indicators. On March 19, 2021, the bacterial identification test was carried out at the China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges.

[0054] Limosilactobacillus fermentum IOB802 was screened for self-fermented kimchi. This strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on August 5, 2021. Its deposit number is CGMCC No. 23120, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. This strain has been tested for strain preservation and physical and chemical indicators. On April 3, 2023, bacterial identification was carried out at China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges.

[0055] Bifidobacterium animalis subsp. lactis IOB-LO7 was screened from the feces of healthy infants. This strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) on December 23, 2021, and its deposit number is CGMCC No. 24185. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0056] The Lactobacillus 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 the General Microbiology Center of the China National Microbiological Culture Collection Administration, and a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0057] The fermented mucus lactobacillus IOB802, strain name: IOB802, classification name: fermented mucus lactobacillus (Limosilactobacillus fermentum), preservation number: CGMCC No.23120, preservation date: August 5, 2021, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing;

[0058] The animal Bifidobacterium lactis subsp. lactis IOB-LO7, the strain name is: IOB-LO7, the classification name is: Bifidobacterium animalis subsp. lactis, the preservation number is: CGMCC No. 24185, the preservation date is: December 23, 2021, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

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

[0060] Example 1

[0061] A method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination, in this embodiment, is a method for preparing postbiotics of Lactobacillus paracasei IOB413, comprising the following steps:

[0062] S1. Strain activation: Take out Lactobacillus paracasei IOB413 from the strain freezing tube, inoculate it into the activation medium at a ratio of 1:20, and culture it at 37°C for 22 hours to obtain the first-level seed solution. Inoculate the first-level seeds into the activation medium at a ratio of 1:25, and culture it at 37°C for 22 hours to obtain the second-level seed solution.

[0063] The activation medium is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2 .0g, CH 3 COONa·3H 2 O 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4`7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2, sterilize at 121℃ for 15min.

[0064] S2, fermentation: the activated Lactobacillus paracasei IOB413 secondary seed liquid is inoculated into the sterilized chickpea solid fermentation substrate at a solid-liquid ratio of 1:1.75, the culture temperature is 37°C, the ventilation volume is 0.2m³ / min, and the culture is statically cultured for 24 hours to obtain a fermentation product of Lactobacillus paracasei IOB413.

[0065] S3, high-temperature inactivating the fermented product of Lactobacillus paracasei IOB413 at 75° C., drying at 55° C., and pulverizing to obtain postbiotics of Lactobacillus paracasei IOB413.

[0066] The Lactobacillus paracasei IOB413 fermentation product comprises fermented Lactobacillus paracasei IOB413 bacterial components, metabolites and fermentation substrates.

[0067] Example 2

[0068] A method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination, this embodiment is a method for preparing postbiotics by fermenting Lactobacillus mucilaginosus IOB802, comprising the following steps:

[0069] S1. Strain activation: Take out the fermented mucus Lactobacillus IOB802 from the strain freezing tube, inoculate it into the activation medium at a ratio of 1:20, and culture it at 37°C for 22 hours to obtain the first-level seed solution. Inoculate the first-level seeds into the activation medium at a ratio of 1:25, and culture it at 37°C for 22 hours to obtain the second-level seed solution.

[0070] The activation medium is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2 .0g, CH 3 COONa·3H 2 O 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4 7H 2 O 0.2g, MnSO 4 ·4H 2O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2, sterilize at 121℃ for 15min.

[0071] S2, fermentation: the activated fermented Lactobacillus mucilaginosus IOB802 secondary seed liquid is inoculated into the sterilized chickpea solid fermentation substrate at a solid-liquid ratio of 1:1.75, the culture temperature is 37° C., and the culture is statically cultured for 24 hours to obtain the fermented Lactobacillus mucilaginosus IOB802 fermentation product.

[0072] S3, high-temperature inactivating the fermented product of Lactobacillus mucilaginosus IOB802 at 75° C., drying at 55° C., and pulverizing to obtain postbiotics of Lactobacillus mucilaginosus IOB802.

[0073] The fermented product of Lactobacillus mucilaginosus IOB802 comprises fermented Lactobacillus mucilaginosus IOB802 bacterial components, metabolites and fermentation substrates.

[0074] Example 3

[0075] A method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination, in this embodiment, is a method for preparing postbiotics of Bifidobacterium animalis subspecies lactis IOB-LO7, comprising the following steps:

[0076] S1. Strain activation: Take out animal Bifidobacterium lactis subsp. IOB-LO7 from the strain freezing tube, inoculate it into the activation medium at a volume ratio of 1:20, activate it at 37°C in a closed anaerobic environment, activate it for 20 hours, and obtain the first-level seed solution. Inoculate the seed solution into the activation medium at a volume ratio of 1:25, activate it at 37°C in a closed anaerobic environment, activate it for 24 hours, and obtain the second-level seed solution.

[0077] The activation culture medium is a modified TPY liquid culture medium, and its ingredients include: 10.0g of hydrolyzed casein, 5.0g of plant peptone, 2.0g of yeast powder, 5.0g of glucose, 1.0g of L-cysteine, 2.0g of dipotassium hydrogen phosphate, the volume is fixed to 1L, the pH is adjusted to 6.5, and sterilized at 121°C for 15min.

[0078] S2, fermentation: The activated animal Bifidobacterium lactis subsp. IOB-LO7 secondary seed liquid was inoculated into the chickpea solid fermentation substrate at a solid-liquid ratio of 1:1.75, the culture temperature was 37°C, and the nitrogen ventilation volume was 0.1m 3 / min, in a closed anaerobic environment, and cultured for 24 hours to obtain the fermentation product of Bifidobacterium animalis subsp. lactis IOB-LO7.

[0079] S3. The fermentation product of Bifidobacterium animalis subsp. lactis IOB-LO7 is inactivated at 75° C., dried at 55° C., and crushed to obtain Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics.

[0080] The fermentation product of Bifidobacterium animalis subspecies lactis IOB-LO7 comprises fermented bacterial components, metabolites and fermentation substrates of Bifidobacterium animalis subspecies lactis IOB-LO7.

[0081] Example 4

[0082] A method for preparing probiotics by segmented solid-state fermentation, comprising the following steps:

[0083] S1. Activation method of Lactobacillus paracasei IOB413 strain: take out Lactobacillus paracasei IOB413 from the strain freezing tube, inoculate it into activation culture medium at a ratio of 1:20, and culture it at 37°C for 22 hours to obtain a first-level seed solution; inoculate the first-level seeds into activation culture medium at a ratio of 1:25, and culture it at 37°C for 22 hours to obtain a second-level seed solution.

[0084] The activation medium is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2 .0g, CH 3 COONa·3H 2 O 5.0g,C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4` 7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2, sterilize at 121℃ for 15min.

[0085] S2. Activation method of fermented mucus Lactobacillus IOB802 strain: take out fermented mucus Lactobacillus IOB802 from the strain freezing tube, inoculate it into activation culture medium at a ratio of 1:20, and culture it at 37°C for 22 hours to obtain first-level seed solution; inoculate the first-level seeds into activation culture medium at a ratio of 1:25, and culture it at 37°C for 22 hours to obtain second-level seed solution.

[0086] The activation medium is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2 .0g, CH 3 COONa·3H2 O 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4` 7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2, sterilize at 121℃ for 15min.

[0087] S3. Activation method of animal Bifidobacterium lactis subsp. IOB-LO7 strain: take out animal Bifidobacterium lactis subsp. IOB-LO7 from the strain freezing tube, inoculate it into activation culture medium at a volume ratio of 1:20, activate it at 37°C in a closed anaerobic environment for 20 hours to obtain a first-level seed solution, inoculate the seed solution into activation culture medium at a ratio of 1:25, activate it at 37°C in a closed anaerobic environment for 24 hours to obtain a second-level seed solution.

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

[0089] S4, the first stage of aerobic fermentation: the activated Lactobacillus paracasei IOB413 secondary seed liquid is inoculated into the sterilized chickpea solid fermentation substrate at a solid-liquid ratio of 1:1.75, the culture temperature is 37°C, the ventilation volume is 0.2m³ / min, and the culture is statically cultured for 12 hours to obtain the Lactobacillus paracasei IOB413 fermentation product.

[0090] The fermentation product of Lactobacillus paracasei IOB413 is subjected to low-temperature drying treatment at -25°C to obtain the postbiotics of Lactobacillus paracasei IOB413.

[0091] The Lactobacillus paracasei IOB413 fermentation product comprises fermented Lactobacillus paracasei IOB413 bacterial components, metabolites and fermentation substrates.

[0092] S5, the second stage of facultative anaerobic fermentation: the activated fermented Lactobacillus mucilaginosus IOB802 secondary seed liquid is inoculated into the Lactobacillus paracasei IOB413 postbiotic at a solid-liquid ratio of 1:1.75, the culture temperature is 37°C, and the culture is statically cultured for 12 hours to obtain a composite fermentation product of Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802.

[0093] The composite fermentation product of Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802 is low-temperature dried and treated at -25°C to obtain the composite postbiotic of Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802.

[0094] The fermented product of Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802 comprises fermented Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802 bacterial components, metabolites and fermentation substrates.

[0095] S6. The third stage of strict anaerobic fermentation: The activated animal Bifidobacterium lactis subsp. lactis IOB-LO7 secondary seed liquid is inoculated into the composite postbiotics of Lactobacillus paracasei IOB413 and Lactobacillus mucilaginosus IOB802 at a solid-liquid ratio of 1:1.75. The culture temperature is 37°C, the nitrogen ventilation volume is 0.1m³ / min, and the closed anaerobic environment is used for 12 hours to obtain the probiotic combination segmented solid-state fermentation product.

[0096] The probiotic combination segmented solid-state fermentation product was high-temperature inactivated at 75°C, dried at 55°C, and crushed to obtain three probiotic combination segmented solid-state fermentation postbiotics, namely, Lactobacillus paracasei IOB413, Lactobacillus mucilaginosus fermentation IOB802, and Bifidobacterium animalis subspecies lactis IOB-LO7.

[0097] The probiotic combination segmented solid fermentation product comprises fermented Lactobacillus paracasei IOB413, fermented Lactobacillus mucilaginosus IOB802, animal Bifidobacterium lactis subspecies IOB-LO7 bacterial components, metabolites and solid fermentation substrates.

[0098] Example 5

[0099] A method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination. This embodiment is a method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination of chickpeas, comprising the following steps:

[0100] S1. Activation method of Lactobacillus paracasei IOB413 strain:

[0101] Take out Lactobacillus paracasei IOB413 from the strain freezing tube, inoculate it into the activation culture medium at a ratio of 1:20, and culture it at 37°C for 22 hours to obtain the first-level seed solution. Inoculate the first-level seeds into the activation culture medium at a ratio of 1:25, and culture it at 37°C for 22 hours to obtain the second-level seed solution.

[0102] The activation medium is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H2 O 2 .0g, CH 3 COONa·3H 2 O 5.0g, C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO4·7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2, sterilize at 121℃ for 15min.

[0103] S2. Activation method of fermented Lactobacillus mucilaginosus IOB802 strain:

[0104] The fermented mucus Lactobacillus IOB802 was taken out from the freezing tube of the strain, inoculated into the activation culture medium at a ratio of 1:20, and cultured at 37°C for 22 hours to obtain the first-level seed solution. The first-level seeds were inoculated into the activation culture medium at a ratio of 1:25, and cultured at 37°C for 22 hours to obtain the second-level seed solution.

[0105] The activation medium is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2 HPO·7H 2 O 2.0g, CH 3 COONa·3H 2 O 5.0g,C 6 H 5 O 7 (NH 4 ) 3 2.0 g, MgSO 4 7H 2 O 0.2g, MnSO 4 ·4H 2 O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2, sterilize at 121℃ for 15min.

[0106] S3. Activation method of Bifidobacterium animalis subspecies lactis IOB-LO7 strain:

[0107] The animal Bifidobacterium lactis subsp. IOB-LO7 was taken out from the strain freezing tube, inoculated into the activation medium at a volume ratio of 1:20, activated at 37°C in a closed anaerobic environment for 20 hours to obtain the first-level seed solution, and the seed solution was inoculated into the activation medium at a volume ratio of 1:25, activated at 37°C in a closed anaerobic environment for 24 hours to obtain the second-level seed solution.

[0108] The activation culture medium is a modified TPY liquid culture medium, and its ingredients include: 10.0g of hydrolyzed casein, 5.0g of plant peptone, 2.0g of yeast powder, 5.0g of glucose, 1.0g of L-cysteine, 2.0g of dipotassium hydrogen phosphate, the volume is fixed to 1L, the pH is adjusted to 6.5, and sterilized at 121°C for 15min.

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

[0110] The activated fermented mucus Lactobacillus IOB802 secondary seed liquid was inoculated into the chickpea solid fermentation substrate (the culture medium was a culture medium containing IOB413) at a solid-liquid ratio of 1:1.75, the culture temperature was 37°C, and the culture was statically cultured for 15 hours.

[0111] The activated animal Bifidobacterium lactis subsp. IOB-LO7 secondary seed liquid was inoculated into the chickpea solid fermentation substrate (the culture medium was a culture medium containing IOB413 and IOB802) at a solid-liquid ratio of 1:1.75, the culture temperature was 37°C, the nitrogen ventilation rate was 0.1m³ / min, and the closed anaerobic environment was used for 15 hours to obtain the fermentation product.

[0112] The fermented product was inactivated at 75°C, dried at 55°C, and crushed to obtain chickpea postbiotics from a combination of three probiotics, namely, Lactobacillus paracasei IOB413, Lactobacillus mucilaginosus fermentation IOB802, and Bifidobacterium animalis subsp. lactis IOB-LO7.

[0113] Example 6

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

[0115] 1. Test materials

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

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

[0118] Cells: Mouse mononuclear macrophage leukemia cells (RAW264.7)

[0119] Experimental samples: postbiotics from Lactobacillus paracasei IOB413, postbiotics from fermented Lactobacillus mucilaginosus IOB802, postbiotics from Bifidobacterium animalis subspecies lactis IOB-LO7, postbiotics from multi-probiotic segmented solid-state fermentation, and postbiotics from multi-probiotic segmented fermentation of chickpeas.

[0120] Experimental groups: blank control group, experimental group 1 (postbiotics from Lactobacillus paracasei IOB413), experimental group 2 (postbiotics from fermented Lactobacillus mucilaginosus IOB802), experimental group 3 (postbiotics from Bifidobacterium animalis subsp. lactis IOB-LO7), experimental group 4 (postbiotics from multi-probiotics solid-state fermentation), and experimental group 5 (postbiotics from multi-probiotics chickpea fermentation).

[0121] 2. Detection method

[0122] ①Cell proliferation assay

[0123] RAW267.4 cells were seeded at 1×104 / well with 100 μL per well and placed at 37°C with 5% CO 2 The blank control group was cultured in DMEM-High medium, the positive control group was cultured in 10 μg / mL LPS, and the supernatant of the culture medium was removed from the experimental group 1, and 100 μL of Lactobacillus paracasei IOB413 postbiotics diluted in DMEM-High medium was added and placed at 37°C with 5% CO 2 In the experimental group 2, the supernatant of the culture medium was removed, and 100 μL of fermented Lactobacillus mucilaginosus IOB802 postbiotics diluted in DMEM-High medium was added and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 3, and the postbiotics of Bifidobacterium animalis subsp. lactis IOB-LO7 diluted in DMEM-High medium were added and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 4, and the probiotics after segmented solid-state fermentation diluted in DMEM-High medium were added and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 5, and chickpea postbiotics diluted with various probiotics in DMEM-High medium were added and incubated at 37°C with 5% CO 2 incubator for 24 h.

[0124] Add 10 μL of 5 mg / mL MTT solution to the cell culture supernatant, protect from light, and culture at 37°C for 4 h. Remove the culture supernatant, add 150 μL DMSO, shake for 10 min, aspirate 100 μL of the solution into a new 96-well plate, and measure the light absorption at 490 nm.

[0125] ② Detection of NO

[0126] RAW264.7 cells were cultured at a rate of 1 × 10 4 Each well was inoculated with 100 μL of culture medium and placed at 37°C with 5% CO 2 Incubator for 24 h. The supernatant of the culture medium was removed in experimental group 1, and 100 μL of Lactobacillus paracasei IOB413 postbiotics diluted in DMEM-High medium was added. In experimental group 2, the supernatant of the culture medium was removed, and 100 μL of Lactobacillus fermented mucus IOB802 postbiotics diluted in DMEM-High medium was added. In experimental group 3, the supernatant of the culture medium was removed, and postbiotics of Bifidobacterium animalis subsp. lactis IOB-LO7 were added in DMEM-High medium. In experimental group 4, the supernatant of the culture medium was removed, and postbiotics of multiple probiotics segmented solid fermentation were added in DMEM-High medium. In experimental group 5, the supernatant of the culture medium was removed, and postbiotics of multiple probiotics segmented fermentation chickpea were added in DMEM-High medium. The cells were placed at 37°C with 5% CO 2 The cells were cultured in an incubator for 24 h. 50 μL of cell culture supernatant was taken out 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 cultured at a rate of 1 × 10 4 / well, and 100 μL was inoculated into each well and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 1, and 100 μL of Lactobacillus paracasei IOB413 postbiotics diluted in DMEM-High medium was added and placed at 37°C with 5% CO 2 In the experimental group 2, the supernatant of the culture medium was removed, and 100 μL of fermented Lactobacillus mucilaginosus IOB802 postbiotics diluted in DMEM-High medium was added and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 3, and the postbiotics of Bifidobacterium animalis subsp. lactis IOB-LO7 diluted in DMEM-High medium were added and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 4, and the probiotics after segmented solid-state fermentation diluted in DMEM-High medium were added and placed at 37°C with 5% CO 2 The supernatant of the culture medium was removed from the experimental group 5, and chickpea postbiotics diluted with various probiotics in DMEM-High medium were added and fermented in a 37°C incubator containing 5% CO.2 Culture in an incubator for 24 h. Take 100 μL of cell culture supernatant and use an ELISA kit for IL-6 to detect the amount of IL-6 produced. Take 100 μL of cell culture supernatant and use an ELISA kit for TNF-α to detect the amount of TNF-α produced.

[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 figure, the MTT test results showed that compared with the model group, after intervention with Lactobacillus paracasei IOB413 postbiotics, Lactobacillus mucilaginosus IOB802 postbiotics, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics, multiple probiotics segmented solid fermentation postbiotics, and multiple probiotics segmented fermentation chickpea postbiotics, there was a significant effect on the relative activity of macrophagocyte RAW264.7 proliferation, and the effect of multiple probiotics segmented solid fermentation postbiotics was more obvious. This shows that multiple probiotics segmented solid fermentation postbiotics can activate macrophagocytes RAW264.7, promote the increase of macrophagocyte RAW264.7 enzyme activity, and improve the phagocytic ability of macrophagocytes RAW264.7, thereby enhancing the body's immunity.

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

[0133] The production of NO is one of the important characteristics of macrophage activation upon stimulation. It is the second step for cells to resist infection by external pathogens. Its release level is usually used to evaluate the immunomodulatory activity of macrophages. Fig. 9 As shown, according to the NO detection results, compared with the blank group, after intervention with Lactobacillus paracasei IOB413 postbiotics, Lactobacillus fermented mucus IOB802 postbiotics, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics, and multiple probiotics segmented solid fermentation postbiotics, the release of NO by macrophagocytes RAW264.7 increased significantly, and the increase in the multiple probiotics segmented solid fermentation postbiotic group was more significant, increasing by 19.14 times, and in a dose-dependent effect. This shows that multiple probiotics segmented solid fermentation postbiotics have the ability to activate macrophages RAW264.7 to release NO, thereby playing an immunomodulatory role.

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

[0135] IL-6 is an important pro-inflammatory cytokine that stimulates B cell terminal differentiation and antibody production, and can also promote lymphocyte proliferation, which helps stimulate the body's normal immune function and resist infection. The ELISA IL-6 detection kit was used to detect the effects of the blank group, model group, Lactobacillus paracasei IOB413 postbiotics, fermented Lactobacillus mucilaginosus IOB802 postbiotics, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics, and multiple probiotics segmented solid-state fermentation postbiotics on the release of IL-6 in RAW264.7 macrophages.

[0136] like Fig.10 As shown, compared with the blank group, the postbiotics of Lactobacillus paracasei IOB413, fermented Lactobacillus mucus IOB802, Bifidobacterium animalis subsp. lactis IOB-LO7, and multiple probiotics segmented solid-state fermentation postbiotics significantly increased the release level of IL-6, and the multiple probiotics segmented solid-state fermentation postbiotic group had a higher increase in the release level of IL-6, indicating that multiple probiotics segmented solid-state fermentation postbiotics can activate macrophages, increase the release of IL-6 by macrophages, and thus exert 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 for information transmission between cells and are important indicators of the body's immune response. When macrophages are stimulated and activated, the various cytokines they secrete play an important role in various immune responses. TNF-α can kill or inhibit the growth of tumor cells and enhance the phagocytic and differentiation abilities of neutrophils.

[0139] like Fig.11 As shown, compared with the blank group, the release level of TNF-α by Lactobacillus paracasei IOB413 postbiotics, fermented Lactobacillus mucus IOB802 postbiotics, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics, and multiple probiotics segmented solid-state fermentation postbiotics significantly increased the release level of TNF-α, and the multiple probiotics segmented solid-state fermentation postbiotic group had a higher increase in the release level of TNF-α, indicating that multiple probiotics segmented solid-state fermentation postbiotics can activate macrophages, increase the release of TNF-α by macrophages, and thus achieve the effect of improving the body's immunity.

[0140] Example 7

[0141] A method for preparing postbiotics from segmented solid-state fermentation of a probiotic combination, this embodiment is to verify the effect of postbiotics from segmented solid-state fermentation of a probiotic combination on improving the immune function of mice, comprising:

[0142] 1. Experimental Materials

[0143] Experimental animals: Specific pathogen-free (SPF) male BALB / c mice.

[0144] Experimental materials: postbiotics from Lactobacillus paracasei IOB413, postbiotics from fermented Lactobacillus mucilaginosus IOB802, postbiotics from Bifidobacterium animalis subspecies lactis IOB-LO7, postbiotics from multi-probiotic segmented solid-state fermentation, and postbiotics from multi-probiotic segmented fermentation of chickpeas.

[0145] 2. Breeding environment

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

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

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

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

[0150] Experimental group 1: intraperitoneal injection of cyclophosphamide + Lactobacillus paracasei IOB413 postbiotic group, gavage volume 100μl / head, gavage concentration 300μg / mL.

[0151] Experimental group 2: intraperitoneal injection of cyclophosphamide + fermented Lactobacillus mucinus IOB802 postbiotic group, gavage volume 100μl / head, gavage concentration 300μg / mL.

[0152] Experimental group 3: intraperitoneal injection of cyclophosphamide + animal Bifidobacterium lactis subsp. IOB-LO7 postbiotics, gavage volume 100μl / mouse, gavage concentration 300μg / mLl.

[0153] Experimental group 4: intraperitoneal injection of cyclophosphamide + multiple probiotics segmented solid-state fermentation postbiotics, 0.5 ml / head, gavage volume 100 μl / head, gavage concentration 300 μg / mL.

[0154] Experimental group 5: intraperitoneal injection of cyclophosphamide + chickpea postbiotics fermented by multiple probiotics, 0.5 ml / head, gavage volume 100 μl / head, gavage concentration 300 μg / mL.

[0155] After one week of adaptive feeding, the mice were gavaged for 14 consecutive days, and intraperitoneally injected with cyclophosphamide at a concentration of 80 mg / kg / mouse, 100 μl, and the injection volume was controlled to be 100 μL / mouse. The mice were killed on the 15th day.

[0156] 4. Detection indicators

[0157] 4.1 Isolation of mouse spleen lymphocytes

[0158] At the end of the experiment, BALB / c mice were euthanized, soaked in 75% ethanol for 3 min, and then transferred to a clean bench, and the spleen was removed aseptically. A notch was cut in the mouse spleen with ophthalmic scissors and placed on a cell screen. PBS was dripped while the tail of a 1mL syringe was gently ground, and the sieved liquid was collected in a 50mL centrifuge tube. Centrifuge at 1000 rpm / min for 5 min, collect the cell pellet, add 2 mL of red blood cell lysis buffer, lyse for 3 min, and then quickly add 3 times the volume of PBS to dilute the red blood cell lysis buffer, and then centrifuge at 1000 rpm / min for 5 min to collect the cell pellet, which is the mouse spleen lymphocyte.

[0159] 4.2 Inoculation of mouse spleen lymphocytes

[0160] The sterile spleen lymphocyte pellet was resuspended in RPMI-1640 complete medium, the number of live cells was counted using trypan blue staining, and the concentration of mouse spleen lymphocyte live cells was adjusted to 2×10 6 pc / mL were inoculated into 96-well plates and used directly in the experiment.

[0161] 4.3 Flow cytometry of mouse spleen lymphocytes

[0162] The spleen was extracted as above, and the spleen lymphocytes of each group of mice were resuspended with 100 μL of PBS containing four flow cytometry antibodies: PE anti-mouseCD4, PE / Cyanine5 anti-mouse CD8a, APC anti-mouse CD19, and FITC anti-mouse CD3, and incubated in the dark for 45 min. The cell pellet was then collected by centrifugation, and the cells were 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. Subsequently, 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, in order to distinguish CD3+CD4+ helper T cells and CD3+CD8+ cytotoxic T cells.

[0163] 5. Experimental results

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

[0165] like Fig.12 As shown, compared with the spleen coefficient of the blank group mice, the spleen coefficient of the model group mice was significantly different, indicating that CTX treatment of mice can affect the spleen. Compared with the spleen coefficient of the model group mice, the spleen coefficients of the Lactobacillus paracasei IOB413 postbiotic group, the fermented Lactobacillus mucus IOB802 postbiotic group, the animal Bifidobacterium lactis subspecies IOB-LO7 postbiotic group, the multi-probiotic segmented solid fermentation postbiotic group, and the multi-probiotic segmented fermentation chickpea postbiotic group were significantly different, and the spleen coefficient of the multi-probiotic segmented solid fermentation postbiotic group was significantly restored, indicating that the multi-probiotic segmented solid fermentation postbiotics were able to restore the decrease in the spleen coefficient caused by CTX, which helped to improve the body's immunity.

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

[0167] like Fig.13As shown in the figure, compared with the splenic lymphocytes of the blank group mice, the splenic lymphocytes of the model group mice were significantly different, indicating that after CTX treatment of mice, the proliferation of their splenic lymphocytes was inhibited. Compared with the blank group, after intervention with Lactobacillus paracasei IOB413 postbiotics, fermented Lactobacillus mucus IOB802 postbiotics, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics, multiple probiotics segmented solid fermentation postbiotics, and multiple probiotics segmented fermentation chickpea postbiotics, the proliferation of splenic cells in CTX-treated mice was not significantly different from that in the blank group, and multiple probiotics segmented solid fermentation postbiotics had a significant promoting effect on the proliferation of splenic cells in CTX-treated mice. This shows that multiple probiotics segmented solid fermentation postbiotics can restore the inhibitory effect of CTX-induced splenic lymphocyte proliferation and have a certain protective effect on CTX-induced spleen damage.

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

[0169] CD3 is a marker for T cells. CD4 is expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells, and plays an important role in the development and activation of T cells. T cells expressing CD8 usually differentiate into cytotoxic T cells (CTLs) after activation, which can specifically kill target cells. Flow cytometry was used to measure CD4 in spleen lymphocytes. 3+ CD 4+ , CD 3+ CD 8+ The proportion of cells. 3+ CD 4+ / CD 3+ CD 8+ CD represents the ratio of T helper cells to T suppressor cells. 3 + CD 4+ / CD 3+ CD 8+ A decrease in the ratio indicates that the body has immunodeficiency disease or viral infection, and the risk of infection increases. 3+ CD 4+ / CD 3+ CD 8+ An increase in the ratio indicates that the body's immune function is hyperactive, and autoimmune diseases often occur.

[0170] like Fig.14 As shown, compared with the blank group, the CD 3+ CD 4+ / CD 3+ CD 8+Compared with the model group, CD of CTX-treated mice was significantly decreased after oral administration of Lactobacillus paracasei IOB413 postbiotics, Lactobacillus mucilaginosus fermentation IOB802 postbiotics, Bifidobacterium animalis subsp. lactis IOB-LO7 postbiotics, multiple probiotics solid fermentation postbiotics, and multiple probiotics fermentation chickpea postbiotics. 3+ CD 4+ / CD 3+ CD 8+ All of them increased, and the increase of the postbiotic group after multiple probiotics solid-state fermentation was more obvious, with significant differences. This shows that postbiotics after multiple probiotics solid-state fermentation can improve the body's immune system damage and further improve the body's immune function.

[0171] 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 postbiotics by segmented solid-state fermentation of a probiotic combination, characterized in that: The steps include: S1, strain activation: fermented Lactobacillus mucilaginosus IOB802, Lactobacillus paracasei IOB413, and Bifidobacterium animalis subspecies lactis IOB-LO7 were activated and cultured to obtain fermented Lactobacillus mucilaginosus IOB802 secondary seed liquid, Lactobacillus paracasei IOB413 secondary seed liquid, and Bifidobacterium animalis subspecies lactis IOB-LO7 secondary seed liquid; S2, aerobic fermentation: the activated cultured Lactobacillus paracasei IOB413 secondary seed liquid is inoculated into the sterilized solid fermentation substrate at a material-liquid ratio of 1:1.5-1:1.75 for aerobic fermentation, followed by low-temperature drying to obtain Lactobacillus paracasei IOB413 postbiotics; S3, facultative anaerobic fermentation: the activated cultured fermented Lactobacillus mucilaginosus IOB802 secondary seed liquid is inoculated into the Lactobacillus paracasei IOB413 postbiotic at a material-liquid ratio of 1:1.5-1:1.75 for facultative anaerobic fermentation, followed by low-temperature drying to obtain a composite postbiotic of Lactobacillus paracasei IOB413 and fermented Lactobacillus mucilaginosus IOB802; S4. Strict anaerobic fermentation: The activated cultured animal Bifidobacterium lactis subsp. IOB-LO7 secondary seed liquid is inoculated into the composite postbiotics of Lactobacillus paracasei IOB413 and Lactobacillus mucilaginosus IOB802 at a solid-liquid ratio of 1:1.5-1:1.75 for strict anaerobic fermentation, followed by high-temperature inactivation, drying, and pulverization to obtain the probiotic combination segmented solid-state fermentation postbiotics.

2. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: The Lactobacillus 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 the General Microbiology Center of the China National Microbiological Culture Collection Administration, and a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; The fermented mucus lactobacillus IOB802, strain name: IOB802, classification name: fermented mucus lactobacillus (Limosilactobacillus fermentum), preservation number: CGMCC No.23120, preservation date: August 5, 2021, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; The animal Bifidobacterium lactis subsp. lactis IOB-LO7, the strain name is: IOB-LO7, the classification name is: Bifidobacterium animalis subsp. lactis, the preservation number is: CGMCC No. 24185, the preservation date is: December 23, 2021, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

3. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: In step S1, the activation method of the Lactobacillus paracasei IOB413 strain is as follows: taking out the Lactobacillus paracasei IOB413 from the strain freezing tube, inoculating it into an activation medium at a volume ratio of 1:20, culturing it at 37±2°C for 22h±2h to obtain a primary seed solution, inoculating the primary seed solution into an activation medium at a volume ratio of 1:25, culturing it at 37±2°C for 22±2h to obtain a secondary seed solution; The activation medium of Lactobacillus paracasei IOB413 is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2.0 g K2HPO4·7H2O, 5.0 g CH3COONa·3H2O, 2.0 g C6H5O7 (NH4)3, 2.0 g MgSO 4` 7H2O 0.2g, MnSO4·4H2O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2±0.2, sterilize at 121℃ for 15min; The activation method of the fermented mucus lactobacillus IOB802 strain is as follows: taking the fermented mucus lactobacillus IOB802 from a strain freezing tube, inoculating it into an activation medium at a volume ratio of 1:20, culturing it at 37±2°C for 22±2h, to obtain a first-level seed liquid, inoculating the first-level seed liquid into an activation medium at a volume ratio of 1:25, culturing it at 37±2°C for 22±2h, to obtain a second-level seed liquid; The activation medium for fermenting Lactobacillus mucilaginosus IOB802 is MRS medium, which includes: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 1.0 g Tween 80, 2.0 g K2HPO4·7H2O, 5.0 g CH3COONa·3H2O, 2.0 g C6H5O7 (NH4)3, 2.0 g MgSO 4` 7H2O 0.2g, MnSO4·4H2O 0.05g, agar 15.0g, dilute to 1L, adjust pH = 6.2±0.2, sterilize at 121℃ for 15min; The method for activating the animal Bifidobacterium lactis subspecies IOB-LO7 strain is as follows: taking out the animal Bifidobacterium lactis subspecies IOB-LO7 from a strain freezing tube, inoculating it into an activation medium at a volume ratio of 1:20, activating it in a closed anaerobic environment at 37±2°C for 20±2h to obtain a first-level seed liquid, inoculating the first-level seed liquid into an activation medium at a volume ratio of 1:25, activating it in a closed anaerobic environment at 37°C±2°C for 24±2h to obtain a second-level seed liquid; Among them, the activation culture medium of animal Bifidobacterium lactis subspecies IOB-LO7 is a modified TPY liquid culture medium, the ingredients of which include: 10.0g hydrolyzed casein, 5.0g plant peptone, 2.0g yeast powder, 5.0g glucose, 1.0g L-cysteine, 2.0g dipotassium hydrogen phosphate, fixed volume to 1L, adjusted pH 6.5±0.1, and sterilized at 121°C for 15min.

4. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: In step S2, the solid-state fermentation substrate is chickpea.

5. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: In step S2, the conditions for aerobic fermentation are: culture temperature of 37±2°C, ventilation volume of 0.2 m³ / min, static culture for 12±2h, and obtaining a fermentation product of Lactobacillus paracasei IOB413.

6. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: In step S3, the conditions for facultative anaerobic fermentation are: the culture temperature is 37±2° C., and the culture is statically cultured for 12±2 hours to obtain a composite fermentation product of Lactobacillus paracasei IOB413 and Lactobacillus mucilaginosus IOB802.

7. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: In step S4, the conditions for strict anaerobic fermentation are: culture temperature of 37±1°C, nitrogen ventilation of 0.1m³ / min, closed anaerobic environment, culture for 12±2h, and obtaining the probiotic combination segmented solid fermentation product.

8. The method for preparing postbiotics by segmented solid-state fermentation of a probiotic combination according to claim 1, characterized in that: In step S4, the high temperature inactivation temperature is 75±2°C, and the drying temperature is 55±2°C.

9. A probiotic combination segmented solid-state fermentation postbiotic according to any one of claims 1 to 8, prepared by the method according to claim 1.

10. Use of the probiotic combination segmented solid-state fermentation postbiotics according to claim 9 in the preparation of immunomodulatory preparations.

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