Preparation method of lactobacillus reuteri probiotics targeting mucosal immunity

Abstract

The present application relates to the technical field of probiotic postbiotic preparation, and particularly relates to a preparation method of a Lactobacillus reuteri postbiotic with targeted mucosal immunity, comprising the following steps: recovering and activating the Lactobacillus reuteri after thawing to obtain activated strains; inoculating the activated strains into multiple shaking bottles containing liquid culture medium for culture and adding tryptophan into the shaking bottles to screen target inducing strains; inoculating the target inducing strains into a biological fermentation tank for amplification culture to obtain amplified strains; sequentially performing heat treatment inactivation and activity detection on the amplified strains, and outputting the Lactobacillus reuteri basic postbiotic when the activity of the amplified strains meets the requirements; and sequentially performing centrifugal washing, dialysis purification and other operations on the Lactobacillus reuteri basic postbiotic to output the Lactobacillus reuteri postbiotic with targeted mucosal immunity function. The present application realizes the improvement of the precision of the Lactobacillus reuteri postbiotic preparation process and the improvement of the targeted mucosal immunity ability.

Description

Technical Field

[0001] This invention relates to the field of probiotic postbiotic preparation technology, and in particular to a method for preparing a Lactobacillus reuteri postbiotic that targets mucosal immunity. Background Technology

[0002] In existing technologies, metabiotics have similar beneficial effects on the host to probiotics, and their stability and safety are far superior to those of probiotics. Therefore, they have a wide range of cutting-edge applications in the health field of targeting mucosal immunity. For example, they can increase the expression of intestinal tight junction proteins, repair damaged intestinal mucosa, and prevent harmful substances from entering the bloodstream; by regulating immune responses and inhibiting pro-inflammatory factors, they have significant protective and alleviating effects on intestinal and liver damage caused by necrotizing enterocolitis, alcoholic liver injury, etc.; and regulate the balance of gut microbiota and optimize the intestinal microecological environment.

[0003] Chinese Patent Publication No. CN117603851A discloses a method for preparing a special postbiotic strain of *Lactobacillus reuteri*. The specific components include: bacterial cells: teichoic acid, teichoic acid, peptidoglycan, cell surface proteins, polysaccharides, cell membrane proteins, and extracellular polysaccharides; metabolites: vitamins, lipids, proteins, peptides, organic acids, and intracellular polysaccharides. The specific processing steps of the bacterial strain are as follows: S1: Initial selection of the original strain; S2: Culture induction; S3: Screening; S4: Multiple passages; S5: Inactivation; S6: Chemical castration; S7: Five-layer encapsulation. This invention provides a method for preparing a special postbiotic strain of *Lactobacillus reuteri* using *Lactobacillus reuteri* as the initial strain, utilizing multiple processing techniques to facilitate its transportation and storage, reduce external environmental requirements, and employing multi-layer encapsulation to facilitate its arrival in the intestines.

[0004] Therefore, it can be seen that the method for preparing postbiotics of Lactobacillus reuteri has the following problems: when the density of Lactobacillus reuteri is too high during the induction and high-density fermentation process, it leads to competition for nutrients and stress on the development space within the bacteria, resulting in reduced bacterial survival activity and premature decay of bacterial activity during inactivation, leading to metabolic disorders and inactivation failure. Summary of the Invention

[0005] Therefore, this invention provides a method for preparing a mucosal immune-targeting Lactobacillus reuteri metabiotic, which overcomes the problems in the prior art where excessively high density of Lactobacillus reuteri during culture induction and high-density fermentation leads to competition for nutrients and stress on development space within the bacteria, resulting in reduced bacterial survival activity and inaccurate inactivation due to premature decay of bacterial activity during inactivation, as well as a decline in the quality of metabiotic preparation.

[0006] To achieve the above objectives, the present invention provides a method for preparing a mucosal immune-targeting Lactobacillus reuteri postbiotic, comprising: The thawed Lactobacillus reuteri was revived and activated to export the activated strain; The activated strain was inoculated into multiple shake flasks containing liquid culture medium and cultured. Tryptophan was added to a shake flask that met the induction conditions to induce the activated strain inoculated in liquid culture medium; At the end of induction, the production of indole-3-lactic acid by the activated strain in the shake flask was measured to screen for target induced strains; The target induced strain was inoculated into a bio-fermenter for amplification culture to output the amplified strain; The temperature of the amplification culture process is determined based on the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain. The amplified strain was subjected to heat treatment for inactivation and activity detection in sequence. When the activity of the amplified strain met the requirements, the basic metabiotic of Lactobacillus reuteri was output. The inactivation time of the amplified strain during the heat treatment inactivation process is determined based on the interval between the time when the target induced strain exhibits a stress state and the time when it recovers to the standard metabolic rate. The reuteri lactic acid bacteria basal metabiotic was subjected to centrifugation, washing, dialysis purification, encapsulation, and freeze-drying in sequence to output a reuteri lactic acid bacteria metabiotic with targeted mucosal immune function.

[0007] Furthermore, the revival and activation process of thawed Lactobacillus reuteri includes: Thawed Lactobacillus reuteri was aspirated from the biosafety cabinet and added dropwise to the first liquid culture medium; Under predetermined temperature conditions, the first liquid culture medium containing thawed Lactobacillus reuteri was placed on a shaker for resuscitation to obtain the first revived strain. The first revived strain was transferred to the second liquid culture medium and cultured in a shaker at the predetermined temperature to obtain the second revived strain. The second revived strain was transferred to a third liquid culture medium and cultured in a shaker at the predetermined temperature to obtain an activated strain.

[0008] Furthermore, the CFU value of the activated strain is greater than that of the second resuscitated strain, and the CFU value of the second resuscitated strain is greater than that of the first resuscitated strain; the pH value of the activated strain is less than that of the second resuscitated strain, and the pH value of the second resuscitated strain is less than that of the first resuscitated strain; the first liquid culture medium, the second liquid culture medium, and the third liquid culture medium have the same composition.

[0009] Furthermore, the predetermined temperature condition is that the temperature inside the shaker is 37°C.

[0010] Furthermore, the induction conditions are that the OD600 value of the liquid culture medium inoculated with the activated strain is greater than or equal to 0.4 and less than or equal to 0.6.

[0011] Furthermore, the production of indole-3-lactic acid by the activated strains in shake flasks was measured to screen for target induced strains, including: After inducing the activated bacterial strain in the shake flask, the yield of indole-3-lactic acid in the liquid culture medium in the shake flask was measured within a unit period. The activated strains that meet the predetermined production conditions of indole-3-lactic acid are identified as the target inducing strains; The predetermined production condition is that the yield of indole-3-lactic acid is greater than the preset yield.

[0012] Furthermore, based on the fact that the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain is less than a preset time interval, the temperature of the amplification culture process is reduced; Specifically, based on the fact that the production rate of indole-3-lactic acid is less than the preset production rate, it is determined that the induced strain is under stress.

[0013] Furthermore, the process of heat-treating and inactivating the amplified strain includes: Centrifuge the amplified strain and retain the supernatant, then collect the bacterial sludge from the bottom of the centrifuge tube; The centrifuged bacterial sludge was placed into a sterile glass bottle in a sterile phosphate buffer solution to clean the bacterial sludge. Place the sterile glass bottle containing the cleaned bacterial sludge and sterile phosphate buffer in a constant temperature water bath to complete the heat treatment inactivation.

[0014] Furthermore, the heat treatment inactivation temperature is 80°C. Based on the fact that the interval between the moment when the induced strain exhibits a stress state and the moment when it recovers to the standard metabolic rate is longer than a preset interval, the inactivation time of the amplified strain during the heat treatment inactivation process is increased.

[0015] Furthermore, the temperature inside the bio-fermentation tank is 37°C; the pH value of the liquid culture medium inoculated with the target induced strain in the bio-fermentation tank is 5.5-6.5, and the dissolved oxygen concentration is 25%.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: After activating Lactobacillus reuteri, the present invention inoculates it into multiple shake flasks containing liquid culture medium for incubation. When the induction conditions are reached, a certain amount of tryptophan is added to each flask. The strain is screened by detecting the production of indole-3-lactic acid to ensure the stability of the strain and the quality of the subsequent amplified strain. When the stress state of the target induced strain coincides with the reproduction time of the target induced strain, the stress state is easily transmitted to the target induced offspring during the reproduction process, thereby affecting the quality of the target metabolites. Lowering the temperature of the amplification culture process reduces the degree of overlap between the stress state of the target induced strain and the next reproduction time of the target induced strain, thereby reducing the probability of the stress state being transmitted to the offspring of the target induced strain and weakening the influence of the stress state on the strain. Furthermore, target-induced strains subjected to stress exhibit reduced activity and altered stress resistance, making them more susceptible to re-triggering stress during inactivation. This can lead to incomplete inactivation even with conventional inactivation times, and misidentification of stressed or recovering strains as inactivated strains during activity testing. Consequently, errors occur in monitoring the inactivation status of amplified strains. Therefore, increasing the inactivation time reduces inactivation errors and improves inactivation effectiveness.

[0017] Furthermore, this invention obtains activated strains by reviving and activating frozen Lactobacillus reuteri. The activated strains are then inoculated into multiple shake flasks containing liquid culture medium for incubation. When induction conditions are met, an equal amount of tryptophan is added for induction. The target induced strain is screened by detecting the production of indole-3-lactic acid in the liquid culture medium within a unit cycle. Because the revival and activation process of frozen Lactobacillus reuteri exposes it to the external environment, leading to partial cellular functional degeneration, and the freeze-thaw process triggers bacterial oxidative stress causing DNA damage, the screening of activated strains ensures that subsequent amplified strains will not mutate into pathogenic bacteria and maintain high metabolic capacity.

[0018] Furthermore, this invention detects the time interval between the point at which the target induced strain reaches the stress metabolic rate condition and the time of reproduction of the next generation of the target induced strain. Based on this interval, the amplification culture temperature of the target induced strain is reduced, thereby lengthening the reproduction cycle of the target induced strain and extending the interval between the two points. This reduces the stress impact on the offspring strains of the target induced strain. In addition, the low temperature reduces the cell division rate, providing a time window for DNA repair or the excretion of metabolic byproducts. By appropriately reducing the temperature of the amplification process, metabolic errors caused by strain stress are avoided, and the structure of the bacterial remains is altered, such as increased cell wall permeability and exposure of surface proteins, thus resulting in a stronger immunomodulatory capacity than ordinary inactivated bacteria.

[0019] Furthermore, this invention involves taking samples of the amplified strain from the bio-fermentation tank at fixed intervals and detecting the metabolic rate of the amplified strain. The time intervals at which the indole-3-lactic acid stress metabolic rate of the amplified strain decreases to the stress metabolic rate and at which the metabolic rate recovers to the standard metabolic rate are recorded. The duration of the inactivation process is adjusted according to these intervals, showing a positive correlation between the interval duration and the inactivation duration. When facing external stress, the activated strain loses a significant amount of energy, reducing its activity and leading to incomplete inactivation during the inactivation process. Simultaneously, extending the inactivation time helps the cell wall rupture, releasing more immunologically active intracellular substances. Therefore, by adjusting the duration of the inactivation process, the thoroughness of inactivation and the quality of the Lactobacillus reuteri basal metabiotic are improved. Attached Figure Description

[0020] Figure 1 This is an overall flowchart of the preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity according to an embodiment of the present invention; Figure 2 This is a flowchart illustrating step S1 of the method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to an embodiment of the present invention. Figure 3 This is a flowchart illustrating the specific process of step S6, heat inactivation, in the preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity according to an embodiment of the present invention. Figure 4 This is a flowchart illustrating the specific process of screening target inducing strains in step S4 of the method for preparing mucosal immune-targeting Lactobacillus postbiotics according to an embodiment of the present invention. Detailed Implementation

[0021] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0022] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0023] Please see Figure 1 The diagram shown is an overall flowchart of the preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity according to an embodiment of the present invention. The preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity in this embodiment includes: Step S1: The thawed Lactobacillus reuteri is revived and activated to output the activated strain; Step S2: The activated strain is inoculated into multiple shake flasks containing liquid culture medium for cultivation; Step S3: Add tryptophan to a shake flask that meets the induction conditions to induce the activated strain inoculated in liquid culture medium; Step S4: At the end of induction, the production of indole-3-lactic acid by the activated strain in the shake flask is detected to screen for target induced strains; Step S5: The target induced strain is inoculated into a bio-fermenter for amplification culture to output the amplified strain; The temperature of the amplification culture process is determined based on the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain. Step S6: The amplified strain is subjected to heat treatment inactivation and activity detection in sequence. When the activity of the amplified strain meets the requirements, the basic metabiotic of Lactobacillus reuteri is output. The inactivation time of the amplified strain during the heat treatment inactivation process is determined based on the interval between the time when the target induced strain exhibits a stress state and the time when it recovers to the standard metabolic rate. Step S7: The Lactobacillus reuteri basic metabiotic is subjected to centrifugation washing, dialysis purification, encapsulation and freeze drying in sequence to output Lactobacillus reuteri metabiotic with targeted mucosal immune function.

[0024] Please see Figure 2 The diagram shows a detailed flowchart of step S1 in the preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity in this embodiment. Step S1 includes: Step S11: The thawed Lactobacillus reuteri is aspirated from the biosafety cabinet and added dropwise to the first liquid culture medium; Step S12: Under predetermined temperature conditions, the first liquid culture medium containing thawed Lactobacillus reuteri is placed on a shaker for 14 hours to obtain the first revived strain. Step S13: Take the first resuscitated strain and transfer it to the second liquid culture medium. Continue to culture the resuscitated strain in a shaker at the predetermined temperature for 14 hours to obtain the second resuscitated strain. Step S14: The second revived strain is transferred to the third liquid culture medium and cultured in a shaker at the predetermined temperature for 16 hours to obtain the activated strain. In this embodiment, the CFU value of the activated strain is greater than the CFU value of the second revived strain, and the CFU value of the second revived strain is greater than the CFU value of the first revived strain; the pH value of the activated strain is less than the pH value of the second revived strain, and the pH value of the second revived strain is less than the pH value of the first revived strain; the first liquid culture medium, the second liquid culture medium, and the third liquid culture medium have the same composition.

[0025] The components of the first / second / third liquid culture medium are: 10.0 g peptone, 10.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 0.2 g magnesium sulfate, 5.0 g sodium acetate, 2.0 g triammonium citrate, 2.0 g dipotassium hydrogen phosphate, 0.04 g manganese sulfate, 1.0 mL Tween-80, and 1.0 L distilled water.

[0026] In this embodiment, the preset temperature condition is 37°C inside the shaker.

[0027] In this embodiment, step S2 includes: Step S21: Add 50 mL of liquid culture medium to each shake flask; Step S22: Add 1.5 mL of activated bacterial strain to each shake flask containing liquid culture medium; Step S23: Place all the shake flasks in an incubator for incubation. When the liquid culture medium in the shake flask meets the condition of 0.4≤OD600≤0.6, add 0.4g of tryptophan for induction for 18h to induce the production of indole-3-lactic acid.

[0028] Step S24: Sample the liquid culture medium for each shake flask to obtain a sample corresponding to each shake flask. Detect the yield of indole-3-lactic acid in each sample within a unit period using HPLC. The activated strain in the corresponding shake flask with an indole-3-lactic acid yield of [0.40g, 0.50g] within a unit period is identified as the target inducing strain.

[0029] In this embodiment, indole-3-lactic acid exerts a targeted mucosal immune effect, enhancing mucosal barrier function, regulating immune cell activity, and inhibiting excessive inflammatory response.

[0030] In this embodiment, step S5 includes: taking the target induced strain and inoculating it into a bio-fermenter containing liquid culture medium for high-density fermentation to amplify it, thereby obtaining the amplified strain; wherein, the temperature inside the bio-fermenter is 37°C; the pH value of the liquid culture medium in the bio-fermenter is 5.5-6.5 and the dissolved oxygen concentration is 25%.

[0031] In step S5, based on the fact that the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain is less than a preset time interval, the risk of the stress state being transmitted to the next generation of the target induced strain exceeds the allowable range, and the temperature of the amplification culture process is reduced. When the time interval is greater than the preset time interval, the risk of the stress state being transmitted to the next generation of the target induced strain is within the allowable range, and the bio-fermentation tank is controlled to amplify and culture the target induced strain at 37°C.

[0032] By reducing the temperature of the amplification culture process, the reproduction cycle of the activated strain is delayed, thereby increasing the time interval between the moment when the target induced strain reaches a stress state and the next reproduction time of the target induced strain. The temperature of the amplification culture process is at least greater than 30°C to prevent microbial ecological imbalance, which could ultimately lead to fermentation failure. It is understood that those skilled in the art can adaptively adjust the reduction of the temperature of the amplification culture process according to the actual culture objectives and the time interval between the moment when the target induced strain reaches a stress state and the next reproduction time of the target induced strain, which will not be elaborated here.

[0033] The time interval is used to determine the risk of transmission of the stress state to the target induced strain during its reproduction in the fermentation induction process. The smaller the time interval, the greater the risk of transmission of the stress state to the target induced strain during its reproduction in the fermentation induction process; the larger the time interval, the smaller the risk of transmission of the stress state to the target induced strain during its reproduction in the fermentation induction process.

[0034] For example, under the conditions of a bio-fermentation tank with a temperature of 37°C, a pH of 5.5-6.5, and a dissolved oxygen concentration of 25%, the preset time interval can be [30 min, 40 min], with a preferred embodiment being 35 min.

[0035] In this embodiment, the induced strain is determined to be under stress because the production rate of indole-3-lactic acid is less than the preset production rate.

[0036] The production rate of indole-3-lactic acid is defined as the mass of indole-3-lactic acid produced per unit volume of liquid culture medium inoculated with the target induced strain per unit time, and is determined by HPLC.

[0037] In this embodiment, under the conditions of abundant nutrients and suitable living space and environment, the preset production rate can be selected within the range of [2.33 mg / (L / h), 2.85 mg / (L / h)], with the preferred embodiment being 2.61 mg / (L / h).

[0038] In this embodiment, the reproduction time of the target induced strain is determined according to the standard reproduction cycle of the target induced strain, and the reproduction time includes the start time of each standard reproduction cycle; The standard reproduction cycle is the time elapsed from the start of reproduction of the target induced strain to the production of the next generation. The standard reproduction cycle is related to the temperature of the bioreactor, the pH of the liquid culture medium, and the dissolved oxygen concentration of the liquid culture medium. Under the conditions of a bioreactor temperature of 37°C, a pH of 5.5-6.5, and a dissolved oxygen concentration of 25%, the preferred embodiment of the standard reproduction cycle is 45 min. Those skilled in the art can determine and adjust the standard reproduction cycle through adaptation experiments according to specific culture conditions, which will not be elaborated here.

[0039] Please see Figure 3 The diagram shows a flowchart of the heat treatment inactivation process in the preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity according to an embodiment of the present invention. In step S6, the heat treatment inactivation process includes: Centrifuge the amplified strain and collect the bacterial sludge from the bottom of the centrifuge tube; The bacterial sludge was placed into a sterile glass bottle in a sterile phosphate buffer solution to clean it. The sterile glass bottle containing the cleaned bacterial sludge and sterile phosphate buffer was placed in a constant temperature water bath to complete the heat treatment inactivation.

[0040] The strain was washed three times, and the supernatant was discarded after each wash.

[0041] In this embodiment, the preferred heat treatment temperature of the constant temperature water bath is 80°C.

[0042] In this embodiment, the inactivation time of the amplified strain during the heat treatment inactivation process is increased because the time interval between the time when the induced strain reaches a stress state and the time when it recovers to the standard metabolic rate is longer than the preset interval.

[0043] Increasing the inactivation time ensures that the inactivation period of the amplified strain during heat treatment is not overridden by the stress recovery period of the induced strain. This reduces the error in detecting the inactivation of the amplified strain during heat treatment. It is understood that a longer inactivation time minimizes interference from the amplified strain being under stress or recovering from stress during the inactivation activity detection stage. When the amplified strain is under stress or recovering from stress, its activity is lower than that of strains not under stress or recovering from stress. Therefore, when detecting the activity of amplified strains, strains under stress or recovering from stress are easily misdetected as inactivated strains.

[0044] The inactivation time after the increase is determined based on the interval time. Preferably, the inactivation time and the interval time are positively correlated. The preset interval time is the standard inactivation time of the amplified strain. Preferably, the standard inactivation time is 1 hour. The inactivation time after the increase should be greater than 1.2 times and not more than 2 times the standard inactivation time to prevent the degradation of the active ingredients of the amplified strain due to prolonged inactivation treatment, thereby reducing the product efficacy of the Lactobacillus reuteri postbiotic that targets mucosal immunity. Those skilled in the art can make adaptive adjustments to the increase according to the actual preparation requirements, which will not be elaborated here.

[0045] In this embodiment, flow cytometry is used to detect the activity of the inactivated strain. If the area of ​​red fluorescence in the flow cytometry scatter plot is greater than 99%, the inactivation of the strain is determined to be complete. If the area of ​​red fluorescence in the flow cytometry scatter plot is less than or equal to 99%, the inactivation of the strain is determined to be incomplete and requires re-heat treatment for inactivation.

[0046] In this embodiment, the area ratio of red fluorescence is the ratio of the area of ​​red fluorescence in the flow cytometry scatter plot to the total area of ​​the flow cytometry scatter plot; wherein, the fluorescence type of the flow cytometry scatter plot includes red and green; it is understood that the process of using flow cytometry to detect the activity of strains is a prior art well known to those skilled in the art, therefore, the specific detection principle and specific detection process will not be described in detail here.

[0047] It is understandable that the longer the interval, the slower the recovery rate of the target induced strain from the stress state, and the shorter the interval, the faster the recovery rate of the target induced strain from the stress state.

[0048] In this embodiment, in step S7, the process of centrifugation washing and dialysis purification is as follows: the sterile phosphate buffer containing the inactivated amplified strain is cooled down and centrifuged at 12000 rpm at 4°C. The inactivated bacterial sludge is collected, washed three times with sterile distilled water, and the collected precipitate is suspended in sterile distilled water and added to a dialysis bag for purification to obtain the purified inactivated amplified strain. The inactivated amplified strain was purified by ultrasonic disruption, and the incompletely disrupted cell fragments were separated from the lysis buffer containing intracellular active substances by centrifugation again. The lysate was mixed 1:1 with the supernatant collected from the first centrifugation of the amplified strain after fermentation to obtain the epigenetic liquid; the epigenetic liquid was freeze-dried to obtain the Lactobacillus reuteri epigenetic with targeted mucosal immune function.

[0049] Example 1, the preparation method of Lactobacillus reuteri postbiotic targeting mucosal immunity in Example 1, includes: The thawed Lactobacillus reuteri was revived and activated to export the activated strain; The activated bacterial strain was inoculated into 50 shake flasks containing 50 mL of liquid culture medium and cultured. When the liquid culture medium in the shake flask meets the condition of 0.4 ≤ OD600 ≤ 0.6, add 0.4 g of tryptophan and induce for 18 h to produce indole-3-lactic acid; At the end of induction, the production of indole-3-lactic acid by the activated strain in the shake flask was measured to screen for target induced strains; The target induced strain was inoculated into a bio-fermenter for amplification culture to output the amplified strain; The temperature inside the bio-fermentation tank is 37°C, and the pH value of the liquid culture medium inoculated with the target induced strain in the bio-fermentation tank is 5.5-6.5, and the dissolved oxygen concentration is 25%. When the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain is 35 minutes, the bioreactor is controlled to amplify the target induced strain at 37℃ to output the amplified strain. When the time interval between the time when the target induced strain reaches a stress state and the time when it recovers to the standard metabolic rate is 1 hour, the amplified strain is subjected to heat treatment for 1 hour for inactivation and activity detection. When the activity of the amplified strain meets the requirements, the basic metabiotic of Lactobacillus reuteri is output. The reuteri lactic acid bacteria basal metabiotic was subjected to centrifugation, washing, dialysis purification, encapsulation, and freeze-drying in sequence to output a reuteri lactic acid bacteria metabiotic with targeted mucosal immune function.

[0050] In this Example 2, the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain is 28 minutes. The temperature in the bio-fermenter during the amplification culture process is set to 36.5°C. When the time interval between the moment when the target induced strain exhibits a stress state and the moment when it recovers to the standard metabolic rate is 1.2 hours, the inactivation time is set to 1.44 hours. In Example 3, the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain is 24 min. The temperature in the bio-fermenter during the amplification culture process is set to 36.0℃. When the time interval between the moment when the target induced strain exhibits a stress state and the moment when it recovers to the standard metabolic rate is 1.4 h, the inactivation time is 1.68 h. Example 4: Based on Example 2, the time interval between the time when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain is 20 min. The temperature of the amplification culture process is set to 35.7℃. When the time interval between the time when the target induced strain exhibits a stress state and the time when it recovers to the standard metabolic rate is 1.6 h, the inactivation time is 1.92 h.

[0051] Performance tables for various tests in Examples 2, 3, and 4

[0052] From the performance tables of the above embodiments, it can be seen that when the time intervals in Embodiments 2, 3, and 4 are less than the preset time interval, by lowering the temperature during the amplification process, the reproduction rate of the target induced strain decreases, thereby reducing the probability of the target induced strain experiencing living space stress and nutrient stress, and reducing the time interval between the stress state and reproduction time during the amplification process, thus reducing the probability of the stress state being passed on to offspring strains, ultimately increasing the rate of production of the target metabolite and the final yield; furthermore, the time intervals between Embodiments 2, 3, and 4... All inactivation times exceeded the standard inactivation time. Therefore, by increasing the inactivation time, the inactivation error of the amplified strain was reduced. The inactivation effect was detected by flow cytometry. The results showed that the proportion of red fluorescence in the samples detected by flow cytometry in Examples 2, 3, and 4 was 98% or higher, indicating that increasing the inactivation time improved the thoroughness of inactivation of the amplified strain. By lowering the amplification temperature and increasing the inactivation time, the production rate of indole-3-lactic acid was within the preset production rate, and the thoroughness of inactivation was significantly improved, ultimately improving the yield and quality of Lactobacillus reuteri metabiotics that target mucosal immunity.

[0053] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a mucosal immune-targeting Lactobacillus reuteri postbiotic, characterized in that, include: The thawed Lactobacillus reuteri was revived and activated to export the activated strain; The activated strain was inoculated into multiple shake flasks containing liquid culture medium and cultured. Tryptophan was added to a shake flask that met the induction conditions to induce the activated strain inoculated in liquid culture medium; At the end of induction, the production of indole-3-lactic acid by the activated strain in the shake flask was measured to screen for target induced strains; The target induced strain was inoculated into a bio-fermenter for amplification culture to output the amplified strain; The temperature of the amplification culture process is determined based on the time interval between the moment when the target induced strain exhibits a stress state and the next reproduction time of the target induced strain. The amplified strain was subjected to heat treatment for inactivation and activity detection in sequence. When the activity of the amplified strain met the requirements, the basic metabiotic of Lactobacillus reuteri was output. The inactivation time of the amplified strain during the heat treatment inactivation process is determined based on the interval between the time when the target induced strain exhibits a stress state and the time when it recovers to the standard metabolic rate. The reuteri lactic acid bacteria basal metabiotic was subjected to centrifugation, washing, dialysis purification, encapsulation, and freeze-drying in sequence to output a reuteri lactic acid bacteria metabiotic with targeted mucosal immune function.

2. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 1, characterized in that, The resuscitation and activation process of thawed Lactobacillus reuteri includes: Thawed Lactobacillus reuteri was aspirated from the biosafety cabinet and added dropwise to the first liquid culture medium; Under predetermined temperature conditions, the first liquid culture medium containing thawed Lactobacillus reuteri was placed on a shaker for resuscitation to obtain the first revived strain. The first revived strain was transferred to the second liquid culture medium and cultured in a shaker at the predetermined temperature to obtain the second revived strain. The second revived strain was transferred to a third liquid culture medium and cultured in a shaker at the predetermined temperature to obtain an activated strain.

3. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 2, characterized in that, The CFU value of the activated strain is greater than that of the second revived strain, and the CFU value of the second revived strain is greater than that of the first revived strain; the pH value of the activated strain is less than that of the second revived strain, and the pH value of the second revived strain is less than that of the first revived strain; the first liquid culture medium, the second liquid culture medium, and the third liquid culture medium have the same composition.

4. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 3, characterized in that, The predetermined temperature condition is that the temperature inside the shaker is 37°C.

5. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 4, characterized in that, The induction conditions are that the OD600 value of the liquid culture medium inoculated with the activated strain is greater than or equal to 0.4 and less than or equal to 0.

6.

6. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 5, characterized in that, The production of indole-3-lactic acid by activated strains in shake flasks was measured to screen for target induced strains, including: After inducing the activated bacterial strain in the shake flask, the yield of indole-3-lactic acid in the liquid culture medium in the shake flask was measured within a unit period. The activated strains that meet the predetermined production conditions of indole-3-lactic acid are identified as the target inducing strains; The predetermined production condition is that the yield of indole-3-lactic acid is greater than the preset yield.

7. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 6, characterized in that, Based on the fact that the time interval between the time when the target induced strain exhibits a stress state and the time when the target induced strain reproduces is less than a preset time interval, the temperature of the amplification culture process is reduced; Specifically, based on the fact that the production rate of indole-3-lactic acid is less than the preset production rate, it is determined that the induced strain is under stress.

8. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 7, characterized in that, The process of heat-treating to inactivate the amplified strain includes: Centrifuge the amplified strain and retain the supernatant, then collect the bacterial sludge from the bottom of the centrifuge tube; The centrifuged bacterial sludge was placed into a sterile glass bottle in a sterile phosphate buffer solution to clean the bacterial sludge. Place the sterile glass bottle containing the cleaned bacterial sludge and sterile phosphate buffer in a constant temperature water bath to complete the heat treatment inactivation.

9. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 8, characterized in that, The heat treatment inactivation temperature is 80°C. Based on the fact that the interval between the moment when the target induced strain exhibits a stress state and the moment when it recovers to the standard metabolic rate is longer than a preset interval, the inactivation time of the amplified strain during the heat treatment inactivation process is increased.

10. The method for preparing Lactobacillus reuteri postbiotics targeting mucosal immunity according to claim 9, characterized in that, The temperature inside the bio-fermentation tank is 37°C; the pH value of the liquid culture medium inoculated with the target induced strain in the bio-fermentation tank is 5.5-6.5, and the dissolved oxygen concentration is 25%.

Images