An Anti-inflammatory lactobacillus acidophilus composition for regulating immune responses

Abstract

The present invention discloses an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses, belonging to the fields of microbial technology and biomedicine. The composition comprises live lyophilized powders of Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium longum, together with homologous postbiotic lyophilized powders of the same strains. By combining functionally complementary probiotics with their homologous postbiotics, a full-chain anti-inflammatory system is constructed, covering immune regulation, inflammation suppression, and intestinal barrier repair. The live bacteria enable long-term colonization and microecological regulation, while the postbiotics act rapidly, producing a synergistic effect. The composition is prepared solely from strain-derived fermentation products without exogenous additives, ensuring safety and purity, and provides a stable and efficient solution for inflammation relief and immune function optimization.

Description

[0001] DESCRIPTION

[0002] An anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses

[0003] TECHNICAL FIELD

[0004] The present invention relates to the fields of microbial technology and biomedicine, and particularly to an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses.

[0005] BACKGROUND ART

[0006] The present invention relates to the technical field of microbial preparations and functional health products, and particularly concerns technologies associated with regulation of the intestinal microecology, immune regulation of the body, and inflammatory intervention. Chronic low-grade inflammation is one of the core inducements of multisystem health problems. As the largest immune organ of the body, the intestine directly determines the orderliness of immune responses and the occurrence and development of inflammatory reactions through the integrity of its barrier function and the stability of its microecology. Dysbiosis of the intestinal microecology directly damages the intestinal barrier, triggers persistent activation of endogenous inflammatory signals, and further leads to disordered immune function and aggravated systemic inflammatory cascades, thereby exerting sustained adverse effects on overall health. With the increasing recognition of microecological health and the deepening of related studies, the physiological functions of probiotics and their related metabolites in regulating immune responses, suppressing inflammation, and repairing the intestinal barrier have been widely confirmed, making them a research and application hotspot in the fields of inflammatory intervention and immune health. Precise combination of function-specific strains and synergistic application of live bacteria and postbiotics have also become core directions of technological research and development in this field and have high application value and broad market prospects.

[0007] Existing probiotic-related preparations for anti-inflammatory and immune- regulating purposes still present many non-negligible defects and limitations in research, development, and practical application. Most existing preparations employ a single strain or a randomly combined multistrain system, without i DESCRIPTION precise matching and synergistic design of strain functions for the full-chain targets involved in the occurrence and development of inflammation, and therefore cannot achieve full-dimensional coverage of immune regulation, inflammation suppression, and intestinal barrier repair. Synergy among strains is insufficient, making it difficult to achieve an ideal inflammatory intervention effect. Most conventional preparations rely on live bacteria as the core active component, but live bacteria themselves have poor environmental tolerance and insufficient storage stability, and are readily inactivated by the acidic and alkaline environment of the digestive tract, making it difficult for them to stably reach and colonize the intestine and exert physiological effects, so that the action of the preparation is highly unstable. At the same time, most existing preparations add various exogenous excipients and other active ingredients, which not only substantially increase the risks of sensitization and metabolic burden, but also interfere with the physiological activity of the core strains. Some strains used in certain preparations have not undergone systematic safety evaluation, resulting in potential safety risks. These defects cannot satisfy the demand for long-term safe conditioning and also restrict the scope of application and wider promotion of such products.

[0008] SUMMARY OF THE INVENTION

[0009] An object of the present invention is to overcome the deficiencies of the prior art by providing an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses. The method achieves highly sensitive and specific detection by designing a specific forward primer directed to the CDR3 region of a patient's monoclonal VDJ rearrangement sequence, in combination with a universal reverse primer and a fluorescent probe directed to the conserved sequence of the IGKJ1-5 gene, strictly standardizing nucleic-acid extraction from samples, establishing a primer validation procedure and a standardized digital PCR reaction system, and effectively reducing the risks of false positives and false negatives through increased loading amount in the retest stage, repeated-well settings, and threshold analysis based on six negative controls.

[0010] To solve the above technical problems, the present invention provides the DESCRIPTION following technical solutions. In one aspect, the present invention provides an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses, wherein the composition consists only of live-bacteria lyophilized powders of Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium longum and homologous postbiotic lyophilized powders of the corresponding strains, and the mass ratio of the live-bacteria lyophilized powders to the postbiotic lyophilized powders is 1 :2.

[0011] Further, the Lactobacillus acidophilus is Lactobacillus acidophilus LA-06, the Lactobacillus plantarum is Lactobacillus plantarum LPL28, and the Bifidobacterium longum is Bifidobacterium longum BL21; the three strains are all preserved strains subjected to whole-genome sequencing and contain no acquired drug-resistance genes or virulence genes.

[0012] Furthermore, in the live-bacteria lyophilized powders, the viable-count mass ratio of Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21 is 2:3:5, and the total viable count of the live- bacteria lyophilized powders is >=1 x 10Al 0 CFU / g.

[0013] Furthermore, in the postbiotic lyophilized powders, the mass ratio of inactivated cells of Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21 is 2:3:5, and the postbiotic lyophilized powders are products obtained by thermal inactivation in a constant-temperature water bath at 65 C for 30 min.

[0014] Furthermore, the live-bacteria lyophilized powders use a five-fold concentrated sterile supernatant of the fermentation broth of the corresponding strain from the same batch as the sole lyophilization protectant, and the composition contains no exogenous strains, prebiotics, traditional Chinese medicine extracts, food additives, or pharmaceutical excipients.

[0015] In another aspect, the present invention provides a preparation method of the antiinflammatory Lactobacillus acidophilus composition for regulating immune responses, comprising the following steps:

[0016] SI 00. Strain activation and seed preparation: separately subculturing and activating the three target strains to prepare seed cultures of the respective strains; DESCRIPTION

[0017] S200. Stepwise independent fermentation: inoculating the seed cultures of the respective strains into their dedicated fermentation media and carrying out independent static fermentation to obtain single-strain fermentation broths;

[0018] S300. Preparation of live-bacteria component: equally dividing each single-strain fermentation broth at a volume ratio of 1:2, and subjecting one-third of the broth to centrifugation, washing, resuspension, and vacuum freeze-drying to obtain a single- strain live-bacteria lyophilized powder;

[0019] S400. Preparation of postbiotic component: subjecting the remaining two-thirds of the broth to thermal inactivation, sterility verification, centrifugation and mixing, and vacuum freeze-drying to obtain a single-strain postbiotic lyophilized powder; S500. Secondary compounding and sample preparation: separately premixing the respective single-strain live-bacteria lyophilized powders and single-strain postbiotic lyophilized powders, then mixing the two premixes and carrying out aseptic packaging to obtain the final composition.

[0020] Furthermore, the strains are subcultured and activated for 2-3 generations; the liquid activation culture time for Lactobacillus acidophilus and Lactobacillus plantarum is 18-24 h, the liquid activation culture time for Bifidobacterium longum is 24-36 h; 0.5 g / L of L-cysteine hydrochloride is added to the activation medium for Bifidobacterium longum to lower the redox potential and create an anaerobic microenvironment; and the activation culture temperature is 37 C.

[0021] Furthermore, the inoculation volume of the seed culture is 3%-5% of the volume of the dedicated fermentation medium for the corresponding strain; the three strains are fermented independently in separate batches to avoid crosscontamination; the fermentation time for Lactobacillus acidophilus and Lactobacillus plantarum is 36 h, with the pH maintained at 5.5-6.0 during fermentation; the fermentation time for Bifidobacterium longum is 48 h, with the pH maintained at 6.0-6.5 during fermentation, and the anaerobic degree of the fermentation environment being >=99.9%; the fermentation culture temperature is 37 C; the growth status of the strains is sampled and tested every 12 h throughout fermentation; and, after completion of fermentation, the broth is immediately cooled to 4 C for temporary storage. DESCRIPTION

[0022] Furthermore, the centrifugation parameters are a temperature of 4 C, a rotational speed of 6000 r / min, and a centrifugation time of 10-15 min; resuspension washing with 4 C precooled sterile normal saline is carried out 2-3 times, and the bacteria are collected by centrifugation under the same parameters each time; the vacuum freeze-drying parameters are a cold-trap temperature of -55 C, a vacuum degree of 10 Pa, and a lyophilization time of 24 h; and the five-fold concentrated sterile supernatant of the fermentation broth from the same batch is used as the lyophilization protectant, followed by prefreezing at -45 C for 6 h, vacuum sublimation at -10 C for 12 h, and analytical drying at 25 C for 6 h to obtain a single-strain live-bacteria lyophilized powder having a moisture content of <=3.0%.

[0023] Furthermore, thermal inactivation is carried out by transferring the fermentation broth to a sterile water-bath inactivation tank, controlling the water-bath temperature at 65 C, and carrying out inactivation with heat preservation for 30 min under uniform stirring; samples are spread on the corresponding MRS solid media, with Lactobacillus acidophilus LA-06 and Lactobacillus plantarum LPL28 cultured at 37 C for 48 h and Bifidobacterium longum BL21 cultured anaerobically at 37 C for 72 h, and after verification that no viable bacteria grow, centrifuging at 4 C and 6000 r / min for 10-15 min, collecting all bacterial precipitates and supernatants together, and subjecting the mixture to vacuum freeze-drying using the same parameters as those used for the live-bacteria lyophilized powder, thereby obtaining a single-strain postbiotic lyophilized powder having a moisture content of <=3.0%.

[0024] Furthermore, the premixing time for the live-bacteria lyophilized powders and the premixing time for the postbiotic lyophilized powders are both 30 min; the mixing time for the live-bacteria premix and the postbiotic premix is 40 min; and mixing is carried out by a three-dimensional motion mixer at a rotational speed of 15 r / min.

[0025] Compared with the prior art, the anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses provided by the present invention has the following beneficial effects: DESCRIPTION

[0026] I. By scientifically combining three functionally complementary probiotic live strains with their corresponding homologous postbiotics, the present invention constructs a full-chain anti-inflammatory and immune-regulating system and achieves deep synergistic enhancement of strain efficacy. The ordered regulation of immune responses by Lactobacillus acidophilus balances the immune state at the source and avoids inflammatory cascade reactions induced by abnormal immune responses; the anti-inflammatory action of Lactobacillus plantarum directly suppresses excessive activation of inflammatory pathways and effectively relieves inflammation; and the repair effect of Bifidobacterium longum on the intestinal barrier strengthens the core protective barrier of the body and reduces persistent stimulation by endogenous inflammatory inducers. The three together form a complete functional loop from immune regulation to inflammation suppression to barrier repair. In combination with the complementary advantages of live bacteria and postbiotics, the composition provides both immediacy and long-term efficacy, adapts to the conditioning needs of different physiological states, offers sustained health protection, and steadily improves overall health.

[0027] II. Through the integrated fermentation preparation of homologous strains and the precise compounding system, the present invention achieves highly simplified ingredients and controllable safety. The whole preparation process relies on the strains' own fermentation products and does not require exogenous additives, thereby avoiding the body burden and sensitization risks potentially caused by various excipients and additional additives. By rationally combining live bacteria and postbiotics, the composition not only preserves the intestinal colonization and long-term proliferation effects of live bacteria, which continuously regulate the intestinal microecological balance, but also takes advantage of the high stability and high bioavailability of postbiotics to overcome the environmental limitations of live-bacteria applications and rapidly exert physiological activity. The two synergistically adapt to different intestinal environments and stably exert antiinflammatory and immune-regulating effects. While realizing comprehensive multi-target conditioning, the composition broadens the scope of application and improves stability of action, thereby providing a safe and efficient solution for DESCRIPTION relief of inflammation and improvement of health status.

[0028] Other advantages, objectives, and features of the present invention will be set forth in part in the following description and in part will become apparent to those skilled in the art upon examination of the following disclosure or may be learned from practice of the invention.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To describe the technical solutions in the embodiments of the present invention or in the prior art more clearly, the drawings required for describing the embodiments or the prior art are briefly introduced below. Obviously, the drawings in the following description merely illustrate certain embodiments of the present invention, and those of ordinary skill in the art can derive other drawings therefrom without inventive effort.

[0031] Figure 1 is a framework diagram of the anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses;

[0032] Figure 2 is a flow chart of the anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses;

[0033] Figure 3 is a flow chart of the stepwise independent fermentation preparation process.

[0034] DETAILED DESCRIPTION

[0035] In order to further illustrate the technical means and effects adopted by the present invention to achieve the intended objectives, the specific implementations, structures, features, and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0036] The strains used in the present invention include Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21. The three strains have all undergone whole-genome sequencing and have been deposited in nationally recognized microbial preservation institutions. Bioinformatic analysis of the whole-genome sequences confirms that none of the three strains carries acquired drug-resistance genes or virulence genes, and none has potential pathogenicity. Accordingly, they exhibit good biosafety and are suitable for DESCRIPTION development and application in foods and related functional products.

[0037] EXAMPLE 1:

[0038] This example provides an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses. The composition consists only of live-bacteria lyophilized powders of Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium longum, together with homologous postbiotic lyophilized powders of the corresponding strains. The mass ratio of the live- bacteria lyophilized powders to the postbiotic lyophilized powders is 1:2.

[0039] Specifically, as shown in Figure 1, the composition of this example is prepared by the following steps.

[0040] SI 00. Strain activation and seed preparation: the three target strains are separately subcultured and activated to prepare seed cultures of the respective strains.

[0041] Specifically, the strains are subcultured and activated for 2 generations. MRS liquid medium is used for activation. The liquid activation culture time for Lactobacillus acidophilus and Lactobacillus plantarum is 20 h, and the liquid activation culture time for Bifidobacterium longum is 30 h. 0.5 g / L of L-cysteine hydrochloride is added to the activation medium for Bifidobacterium longum to lower the redox potential of the medium and create an anaerobic microenvironment suitable for growth of the strain. The activation culture temperature for all strains is 37 C.

[0042] More specifically, the detailed operation of single-strain activation is as follows. The preserved glycerol stock of the target strain is taken and streak-inoculated onto the corresponding MRS solid-medium plate under sterile conditions. The inoculated plates of Lactobacillus acidophilus and Lactobacillus plantarum are cultured in a constant-temperature incubator at 37 C for 24 h. The inoculated plate of Bifidobacterium longum is cultured in an anaerobic incubator at 37 C for 36 h. After cultivation, single colonies on the plates that are morphologically full and uniform in size are selected and inoculated into the corresponding MRS liquid medium, where first-generation activation culture is completed under the corresponding conditions. The bacterial liquid obtained after first-generation activation culture is transferred at an inoculation amount of 2% by volume to DESCRIPTION fresh corresponding MRS liquid medium for second-generation activation culture, thereby obtaining a seed culture of the corresponding strain.

[0043] S200. Stepwise independent fermentation: the seed cultures of the respective strains are inoculated into their dedicated fermentation media for independent static fermentation to obtain single-strain fermentation broths, as shown in Figure 3.

[0044] Specifically, the inoculation volume of the seed culture is 4% of the volume of the dedicated fermentation medium for the corresponding strain. The three strains are fermented independently in separate batches to avoid cross-contamination among different strains and to ensure the purity of the single-strain fermentation broth and the stability of strain metabolites. The fermentation time for Lactobacillus acidophilus and Lactobacillus plantarum is 36 h, with the pH maintained at 5.5 to 6.0 during fermentation. The fermentation time for Bifidobacterium longum is 48 h, with the pH maintained at 6.0 to 6.5 during fermentation, the anaerobic degree of the fermentation environment being >=99.9%, and the fermentation culture temperature for all strains being 37 C. Samples are taken every 12 h during the entire fermentation process to detect the strain growth status, and, upon completion of fermentation, the broth is immediately cooled to 4 C for temporary storage.

[0045] Specifically, the dedicated fermentation medium for Lactobacillus acidophilus and Lactobacillus plantarum is an MRS basic fermentation medium containing peptone, beef extract, yeast extract powder, glucose, anhydrous sodium acetate, diammonium hydrogen citrate, Tween 80, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, and deionized water as the solvent. The dedicated fermentation medium for Bifidobacterium longum is prepared by additionally adding 0.5 g / L of L-cysteine hydrochloride to the above MRS basic fermentation medium, so as to reduce the oxidation-reduction potential of the medium and maintain a stable anaerobic growth environment.

[0046] S300. Preparation of live-bacteria component: each single-strain fermentation broth is equally divided at a volume ratio of 1:2, and one-third of the broth is centrifuged, washed, resuspended, and vacuum freeze-dried to obtain a single- DESCRIPTION strain live-bacteria lyophilized powder.

[0047] Specifically, the single-strain fermentation broth prepared in the same batch is equally divided at a volume ratio of 1:2, wherein one-third of the broth is used for preparing the single-strain live-bacteria lyophilized powder of the corresponding strain and the remaining two-thirds of the broth from the same batch is used for subsequently preparing the single-strain postbiotic lyophilized powder of the corresponding strain.

[0048] Specifically, the centrifugation parameters are a temperature of 4 C, a rotational speed of 6000 r / min, and a centrifugation time of 12 min. After centrifugation, the supernatant is discarded and the bacterial precipitate is collected, resuspended, and washed with 4 C precooled sterile normal saline. The washing operation is repeated three times, and after each washing the bacteria are collected by centrifugation under the same parameters. After washing, the five-fold concentrated sterile supernatant of the fermentation broth of the corresponding strain from the same batch is used as the lyophilization protectant to resuspend the bacteria, thereby obtaining a bacterial suspension for lyophilization.

[0049] Specifically, the preparation method of the five-fold concentrated sterile supernatant of the fermentation broth from the same batch is as follows. The single-strain fermentation broth from the same batch is subjected to centrifugation using the same parameters as above, and the supernatant is collected. The supernatant is sterilized by filtration through a 0.22 um sterile membrane to obtain a sterile supernatant. The sterile supernatant is then concentrated five-fold by means of low-temperature vacuum concentration equipment at 40 C and a vacuum degree of 0.09 MPa, thereby obtaining a concentrated sterile supernatant for use as the lyophilization protectant.

[0050] Specifically, the operating procedure of vacuum freeze-drying is as follows. The prepared bacterial suspension is placed in a sterile freeze-drying tray with a material thickness of 8 mm and first prefrozen at -45 C for 6 h. After prefreezing is completed, the material is transferred to a vacuum freeze dryer, where, under a cold-trap temperature of -55 C and a vacuum degree of 10 Pa, it undergoes vacuum sublimation at -10 C for 12 h followed by analytical drying at 25 C for 6 DESCRIPTION h. After sterile discharge, a single-strain live-bacteria lyophilized powder having a moisture content of <=3.0% is obtained.

[0051] S400. Preparation of postbiotic component: the remaining two-thirds of the broth is subjected to thermal inactivation, sterility verification, centrifugation and mixing, and vacuum freeze-drying to obtain a single-strain postbiotic lyophilized powder.

[0052] Specifically, thermal inactivation is carried out as follows. The remaining two- thirds of the single-strain fermentation broth from the same batch is transferred to a sterile water-bath inactivation tank, the water-bath temperature is controlled at 65 C, and thermal inactivation is performed under uniform stirring with heat preservation for 30 min. After inactivation is completed, samples are taken for sterility verification.

[0053] Specifically, sterility verification is carried out as follows. The inactivated bacterial liquid is spread onto the corresponding MRS solid-medium plates, wherein the plates of Lactobacillus acidophilus LA-06 and Lactobacillus plantarum LPL28 are cultured in a constant-temperature incubator at 37 C for 48 h, and the plates of Bifidobacterium longum BL21 are cultured anaerobically at 37 C for 72 h. After culture is completed, the absence of viable bacterial growth on the plates confirms successful inactivation and good sterility of the postbiotic component.

[0054] Specifically, after sterility verification is completed, the inactivated fermentation broth is centrifuged at a temperature of 4 C and a rotational speed of 6000 r / min for 12 min. After centrifugation, all bacterial precipitates and supernatants are collected and thoroughly mixed to obtain a postbiotic suspension. The postbiotic suspension is freeze-dried using vacuum freeze-drying parameters identical to those of the live-bacteria lyophilized powder, thereby obtaining a single-strain postbiotic lyophilized powder having a moisture content of <=3.0%.

[0055] S500. Secondary compounding and sample preparation: the respective singlestrain live-bacteria lyophilized powders and single-strain postbiotic lyophilized powders are separately premixed, then the two premixes are mixed, and the final composition is prepared by aseptic packaging. DESCRIPTION

[0056] Specifically, the single-strain live-bacteria lyophilized powders of Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21 are first weighed in accordance with a viable-count mass ratio of 2:3:5 and placed in a three-dimensional motion mixer for premixing at a rotational speed of 15 r / min for 30 min, thereby obtaining a live-bacteria premix.

[0057] Specifically, the single-strain postbiotic lyophilized powders of Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21 are then weighed in accordance with a mass ratio of 2:3:5 based on inactivated cells and metabolites and placed in a three-dimensional motion mixer for premixing at a rotational speed of 15 r / min for 30 min, thereby obtaining a postbiotic premix.

[0058] Specifically, the prepared live-bacteria premix and postbiotic premix are weighed in a mass ratio of 1:2 and placed in a three-dimensional motion mixer for final blending at a rotational speed of 15 r / min for 40 min. After mixing is completed, aseptic packaging is carried out in a Class 10,000 sterile environment to obtain the final composition.

[0059] The composition prepared in this example contains no exogenous strains, prebiotics, traditional Chinese medicine extracts, food additives, or pharmaceutical excipients, and all active ingredients originate from the live bacterial cells and homologous inactivated cells and metabolites of the three target strains.

[0060] EXAMPLE 2:

[0061] This example provides an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses. The composition consists only of live-bacteria lyophilized powders of Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium longum, together with homologous postbiotic lyophilized powders of the corresponding strains. The mass ratio of the live- bacteria lyophilized powders to the postbiotic lyophilized powders is 1:2.

[0062] Specifically, as shown in Figure 2, the composition of this example is prepared by the following steps.

[0063] SI 00. Strain activation and seed preparation: the three target strains are separately DESCRIPTION subcultured and activated to prepare seed cultures of the respective strains.

[0064] Specifically, the strains are subcultured and activated for 2 generations. MRS liquid medium is used for activation. The liquid activation culture time for Lactobacillus acidophilus and Lactobacillus plantarum is 18 h, and the liquid activation culture time for Bifidobacterium longum is 24 h. 0.5 g / L of L-cysteine hydrochloride is added to the activation medium for Bifidobacterium longum to lower the redox potential of the medium and create an anaerobic microenvironment suitable for growth of the strain. The activation culture temperature for all strains is 37 C.

[0065] More specifically, the detailed operation of single-strain activation is the same as that of Example 1.

[0066] S200. Stepwise independent fermentation: the seed cultures of the respective strains are inoculated into their dedicated fermentation media for independent static fermentation to obtain single-strain fermentation broths.

[0067] Specifically, the inoculation volume of the seed culture is 3% of the volume of the dedicated fermentation medium for the corresponding strain. The three strains are fermented independently in separate batches to avoid cross-contamination among different strains and to ensure the purity of the single-strain fermentation broth and the stability of strain metabolites. The fermentation time for Lactobacillus acidophilus and Lactobacillus plantarum is 36 h, with the pH maintained at 5.5 to 6.0 during fermentation. The fermentation time for Bifidobacterium longum is 48 h, with the pH maintained at 6.0 to 6.5 during fermentation, the anaerobic degree of the fermentation environment being >=99.9%, and the fermentation culture temperature for all strains being 37 C.

[0068] Specifically, the dedicated fermentation media are the same as those of Example 1. S300. Preparation of live-bacteria component: each single-strain fermentation broth is equally divided at a volume ratio of 1:2, and one-third of the broth is centrifuged, washed, resuspended, and vacuum freeze-dried to obtain a singlestrain live-bacteria lyophilized powder.

[0069] Specifically, the division of the broth is the same as in Example 1.

[0070] Specifically, the centrifugation parameters are a temperature of 4 C, a rotational DESCRIPTION speed of 6000 r / min, and a centrifugation time of 10 min. After centrifugation, the supernatant is discarded and the bacterial precipitate is collected, resuspended, and washed with 4 C precooled sterile normal saline. The washing operation is repeated twice, and after each washing the bacteria are collected by centrifugation under the same parameters. After washing, the five-fold concentrated sterile supernatant of the fermentation broth of the corresponding strain from the same batch is used as the lyophilization protectant to resuspend the bacteria.

[0071] The preparation method of the concentrated sterile supernatant and the operating procedure of vacuum freeze-drying are the same as those of Example 1.

[0072] S400. Preparation of postbiotic component: the remaining two-thirds of the broth is subjected to thermal inactivation, sterility verification, centrifugation and mixing, and vacuum freeze-drying to obtain a single-strain postbiotic lyophilized powder.

[0073] Specifically, thermal inactivation and sterility verification are the same as those of Example 1.

[0074] Specifically, after sterility verification is completed, the inactivated fermentation broth is centrifuged at a temperature of 4 C and a rotational speed of 6000 r / min for 10 min. After centrifugation, all bacterial precipitates and supernatants are collected and thoroughly mixed to obtain a postbiotic suspension. The postbiotic suspension is freeze-dried using vacuum freeze-drying parameters identical to those of the live-bacteria lyophilized powder, thereby obtaining a single-strain postbiotic lyophilized powder having a moisture content of <=3.0%.

[0075] S500. Secondary compounding and sample preparation: the respective singlestrain live-bacteria lyophilized powders and single-strain postbiotic lyophilized powders are separately premixed, then the two premixes are mixed, and the final composition is prepared by aseptic packaging.

[0076] Specifically, the premixing and final mixing operations are the same as those of Example 1.

[0077] The composition prepared in this example contains no exogenous strains, prebiotics, traditional Chinese medicine extracts, food additives, or pharmaceutical excipients, and all active ingredients originate from the live DESCRIPTION bacterial cells and homologous inactivated cells and metabolites of the three target strains.

[0078] EXAMPLE 3:

[0079] This example provides an anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses. The composition consists only of live-bacteria lyophilized powders of Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium longum, together with homologous postbiotic lyophilized powders of the corresponding strains. The mass ratio of the live- bacteria lyophilized powders to the postbiotic lyophilized powders is 1:2.

[0080] Specifically, the composition of this example is prepared by the following steps. SI 00. Strain activation and seed preparation: the three target strains are separately subcultured and activated to prepare seed cultures of the respective strains.

[0081] Specifically, the strains are subcultured and activated for 3 generations. MRS liquid medium is used for activation. The liquid activation culture time for Lactobacillus acidophilus and Lactobacillus plantarum is 24 h, and the liquid activation culture time for Bifidobacterium longum is 36 h. 0.5 g / L of L-cysteine hydrochloride is added to the activation medium for Bifidobacterium longum to lower the redox potential of the medium and create an anaerobic microenvironment suitable for growth of the strain. The activation culture temperature for all strains is 37 C.

[0082] More specifically, the detailed operation of single-strain activation is the same as that of Example 1.

[0083] S200. Stepwise independent fermentation: the seed cultures of the respective strains are inoculated into their dedicated fermentation media for independent static fermentation to obtain single-strain fermentation broths.

[0084] Specifically, the inoculation volume of the seed culture is 5% of the volume of the dedicated fermentation medium for the corresponding strain. The three strains are fermented independently in separate batches to avoid cross-contamination among different strains and to ensure the purity of the single-strain fermentation broth and the stability of strain metabolites. The fermentation time for Lactobacillus acidophilus and Lactobacillus plantarum is 36 h, with the pH maintained at 5.5 to DESCRIPTION

[0085] 6.0 during fermentation. The fermentation time for Bifidobacterium longum is 48 h, with the pH maintained at 6.0 to 6.5 during fermentation, the anaerobic degree of the fermentation environment being >=99.9%, and the fermentation culture temperature for all strains being 37 C.

[0086] Specifically, the dedicated fermentation media are the same as those of Example 1. S300. Preparation of live-bacteria component: each single-strain fermentation broth is equally divided at a volume ratio of 1:2, and one-third of the broth is centrifuged, washed, resuspended, and vacuum freeze-dried to obtain a singlestrain live-bacteria lyophilized powder.

[0087] Specifically, the division of the broth is the same as in Example 1.

[0088] Specifically, the centrifugation parameters are a temperature of 4 C, a rotational speed of 6000 r / min, and a centrifugation time of 15 min. After centrifugation, the supernatant is discarded and the bacterial precipitate is collected, resuspended, and washed with 4 C precooled sterile normal saline. The washing operation is repeated three times, and after each washing the bacteria are collected by centrifugation under the same parameters. After washing, the five-fold concentrated sterile supernatant of the fermentation broth of the corresponding strain from the same batch is used as the lyophilization protectant to resuspend the bacteria.

[0089] The preparation method of the concentrated sterile supernatant and the operating procedure of vacuum freeze-drying are the same as those of Example 1.

[0090] S400. Preparation of postbiotic component: the remaining two-thirds of the broth is subjected to thermal inactivation, sterility verification, centrifugation and mixing, and vacuum freeze-drying to obtain a single-strain postbiotic lyophilized powder.

[0091] Specifically, thermal inactivation and sterility verification are the same as those of Example 1.

[0092] Specifically, after sterility verification is completed, the inactivated fermentation broth is centrifuged at a temperature of 4 C and a rotational speed of 6000 r / min for 15 min. After centrifugation, all bacterial precipitates and supernatants are collected and thoroughly mixed to obtain a postbiotic suspension. The postbiotic DESCRIPTION suspension is freeze-dried using vacuum freeze-drying parameters identical to those of the live-bacteria lyophilized powder, thereby obtaining a single-strain postbiotic lyophilized powder having a moisture content of <=3.0%.

[0093] S500. Secondary compounding and sample preparation: the respective singlestrain live-bacteria lyophilized powders and single-strain postbiotic lyophilized powders are separately premixed, then the two premixes are mixed, and the final composition is prepared by aseptic packaging.

[0094] Specifically, the premixing and final mixing operations are the same as those of Example 1.

[0095] The composition prepared in this example contains no exogenous strains, prebiotics, traditional Chinese medicine extracts, food additives, or pharmaceutical excipients, and all active ingredients originate from the live bacterial cells and homologous inactivated cells and metabolites of the three target strains.

[0096] COMPARATIVE EXAMPLE:

[0097] The present comparative example provides a probiotic composition whose preparation method is basically the same as that of Example 1, except for the following aspects.

[0098] The composition of the comparative example consists only of live-bacteria lyophilized powders of Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21, without adding homologous postbiotic lyophilized powders of the corresponding strains. During preparation of the live-bacteria lyophilized powders, a 10% skim-milk powder solution is used as the lyophilization protectant instead of the five-fold concentrated sterile supernatant of the fermentation broth from the same batch. During preparation of the composition, only the single-strain live-bacteria lyophilized powders of the three strains are mixed according to a viable-count mass ratio of 2:3:5 to obtain the final composition. In the composition of the comparative example, fructooligosaccharide in a mass percentage of 1% is added as a prebiotic and magnesium stearate in a mass percentage of 0.5% is added as an anti-caking agent. EFFECT VERIFICATION TEST DESCRIPTION

[0099] To verify the immune-regulating and anti-inflammatory effects of the composition of the present invention, a mouse colitis model is used for testing. The test samples include the compositions prepared in Example 1, Example 2, and Example 3, as well as the composition prepared in the comparative example.

[0100] The test animals are SPF-grade male BALB / c mice weighing 20 g to 22 g. After adaptive feeding for 3 days, the mice are randomly divided into six groups, namely a blank control group, a model control group, an Example 1 group, an Example 2 group, an Example 3 group, and a comparative-example group, with 10 mice in each group.

[0101] Except for the blank control group, the mice in all other groups are used to establish an acute colitis model induced by dextran sulfate sodium, with a modeling period of 7 days. During modeling, the mice in the blank control group are allowed free access to sterile distilled water, while the mice in all other groups are allowed free access to a 3% dextran sulfate sodium solution (w / v).

[0102] At the same time as model induction, intragastric administration is started for a dosing period of 7 days. The mice in the blank control group and the model control group are intragastrically administered equal volumes of sterile normal saline every day. The mice in the Example 1 group, Example 2 group, and Example 3 group are intragastrically administered the compositions prepared in the corresponding examples every day at a dose of 10 mg of composition per 10 g body weight per day. The mice in the comparative-example group are intragastrically administered the composition prepared in the comparative example every day at the same dose as that used for the example groups.

[0103] After completion of dosing, the mice are fasted for 12 h but allowed free access to water, then sacrificed by cervical dislocation, and colon tissues and serum samples are collected for determination of relevant indices.

[0104] The specific indices measured include the disease activity index score of the mice, colon length, the contents of tumor necrosis factor-alpha, interleukin-6, and interleukin- 1 beta in serum, and the relative expression levels of occludin and zonula occludens-1 in colon tissue.

[0105] The test results are as follows. DESCRIPTION

[0106] Compared with the blank control group, the model control group shows a significantly increased disease activity index score, a significantly shortened colon length, significantly increased contents of tumor necrosis factor-alpha, interleukin-6, and interleukin- 1 beta in serum, and significantly decreased relative expression levels of occludin and zonula occludens-1 in colon tissue, indicating successful model establishment.

[0107] Compared with the model control group, the groups of the examples and the comparative example all show disease activity index scores reduced to different extents, colon length increased to different extents, inflammatory factor contents in serum reduced to different extents, and relative expression levels of tight- junction proteins in colon tissue increased to different extents. Among them, the improvement effects of all indices in the Example 1 group, Example 2 group, and Example 3 group are significantly better than those in the comparative-example group. There is no significant difference among the indices of the Example 1 group, Example 2 group, and Example 3 group, indicating that the technical solutions within the scope of protection of the claims of the present invention all have stable implementation effects.

[0108] Specifically, compared with the comparative-example group, the reductions in serum tumor necrosis factor-alpha, interleukin-6, and interleukin- 1 beta in the Example 1 group all exceed 40%, and the increases in relative expression levels of tight-junction proteins in colon tissue all exceed 35%, indicating that the composition of the present invention has superior anti-inflammatory and intestinal-barrier-repair effects while also exhibiting good immune-regulating activity.

[0109] Comparison of core parameters and effects:

[0110] For clearer presentation of the core differences and test effects of the examples and comparative example, the core parameters and key test results are summarized below: DESCRIPTION

[0111] Increas

[0112] Mass e in

[0113] Reductio ratio of Type of colonic n in total Core live lyophiliza tight-

[0114] Exogenous serum

[0115] Group composi bacteri tion junctio additives inflamma tion a to protectan n tory postbio t protein factors tics express ion

[0116] Live- bacteria lyophiliz

[0117] Five-fold ed concentrat powders ed sterile of the supernata three

[0118] Example nt of strains + None 68.2% 42.6%

[0119] 1 fermentati homolog on broth ous from the postbioti same c batch lyophiliz ed powders

[0120] Live- bacteria lyophiliz

[0121] Five-fold ed concentrat powders ed sterile of the supernata three

[0122] Example nt of strains + None 65.7% 40.1%

[0123] 2 fermentati homolog on broth ous from the postbioti same c batch lyophiliz ed powders

[0124] Example Live- 1 :2 Five-fold None 67.4% 41.8% DESCRIPTION

[0125] 3 bacteria concentrat lyophiliz ed sterile ed supernata powders nt of of the fermentati three on broth strains + from the homolog same ous batch postbioti c lyophiliz ed powders

[0126] Only live- bacteria No Fructooligosacc

[0127] Compara lyophiliz postbiot skim-milk haride; tive ed ic 32.5% 18.3% powder magnesium example powders compon solution stearate of the ent three strains

[0128] As can be seen from the above tabulated data, the technical solutions of the three examples of the present invention all achieve stable and excellent antiinflammatory and intestinal-barrier-repair effects, and all indices are significantly better than those of the prior-art solution represented by the comparative example. The three examples adjust the relevant operating conditions within the parameter ranges defined by the claims, and the finally obtained compositions show no significant difference in efficacy, indicating that the technical solution of the present invention has good stability and reproducibility and that the scope of protection defined by the claims is reasonable and can be fully implemented. Through scientific combination of homologous live bacteria and postbiotics together with a dedicated preparation process, the present invention achieves synergistic enhancement of immune regulation, inflammation suppression, and intestinal barrier repair without adding any exogenous excipients or active DESCRIPTION ingredients, thereby providing a stable and feasible technical solution for development of related functional products.

[0129] The foregoing are merely preferred embodiments of the present invention and do not limit the present invention in any form. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art may make certain changes or modifications to the disclosed technical contents without departing from the scope of the technical solution of the present invention, and such equivalent embodiments resulting from equivalent changes or modifications shall still fall within the scope of the technical solution of the present invention.

Claims

CLAIMS1. An anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses, characterized in that the composition consists only of live- bacteria lyophilized powders of Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium longum and homologous postbiotic lyophilized powders of the corresponding strains, wherein the mass ratio of the live-bacteria lyophilized powders to the postbiotic lyophilized powders is 1:2.

2. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 1, characterized in that the Lactobacillus acidophilus is Lactobacillus acidophilus LA-06, the Lactobacillus plantarum is Lactobacillus plantarum LPL28, and the Bifidobacterium longum is Bifidobacterium longum BL21; the three strains are all preserved strains subjected to whole-genome sequencing and contain no acquired drug-resistance genes or virulence genes.

3. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 1, characterized in that, in the live-bacteria lyophilized powders, the viable-count mass ratio of Lactobacillus acidophilus LA- 06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21 is 2:3:5, and the total viable count of the live-bacteria lyophilized powders is >=1 x 10A10 CFU / g.

4. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 1, characterized in that, in the postbiotic lyophilized powders, the mass ratio of inactivated cells of Lactobacillus acidophilus LA-06, Lactobacillus plantarum LPL28, and Bifidobacterium longum BL21 is 2:3:5, and the postbiotic lyophilized powders are products obtained by thermal inactivation in a constant-temperature water bath at 65 C for 30 min.

5. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 1, characterized in that the live-bacteria lyophilized powders use a five-fold concentrated sterile supernatant of the fermentation broth of the corresponding strain from the same batch as the sole lyophilization protectant; and the composition contains no exogenous strains, prebiotics, traditional Chinese medicine extracts, food additives, or pharmaceuticalCLAIMS excipients.

6. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 1, characterized in that the preparation method of the composition comprises the following steps:SI 00. Strain activation and seed preparation: separately subculturing and activating the three target strains to prepare seed cultures of the respective strains;S200. Stepwise independent fermentation: inoculating the seed cultures of the respective strains into their dedicated fermentation media and carrying out independent static fermentation to obtain single-strain fermentation broths;S300. Preparation of live-bacteria component: equally dividing each single-strain fermentation broth at a volume ratio of 1 :2, and subjecting one-third of the broth to centrifugation, washing, resuspension, and vacuum freeze-drying to obtain a singlestrain live-bacteria lyophilized powder;S400. Preparation of postbiotic component: subjecting the remaining two-thirds of the broth to thermal inactivation, sterility verification, centrifugation and mixing, and vacuum freeze-drying to obtain a single-strain postbiotic lyophilized powder;S500. Secondary compounding and sample preparation: separately premixing the respective single-strain live-bacteria lyophilized powders and single-strain postbiotic lyophilized powders, then mixing the two premixes and carrying out aseptic packaging to obtain the final composition.

7. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 6, characterized in that, in step SI 00, the strains are subcultured and activated for 2-3 generations; the liquid activation culture time for Lactobacillus acidophilus and Lactobacillus plantarum is 18-24 h, the liquid activation culture time for Bifidobacterium longum is 24-36 h; 0.5 g / L of L-cysteine hydrochloride is added to the activation medium for Bifidobacterium longum; and the activation culture temperature is 37 C.

8. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 6, characterized in that, in step S200, the inoculation volume of the seed culture is 3%-5% of the volume of the dedicated fermentation medium for the corresponding strain; the three strains are fermentedCLAIMS independently in separate batches to avoid cross-contamination; the fermentation time for Lactobacillus acidophilus and Lactobacillus plantarum is 36 h, with the pH maintained at 5.5 -6.0 during fermentation; the fermentation time for Bifidobacterium longum is 48 h, with the pH maintained at 6.0-6.5 during fermentation, and the anaerobic degree of the fermentation environment being >=99.9%; and the fermentation culture temperature is 37 C.

9. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 6, characterized in that, in step S300, the centrifugation parameters are a temperature of 4 C, a rotational speed of 6000 r / min, and a centrifugation time of 10-15 min; resuspension washing with 4 C precooled sterile normal saline is carried out 2-3 times, and the bacteria are collected by centrifugation under the same parameters each time; the vacuum freeze-drying parameters are a cold-trap temperature of -55 C, a vacuum degree of 10 Pa, and a lyophilization time of 24 h; and the five-fold concentrated sterile supernatant of the fermentation broth from the same batch is used as the lyophilization protectant, followed by prefreezing at -45 C for 6 h, vacuum sublimation at -10 C for 12 h, and analytical drying at 25 C for 6 h to obtain a single-strain live-bacteria lyophilized powder having a moisture content of <=3.0%.

10. The anti-inflammatory Lactobacillus acidophilus composition for regulating immune responses according to claim 6, characterized in that, in step S400, thermal inactivation is carried out by transferring the fermentation broth to a sterile waterbath inactivation tank, controlling the water-bath temperature at 65 C, and carrying out inactivation with heat preservation for 30 min under uniform stirring; samples are spread on the corresponding MRS solid media, with Lactobacillus acidophilus LA-06 and Lactobacillus plantarum LPL28 cultured at 37 C for 48 h and Bifidobacterium longum BL21 cultured anaerobically at 37 C for 72 h, and after verification that no viable bacteria grow, centrifuging at 4 C and 6000 r / min for 10- 15 min, collecting all bacterial precipitates and supernatants together, and subjecting the mixture to vacuum freeze-drying using the same parameters as those used for the live-bacteria lyophilized powder, thereby obtaining a single- strain postbiotic lyophilized powder having a moisture content of <=3.0%.

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