Bifidobacterium metagen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly discloses a bifidobacterium metagen as well as a preparation method and application thereof. The preparation method of the bifidobacterium metagen provided by the invention comprises the following steps: carrying out low-temperature gradient inactivation and solid-liquid separation on bifidobacterium liquid, respectively collecting precipitates and a solution, and carrying out sound-sensitive agent assisted ultrasonic treatment on the precipitates or the solution to prepare the corresponding bifidobacterium metagen, wherein the sound-sensitive agent comprises sodium copper chlorophyllin with the final concentration of 0.05 mg / mL to 0.1 mg / mL or riboflavin with the final concentration of 0.01 mg / mL to 0.05 mg / mL. According to the preparation method provided by the invention, through the synergistic effect of low-temperature gradient inactivation and sound-sensitive agent-assisted ultrasound, the overall biological activity of the metagen is improved to the maximum extent, the anti-oxidation and anti-inflammatory capacities of the bifidobacterium metagen are remarkably improved, and a technical support is provided for application of the bifidobacterium metagen in food, health-care food and anti-inflammatory intestinal preparations of the food, the health-care food and the anti-inflammatory intestinal preparations of the bifidobacterium metagen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotics, and relates to a bifidobacterium postbiotic, a preparation method and application thereof. BACKGROUND

[0002] Postbiotics are non-viable probiotic-derived products containing microbial metabolites, cell components or mixtures thereof, which can bring clear health benefits to the host through pathways such as regulating the host intestinal barrier function, inhibiting the proliferation of harmful microorganisms, improving antioxidant activity or regulating immune response, and have increasingly broad application prospects in the fields of food, health products and medicine.

[0003] In the preparation process of postbiotics, the inactivation step of live bacteria is an important link to determine the biological activity and application value of postbiotics. The reason is that the inactivation method can directly affect the composition and retention rate of active ingredients (such as short-chain fatty acids, exopolysaccharides, bacteriocins and intracellular functional proteins) in postbiotics, and different inactivation methods can cause significant differences in the biological activity of postbiotics. The inactivation of probiotics is usually carried out by conventional high-temperature sterilization, among which 121℃ high-pressure steam sterilization is the most widely used method. However, this sterilization method has significant technical defects: some core active ingredients in postbiotics are extremely sensitive to high temperature, and the high-temperature environment of 121℃ can cause thermal denaturation, hydrolysis or oxidation of such ingredients, directly causing the loss of their activity; at the same time, high temperature can also cause excessive cross-linking of bacterial cell components (such as peptidoglycan and teichoic acid), which not only cannot effectively release intracellular active components, but also forms insoluble polymerization products, further reducing the overall biological activity and functional utilization rate of postbiotics, making it difficult to meet the preparation needs of subsequent high-activity postbiotic products.

[0004] To solve the problem of insufficient release of intracellular active components caused by high-temperature inactivation, ultrasonic crushing treatment is often introduced in the prior art to destroy the cell wall of the inactivated bacteria by mechanical vibration and promote the release of intracellular active substances. However, in the ultrasonic treatment process, if a lower ultrasonic power or a shorter treatment time is used, the cell wall of the bacteria is not fully broken, and the release rate of intracellular active components is insufficient, which cannot achieve effective enrichment of active ingredients; if the ultrasonic power or treatment time is increased to improve the crushing efficiency, the ultrasonic energy will directly act on the released active ingredients, causing damage to their spatial structure (such as protein denaturation and polysaccharide chain rupture), and at the same time causing local temperature rise, further intensifying the inactivation of heat-sensitive components; in addition, the ultrasonic environment can also induce free radical oxidation reaction of active ingredients, forming inactive degradation products, ultimately resulting in the biological activity of postbiotics failing to achieve the expected effect. SUMMARY

[0005] The conventional high-temperature sterilization method in the prior art cannot effectively avoid the inactivation of heat-sensitive active ingredients in the preparation process of the postbiotic, and in the subsequent ultrasonic crushing step, the full release and activity retention of intracellular active components cannot be considered when there is no protective measures.

[0006] To achieve the above-mentioned purposes, the embodiments of the present application adopt the following technical solutions: In a first aspect, the present application provides a preparation method of a bifidobacterium postbiotic, comprising the following steps: low-temperature gradient inactivation of a bifidobacterium bacterial solution, solid-liquid separation to collect the precipitate and the solution respectively; and then at least one of the following treatments is performed: The first treatment: resuspend the precipitate, add a sonosensitizer, perform ultrasonic crushing treatment, separate the solid and the liquid, collect the precipitate to obtain a bacterial lysate, and obtain a bifidobacterium postbiotic A; The second treatment: add a sonosensitizer to the solution, perform ultrasonic treatment, and obtain a bifidobacterium postbiotic B; The low-temperature gradient inactivation conditions are: sequentially incubating at 48-52℃, 58-62℃, 68-72℃, 78-82℃ and 98-100℃, and the incubation time at each temperature section is 8-12 min; The sonosensitizer includes at least one of sodium copper chlorophyll or riboflavin; The final concentration of the sodium copper chlorophyll in the system is 0.05-0.1 mg / mL; The final concentration of the riboflavin in the system is 0.01-0.05 mg / mL.

[0007] The preparation method of the bifidobacterium postbiotic provided by the present application maximizes the overall biological activity of the postbiotic through the synergistic effect of low-temperature gradient inactivation and sonosensitizer-assisted ultrasonic treatment, significantly improves the antioxidant and anti-inflammatory abilities of the bifidobacterium postbiotic from the three dimensions of active ingredient retention-release-activity enhancement, and provides technical support for its application in food, health food and anti-inflammatory intestinal preparations.

[0008] The low-temperature gradient inactivation method provided by the present application can not only reduce the thermal damage of heat-sensitive antioxidant components, but also retain as many anti-inflammatory core components as possible, such as bacteriocin and peptidoglycan. Firstly, the advantages of gradient heating in retaining heat-sensitive antioxidant components are as follows: (1) low-temperature pre-adaptation to avoid instantaneous thermal shock: the initial low-temperature section can first inactivate low-heat-resistant miscellaneous bacteria, and at the same time, the bacteria can slowly adapt to temperature changes to avoid rapid shrinkage of the cell wall and destruction of the spatial conformation of intracellular antioxidant enzymes caused by instantaneous high temperature; (2) medium-temperature section for activity preservation to reduce metabolic product degradation: the 68℃~72℃ and 78℃~82℃ medium-temperature sections can further inactivate target bacteria while avoiding the destruction of polysaccharide chains, and the length of the polysaccharide chain directly affects the free radical scavenging capacity; (3) short preservation at high temperature to balance inactivation efficiency and activity retention: the 98℃~100℃ section is only preserved for 8min~12min, which can ensure complete inactivation of live bacteria while minimizing the loss of active substances such as GABA, thereby laying a foundation for subsequent ultrasonic release of intracellular antioxidant components. Secondly, the anti-inflammatory activity of Bifidobacterium mainly depends on bacteriocin (such as nisin analogues to inhibit the proliferation of harmful bacteria), cell wall peptidoglycan (to regulate the release of immune cytokines), and intracellular anti-inflammatory proteins (such as anti-inflammatory factor IL-10 induced proteins), and the tolerance of such components to high temperature is also limited. Gradient heating can retain the bacteriostatic activity of bacteriocin and maintain the structural integrity of peptidoglycan through a mild inactivation mode. The segmented heating sterilization can avoid the denaturation of bacteriocin caused by high temperature, so that the inhibition rate of bacteriocin on harmful bacteria is higher than that of high-temperature sterilization, thereby indirectly reducing the inflammatory response caused by the metabolism of harmful bacteria (such as LPS); in addition, high-temperature sterilization can easily lead to excessive cross-linking of bacterial cell walls and fragmentation of peptidoglycan, while gradient heating can maintain the relatively complete structure of the bacterial cell wall, so that "long-chain peptidoglycan" can be released during subsequent ultrasonic crushing, and long-chain peptidoglycan can more efficiently activate the anti-inflammatory pathways of macrophages, reduce the secretion of pro-inflammatory factors, and significantly improve the anti-inflammatory effect compared with fragmented peptidoglycan.

[0009] The core problem of ultrasonic in the prior art is the insufficient release of intracellular components and the destruction of active components. The present application designs a sonosensitizer-assisted ultrasonic for inactivated bacteria and metabolic products, respectively, and simultaneously realizes efficient release of active components and reduction of active damage through sonosensitizer-mediated precise action, and additionally exerts the biological activity of the sonosensitizer itself. Gradient heating lays a foundation for ultrasonic, and sonosensitizer-assisted ultrasonic enhances the gradient inactivation. For the intact bacteria and active metabolic products retained after gradient inactivation, ultrasonic combined with sonosensitizer realizes precise crushing and activity protection, releases intracellular components, avoids active damage, and further strengthens the overall efficacy of the probiotic by virtue of the antioxidant / anti-inflammatory activity of the sonosensitizer itself.

[0010] Preferably, the conditions of the ultrasonic crushing treatment are: ultrasonic power is 550W-650W, 4s-6s of ultrasonic treatment is followed by 4s-6s of interval, and the total treatment time is 12min-18min.

[0011] Preferably, the conditions of the ultrasonic treatment are: ultrasonic power is 180W-220W, 2s-3s of ultrasonic treatment is followed by 2s-3s of interval, and the total treatment time is 8min-12min.

[0012] Preferably, the preparation method of the Bifidobacterium liquid includes the following steps: culturing Bifidobacterium anaerobically at 36℃-38℃ for 12h-20h to obtain a Bifidobacterium fermentation liquor; adjusting the viable count of the Bifidobacterium fermentation liquor to 10 6 CFU / mL-10 7 CFU / mL to obtain a Bifidobacterium liquid.

[0013] Preferably, the Bifidobacterium includes Bifidobacterium animalis lactis i797.

[0014] The Latin name of the Bifidobacterium animalis lactis i797 in the application is Bifidobacterium animalis subsp. lactis Figure 1 , and the accession number is CGMCC No.18403, which is first disclosed in Chinese patent CN202010259210.8.

[0015] Further preferably, the preparation method of the Bifidobacterium animalis lactis i797 postbiotic specifically includes the following steps: Inoculating Bifidobacterium animalis lactis i797 into MRS culture medium and culturing anaerobically at 36℃-38℃ for 12h-20h to obtain a Bifidobacterium animalis lactis i797 fermentation liquor; Adjusting the bacterial concentration of the Bifidobacterium fermentation liquor to 10 6 CFU / mL-10 7 CFU / mL using DMEM culture medium to obtain a Bifidobacterium animalis lactis i797 liquid; Freezing and gradient inactivating the Bifidobacterium animalis lactis i797 liquid, and collecting the precipitate and the solution after centrifugation, wherein the obtained precipitate is inactivated bacteria, and the obtained solution is inactivated cell-free supernatant; then at least one of the following treatments is performed on the inactivated bacteria and the inactivated cell-free supernatant: The first treatment: resuspending the inactivated bacteria, adding a sonosensitizer, performing ultrasonic crushing treatment, and collecting the precipitate by centrifugation to obtain a Bifidobacterium animalis lactis i797 postbiotic A; The second treatment: adding a sonosensitizer to the inactivated cell-free supernatant, and performing ultrasonic treatment to obtain a Bifidobacterium animalis lactis i797 postbiotic B; The low-temperature gradient inactivation conditions are: sequentially incubating at 48-52 DEG C, 58-62 DEG C, 68-72 DEG C, 78-82 DEG C and 98-100 DEG C, and the incubation time at each temperature section is 8-12 min. The sound-sensitive agent comprises sodium copper chlorophyllin with a final concentration of 0.05-0.1 mg / mL or riboflavin with a final concentration of 0.01-0.05 mg / mL.

[0016] In a second aspect, the present application provides a bifidobacterium postbiotic prepared by the preparation method of the bifidobacterium postbiotic.

[0017] Preferably, the bifidobacterium postbiotic is animal bifidobacterium lactis i797 postbiotic; the animal bifidobacterium lactis i797 postbiotic comprises at least one of the above-mentioned animal bifidobacterium lactis i797 postbiotic A or animal bifidobacterium lactis i797 postbiotic B.

[0018] Compared with live probiotics, the postbiotic is safer and is not inhibited by antibiotics, and can still stably function even in the scene of combination with antibiotics; at the same time, the postbiotic has more relaxed requirements for storage conditions, and does not need to rely on strict storage means such as cold chain, and can realize a long shelf life in a normal temperature and conventional packaging environment, thereby significantly reducing the cost and operation difficulty in the production, circulation and application links.

[0019] In a third aspect, the present application provides application of the above-mentioned bifidobacterium postbiotic in preparation of a product with antioxidant function and / or anti-inflammatory function.

[0020] Further, the product comprises fermented milk.

[0021] The bifidobacterium postbiotic A provided by the present application is a bacterial lysate, and has obvious antioxidant activity; the bifidobacterium postbiotic B is a heat-inactivated cell-free supernatant, and has good anti-inflammatory activity effect. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0023] Figure 1 The free radical scavenging capacity determination results of different groups in the effect example 1 of the present application are as follows, wherein Figure 1 A represents the determination results of hydroxyl radical scavenging capacity, Figure 1 B is the determination results of DPPH radical scavenging capacity,Figure 2 C is the result of superoxide anion radical scavenging capacity determination; Figure 2 The determination results of antioxidant enzyme activities of different groups in the effect example 1 of the present application are shown in Table 1, wherein Figure 2 A is the CAT enzyme activity determination result, Figure 2 B is the GSH-Px enzyme activity determination result, Figure 3 C is the SOD enzyme activity determination result; Figure 3 The determination results of relative expressions of antioxidant enzyme genes of different groups in the effect example 1 of the present application are shown in Table 2, wherein Figure 3 A is the SOD gene relative expression determination result, Figure 3 B is the CAT gene relative expression determination result, Figure 4 C is the GSH-Px gene relative expression determination result; Figure 4 The determination results of relative expressions of inflammatory factor genes of different groups in the effect example 1 of the present application are shown in Table 3, wherein Figure 4 A is the IL-10 gene relative expression determination result, Figure 5 B is the IL-1β gene relative expression determination result; Figure 6 The determination results of DPPH radical scavenging rates of different groups in the effect example 2 of the present application are shown in Table 4. Figure 7 The determination results of SOD enzyme activities of different groups in the effect example 2 of the present application are shown in Table 5. Figure 1 The determination results of relative expressions of IL-10 genes of different groups in the effect example 3 of the present application are shown in Table 6. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0025] The raw materials and reagents used in the present application are all conventional commercially available products unless otherwise specified, and the methods used in the present application are all conventional methods in the art unless otherwise specified.

[0026] Part of the experimental materials used in the present application and the antioxidant capacity and anti-inflammatory activity determination methods are as follows: I. Part of the experimental materials The sodium copper chlorophyllin content in the present application is 98%, which is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.; the riboflavin content is 97.7%, which is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0027] J774A.1 cells and Caco-2 cells were purchased from Taiyuan Rosetta Stone Biotechnology Co., Ltd.; DPPH free radical, hydroxyl radical, and superoxide anion clearance ability kits were purchased from Suzhou Gexisi Biotechnology Co., Ltd.; SOD, CAT, and GSH-px activity kits were purchased from Suzhou Gexisi Biotechnology Co., Ltd.

[0028] II. Antioxidant capacity and anti-inflammatory activity determination methods 1. Antioxidant capacity test (1) Free radical scavenging capacity test: The scavenging capacity of different metaplasms on DPPH free radicals, hydroxyl radicals, and superoxide anions was detected according to the corresponding instructions of the Gexisi kit. The specific method was as follows: 1 mL of prepared metaplasma was added to 1 mL of 80% methanol extract, and under the conditions of power 200 W, frequency 25 kHz, 3 s of ultrasonic treatment and 10 s of intermittent treatment, alarm temperature 28 ℃, 30 cycles of ultrasonic treatment were performed. After ultrasonic treatment, 12000 rpm centrifugation was performed for 10 min, and the supernatant was detected. Subsequent detection was performed according to the instructions. DPPH free radicals were detected at a wavelength of 517 nm; hydroxyl radicals and superoxide anions were detected at a wavelength of 510 nm.

[0029] (2) Caco-2 cell model construction: Caco-2 cells were used for antioxidant research, and DMEM high-sugar medium containing 20% (v / v) fetal bovine serum, 50 μg / mL penicillin, and 50 μg / mL streptomycin was used to culture Caco-2 cells in a 37 ℃, 5% CO2 incubator. The 20th generation of cells was used for the test, and the confluence reached 85%-90% as the normal cell model. Caco-2 cells with a density of 1×10 6 cells / mL were inoculated in a 6-well cell culture plate and cultured overnight. After adhesion, 2 mL of 1 mM H2O2-metaplasma mixture was added to each well, and the Caco-2 cells were incubated for 4 hours. Caco-2 cells treated only with H2O2 (i.e., without metaplasma treatment) were used as the positive control group, and Caco-2 cells cultured in DMEM high-sugar medium were used as the blank control group.

[0030] (3) Antioxidant enzyme gene level expression determination: The RNA of the incubated Caco-2 cells was extracted using the kit, and reverse transcription was performed to obtain cDNA. According to the related kit instructions, the reverse transcription and RT-qPCR parameters were set, and the relative expression of antioxidant-related enzyme genes was detected. The reaction system and primers are shown in Tables 1-2, and the qPCR reaction program is shown in Table 3. GAPDH was used as the internal reference, and the relative transcription level of each gene was calculated using the 2 -ΔΔCT method.

[0031] Table 1 qPCR reaction system

[0032] Table 2 Gene and primer sequence

[0033] Table 3 qPCR reaction program

[0034] (4) Determination of the effect of biofertilizer on antioxidant enzyme activity: according to the method of the instruction, the activities of SOD, CAT and GSH-px in different treatment groups were analyzed using the corresponding kit of Gries.

[0035] 2. Anti-inflammatory ability test (1) Construction of cell model J774A.1 cells were used for inflammation research, and DMEM high-sugar medium containing 10% fetal bovine serum (containing 1% double antibody) was subcultured at 37°C, 5% CO2 incubator, and 20th generation cells were used as normal cell model. J774A.1 cells containing 500 ng / mL LPS and cell concentration of 1×10 6 6-well cell culture plates, and when the confluence was about 85%, the culture medium was removed to construct an inflammatory cell model.

[0036] (2) Determination of anti-inflammatory activity Blank control group: J774A.1 cells without 500 ng / mL LPS and cell concentration of 1×10 6 cells / mL were used as negative control; Biofertilizer treatment group: to the above constructed inflammatory cell model, 1×10 6 cells / well were added, 2 mL of biofertilizer was added, and after removing the original culture medium, they were incubated for 4 hours; LPS group: the inflammatory cell model without adding other components was used as positive control group, and was incubated for 4 hours under the same conditions.

[0037] After incubation, the culture medium was removed, washed twice with PBS, and the cells were recovered with a cell scraper. The total RNA of each group of cells was extracted according to the kit, and then reverse transcribed into cDNA. The relative expression of IL-1β and IL-10 genes was determined by RT-qPCR, and the RT-qPCR reaction system is shown in Table 1 and the program is shown in Table 3. GAPDH was used as an internal reference, and the primer sequence is shown in Table 4.

[0038] Table 4 Gene and primer sequence

[0039] Example 1 The embodiment provides a preparation method of a bifidobacterium probiotic, and the preparation method comprises the following steps: S1. Taking animal bifidobacterium lactis i797 from a frozen storage tube, culturing in MRS medium at 37 DEG C in an anaerobic state for 16 hours, activating 3 times, and obtaining an animal bifidobacterium lactis i797 seed liquid; Taking 0.6 mL of the animal bifidobacterium lactis i797 seed liquid and inoculating into 20 mL of MRS medium, culturing in a 37 DEG C incubator in an anaerobic state for 16 hours, and measuring that the bacterial concentration of the fermentation liquor is 10 9 CFU / mL of the fermentation liquor, gradient diluting to 10 7 CFU / mL by using DMEM medium, and obtaining an animal bifidobacterium lactis i797 bacterial liquid; The animal bifidobacterium lactis i797 bacterial liquid is subjected to low-temperature gradient inactivation at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C and 100 DEG C in sequence, and each temperature is kept for 10 minutes. After the low-temperature gradient inactivation, whether inactivation is complete is verified by using a viable bacterial count method, complete inactivation is confirmed, the sterilized bacterial liquid is divided into centrifuge tubes, 50 mL / tube, and the precipitate is collected after centrifugation at 6000 rpm for 10 minutes, and the obtained precipitate is an inactivated bacterial body; S2. Taking 50 mL of the inactivated bacterial body corresponding to the bacterial liquid, resuspending to a total volume of 50 mL by using PBS buffer with a pH of 7.2-7.4, adding sodium copper chlorophyllin with a final concentration of 0.05 mg / mL, uniformly mixing, and placing in the dark for 10 minutes, and placing in an ultrasonic crusher, setting an ultrasonic power of 600 W and ultrasonic crushing for 15 minutes under the condition of ice bath, 5 seconds of interval for each 5 seconds of ultrasonic crushing, centrifuging at 4 DEG C and 12000 rpm for 30 minutes, and collecting the precipitate to obtain a bacterial lysate, resuspending the bacterial lysate by using DMEM medium to adjust the concentration to 1x10 7 CFU / mL, namely an animal bifidobacterium lactis i797 probiotic A, recorded as probiotic A-SCP1.

[0040] Embodiment 2 The embodiment provides a preparation method of a bifidobacterium probiotic, and the preparation method comprises the following steps: S1. The animal bifidobacterium lactis i797 seed liquid is prepared according to the method recorded in embodiment 1; Taking 0.6 mL of the animal bifidobacterium lactis i797 seed liquid and inoculating into 20 mL of MRS medium, culturing in a 38 DEG C incubator in an anaerobic state for 15 hours, and measuring that the bacterial concentration of the fermentation liquor is 6x10 8 CFU / mL of the fermentation liquor, gradient diluting to 10 7 CFU / mL by using DMEM medium, and obtaining an animal bifidobacterium lactis i797 bacterial liquid; The animal Bifidobacterium lactis i797 bacterial liquid was sequentially subjected to low-temperature gradient inactivation at 48℃, 62℃, 68℃, 78℃ and 98℃, and each temperature was maintained for 9 min. After low-temperature gradient inactivation, whether inactivation was verified by viable cell counting method, and complete inactivation was confirmed. The sterilized bacterial liquid was divided into centrifuge tubes, 50 mL / tube; after centrifugation at 6000 rpm for 10 min, the precipitate was collected, and the obtained precipitate was inactivated bacteria; S2. Take 50 mL of inactivated bacteria corresponding to the bacterial liquid, resuspend in PBS buffer with pH of 7.2-7.4 to a total volume of 50 mL, add sodium copper chlorophyll with a final concentration of 0.1 mg / mL, mix well, and avoid light for 10 min. Place in an ultrasonic crusher, set the ultrasonic power to 550 W, and perform ultrasonic crushing treatment for 18 min under ice bath conditions, with 6 s of ultrasonic and 6 s of intermittent. Centrifuge at 4℃, 12000 rpm for 30 min, collect the precipitate to obtain bacterial lysate, and dilute with DMEM medium to contain 1×10 7 CFU bacteria corresponding to the lysate, namely animal Bifidobacterium lactis i797 metachitona A, is recorded as metachitona A-SCP2.

[0041] Example 3 The present embodiment provides a preparation method of Bifidobacterium metachitona, and the preparation method comprises the following steps: S1. Prepare the animal Bifidobacterium lactis i797 seed liquid according to the method described in Example 1; Take 0.6 mL of animal Bifidobacterium lactis i797 seed liquid and inoculate in 20 mL of MRS medium, and anaerobically culture in a 36℃ incubator for 20 h. The measured bacterial concentration is 2×10 9 CFU / mL of fermentation liquid, and gradient dilute with DMEM medium to 10 7 CFU / mL to obtain animal Bifidobacterium lactis i797 bacterial liquid; The animal Bifidobacterium lactis i797 bacterial liquid was sequentially subjected to low-temperature gradient inactivation at 48℃, 62℃, 68℃, 78℃ and 98℃, and each temperature was maintained for 9 min. After low-temperature gradient inactivation, whether inactivation was verified by viable cell counting method, and complete inactivation was confirmed. The sterilized bacterial liquid was divided into centrifuge tubes, 50 mL / tube; after centrifugation at 6000 rpm for 10 min, the precipitate was collected, and the obtained precipitate was inactivated bacteria; S2. Take 50 mL of the inactivated bacteria corresponding to the bacterial solution, resuspend it in PBS buffer with a pH of 7.2-7.4 to a total volume of 50 mL, add riboflavin at a final concentration of 0.01 mg / mL, mix well, and avoid light for 10 min. Place it in an ultrasonic disrupter, set the ultrasonic power to 650 W under ice bath conditions, and perform ultrasonic disruption treatment for 12 min with 4 s of ultrasonic treatment and 4 s of intermittent treatment. Centrifuge at 4°C and 12000 rpm for 30 min, collect the precipitate to obtain bacterial lysate, and dilute it with DMEM medium to contain 1x10 7 The lysate corresponding to the CFU bacteria, i.e., the animal Bifidobacterium lactis i797 probiotic A, is recorded as probiotic A-RFN1.

[0042] Example 4 The present embodiment provides a preparation method of a Bifidobacterium probiotic, which comprises the following steps: S1. Prepare the animal Bifidobacterium lactis i797 bacterial solution according to the method described in Example 1. The animal Bifidobacterium lactis i797 bacterial solution is sequentially subjected to low-temperature gradient inactivation at 50°C, 62°C, 72°C, 82°C, and 100°C, each temperature for 10 min. After low-temperature gradient inactivation, whether inactivation is complete is verified by viable cell count method, and the inactivated bacterial solution is aliquoted into centrifuge tubes, 50 mL / tube. After centrifugation at 6000 rpm for 10 min, the precipitate is collected, and the obtained precipitate is inactivated bacteria; S2. Take 50 mL of the inactivated bacteria corresponding to the bacterial solution, resuspend it in PBS buffer with a pH of 7.2-7.4 to a total volume of 50 mL, add riboflavin at a final concentration of 0.05 mg / mL, mix well, and avoid light for 10 min. Place it in an ultrasonic disrupter, set the ultrasonic power to 600 W under ice bath conditions, and perform ultrasonic disruption treatment for 14 min with 6 s of ultrasonic treatment and 5 s of intermittent treatment. Centrifuge at 4°C and 12000 rpm for 30 min, collect the precipitate to obtain bacterial lysate, and dilute it with DMEM medium to contain 1x10 7 The lysate corresponding to the CFU bacteria, i.e., the animal Bifidobacterium lactis i797 probiotic A, is recorded as probiotic A-RFN2.

[0043] Example 5 The present embodiment provides a preparation method of a Bifidobacterium probiotic, which comprises the following steps: S1. Prepare the animal Bifidobacterium lactis i797 bacterial solution according to the method described in Example 1. The animal Bifidobacterium lactis i797 bacterial solution was sequentially subjected to low-temperature gradient inactivation at 50℃, 60℃, 70℃, 80℃ and 100℃, and each temperature was maintained for 10 minutes. After low-temperature gradient inactivation, whether inactivation was verified by using viable cell counting method, and complete inactivation was confirmed. The sterilized bacterial solution was divided into centrifuge tubes, 50mL / tube; after centrifugation at 6000rpm for 10 minutes, the supernatant was collected, and the obtained supernatant was inactivated cell-free supernatant; S2. 50mL of the inactivated cell-free supernatant corresponding to the bacterial solution was passed through a 0.22μm filter membrane, and a final concentration of 0.05mg / mL of copper sodium salt of chlorophyll was added. After mixing and avoiding light for 10 minutes, it was placed in an ultrasonic crusher under ice bath conditions, and the ultrasonic power was set to 200W, and ultrasonic treatment was performed for 10 minutes with 2s of intermittent 2s each time. The heat-inactivated cell-free supernatant was obtained, that is, the animal Bifidobacterium lactis i797 postbiotic B was obtained, which was recorded as postbiotic B-SCP1.

[0044] Example 6 The present embodiment provides a preparation method of a Bifidobacterium postbiotic, which comprises the following steps: S1. The animal Bifidobacterium lactis i797 bacterial solution was prepared according to the method described in Example 1; The animal Bifidobacterium lactis i797 bacterial solution was sequentially subjected to low-temperature gradient inactivation at 50℃, 62℃, 72℃, 80℃ and 98℃, and each temperature was maintained for 12 minutes. After low-temperature gradient inactivation, whether inactivation was verified by using viable cell counting method, and complete inactivation was confirmed. The sterilized bacterial solution was divided into centrifuge tubes, 50mL / tube; after centrifugation at 6000rpm for 10 minutes, the supernatant was collected, and the obtained supernatant was inactivated cell-free supernatant; S2. 50mL of the inactivated cell-free supernatant corresponding to the bacterial solution was passed through a 0.22μm filter membrane, and a final concentration of 0.05mg / mL of copper sodium salt of chlorophyll was added. After mixing and avoiding light for 10 minutes, it was placed in an ultrasonic crusher under ice bath conditions, and the ultrasonic power was set to 200W, and ultrasonic treatment was performed for 10 minutes with 2s of intermittent 2s each time. The heat-inactivated cell-free supernatant was obtained, that is, the animal Bifidobacterium lactis i797 postbiotic B was obtained, which was recorded as postbiotic B-SCP1.

[0045] Example 7 The present embodiment provides a preparation method of a Bifidobacterium postbiotic, which comprises the following steps: S1. The animal Bifidobacterium lactis i797 bacterial solution was prepared according to the method described in Example 1; The Bifidobacterium animalis lactis i797 bacterial solution was sequentially subjected to low-temperature gradient inactivation at 52°C, 60°C, 72°C, 80°C and 100°C, and each temperature was maintained for 10 min. After low-temperature gradient inactivation, whether inactivation was complete was verified by viable cell counting, and the inactivated bacterial solution was divided into centrifuge tubes, 50 mL / tube. After centrifugation at 6000 rpm for 10 min, the supernatant was collected, and the obtained supernatant was an inactivated cell-free supernatant; S2. 50 mL of the inactivated cell-free supernatant corresponding to the bacterial solution was passed through a 0.22 μm filter membrane, and riboflavin was added at a final concentration of 0.01 mg / mL. After mixing and standing in the dark for 10 min, the mixture was placed in an ultrasonic cell disruptor, and ultrasonic treatment was performed under ice bath conditions at an ultrasonic power of 220 W, 2 s of ultrasonic treatment and 2 s of intermittent treatment, for 8 min. The heat-inactivated cell-free supernatant was obtained, and a Bifidobacterium animalis lactis i797 postbiotic B was obtained, which was recorded as postbiotic B-RFN1.

[0046] Example 8 The present example provides a preparation method of a Bifidobacterium postbiotic, which comprises the following steps: S1. The Bifidobacterium animalis lactis i797 bacterial solution was prepared according to the method described in Example 1. The Bifidobacterium animalis lactis i797 bacterial solution was sequentially subjected to low-temperature gradient inactivation at 48°C, 58°C, 68°C, 82°C and 100°C, and each temperature was maintained for 10 min. After low-temperature gradient inactivation, whether inactivation was complete was verified by viable cell counting, and the inactivated bacterial solution was divided into centrifuge tubes, 50 mL / tube. After centrifugation at 6000 rpm for 10 min, the supernatant was collected, and the obtained supernatant was an inactivated cell-free supernatant; S2. 50 mL of the inactivated cell-free supernatant corresponding to the bacterial solution was passed through a 0.22 μm filter membrane, and riboflavin was added at a final concentration of 0.05 mg / mL. After mixing and standing in the dark for 10 min, the mixture was placed in an ultrasonic cell disruptor, and ultrasonic treatment was performed under ice bath conditions at an ultrasonic power of 190 W, 3 s of ultrasonic treatment and 3 s of intermittent treatment, for 10 min. The heat-inactivated cell-free supernatant was obtained, and a Bifidobacterium animalis lactis i797 postbiotic B was obtained, which was recorded as postbiotic B-RFN2.

[0047] Example 9 The present example provides a product with antioxidant activity, which comprises the Bifidobacterium postbiotic provided in Examples 1-4. The product can be fermented milk, and specifically a sterilized flavor fermented milk.

[0048] Example 10 The present example provides a product with anti-inflammatory activity, which comprises the Bifidobacterium postbiotic provided in Examples 5-8. The product can be fermented milk, and specifically a sterilized flavor fermented milk.

[0049] Example 11 The present example provides a sterilized fermented milk with both anti- stress activity and anti-inflammatory activity, which comprises postbiotic A-SCP1 prepared according to the method described in Example 1 and postbiotic B-SCP1 prepared according to the method described in Example 5.

[0050] Comparative Example 1 The present comparative example provides a preparation method of a Bifidobacterium postbiotic, which is basically the same as that of Example 1, except that in step S2, “adding sodium copper chlorophyllin with a final concentration of 0.05 mg / mL” is replaced by “adding PBS buffer with pH of 7.2-7.4 in the same mass as sodium copper chlorophyllin”, and the rest of the preparation steps and parameters are the same as those of Example 1. The final postbiotic is denoted as postbiotic A-PBS.

[0051] Comparative Example 2 The present comparative example provides a preparation method of a Bifidobacterium postbiotic, which is basically the same as that of Example 1, except that in step S2, “adding sodium copper chlorophyllin with a final concentration of 0.05 mg / mL” is replaced by “adding sodium copper chlorophyllin with a final concentration of 0.5 mg / mL”, and the rest of the preparation steps and parameters are the same as those of Example 1. The final postbiotic is denoted as postbiotic A-SCP3.

[0052] Comparative Example 3 The present comparative example provides a preparation method of a Bifidobacterium postbiotic, which is basically the same as that of Example 3, except that in step S2, “adding riboflavin with a final concentration of 0.01 mg / mL” is replaced by “adding sodium copper chlorophyllin with a final concentration of 0.1 mg / mL”, and the rest of the preparation steps and parameters are the same as those of Example 3. The final postbiotic is denoted as postbiotic A-RNF3.

[0053] Comparative Example 4 The present comparative example provides a preparation method of a Bifidobacterium postbiotic, which is basically the same as that of Example 5, except that after “taking 50 mL of cell-free supernatant corresponding to the bacterial solution through a 0.22 μm filter membrane” in step S2, instead of adding sodium copper chlorophyllin and standing, it is directly placed in an ultrasonic disrupter for ultrasonic treatment for 10 min. The rest of the preparation steps and parameters are the same as those of Example 5. The final postbiotic is denoted as postbiotic B-ordinary ultrasonic, and the specific preparation method comprises the following steps: S1. preparing an animal Bifidobacterium lactis i797 bacterial solution according to the method described in Example 1; The animal bifidobacterium lactis i797 bacterial solution was sequentially subjected to low-temperature gradient inactivation at 50℃, 60℃, 70℃, 80℃ and 100℃, and each temperature was kept for 10 min. After low-temperature gradient inactivation, whether inactivation was complete was verified by using viable cell counting method, and the inactivated bacteria solution was confirmed to be completely inactivated, and the sterilized bacteria solution was divided into centrifuge tubes, 50 mL / tube; after centrifugation at 6000 rpm for 10 min, the supernatant was collected, and the obtained supernatant was inactivated cell-free supernatant; S2. 50 mL of the inactivated cell-free supernatant corresponding to the bacteria solution was filtered through a 0.22 μm filter membrane, and was placed in an ultrasonic crusher, and was subjected to ultrasonic treatment for 10 min under the condition of ice bath, with the ultrasonic power being set to 200 W and the interval between each ultrasonic being 2 s, to obtain a heat-inactivated cell-free supernatant, that is, the animal bifidobacterium lactis i797 postbiotic B, which was recorded as postbiotic B-ordinary ultrasonic.

[0054] Comparative Example 5 The present comparative example provides a preparation method of a bifidobacterium postbiotic, which is basically the same as that of Example 5, except that in step S2, “adding sodium copper chlorophyllin with a final concentration of 0.05 mg / mL” is replaced by “adding sodium copper chlorophyllin with a final concentration of 0.5 mg / mL”, and the rest of the preparation steps and parameters are the same as those of Example 5. The final obtained postbiotic is recorded as postbiotic B-SCP3.

[0055] Comparative Example 6 The present comparative example provides a preparation method of a bifidobacterium postbiotic, which is basically the same as that of Example 7, except that in step S2, “adding riboflavin with a final concentration of 0.01 mg / mL” is replaced by “adding sodium copper chlorophyllin with a final concentration of 0.1 mg / mL”, and the rest of the preparation steps and parameters are the same as those of Example 7. The final obtained postbiotic is recorded as postbiotic B-RNF3.

[0056] Effect Example 1 The present application investigates the effects of low-temperature gradient inactivation (DZ) and high-temperature inactivation (GW) on the antioxidant capacity and anti-inflammatory capacity of postbiotics. The specific content is as follows: 1. Experimental grouping In the live bacteria group, the live bacteria were animal bifidobacterium lactis i797 bacterial solution with a concentration of 10 7 CFU / mL provided in Example 1.

[0057] Preparation of live bacteria lysate: the concentration of the live bacteria lysate was 10 750 mL of the Bifidobacterium animalis lactis i797 bacteria solution with 10 7 The lysate corresponding to the CFU bacteria is used.

[0058] Preparation of live bacteria cell-free supernatant: 50 mL of the Bifidobacterium animalis lactis i797 bacteria solution with 10 7 The supernatant is obtained by centrifuging 50 mL of the Bifidobacterium animalis lactis i797 bacteria solution with 10

[0059] The low-temperature gradient inactivation method (DZ) is as follows: 50 mL of the Bifidobacterium animalis lactis i797 bacteria solution with 10 7 The bacteria solution is placed in a water bath, and is subjected to low-temperature gradient inactivation at 50°C, 60°C, 70°C, 80°C and 100°C, respectively, for 10 min at each temperature. The high-temperature inactivation method (GW) is as follows: 50 mL of the Bifidobacterium animalis lactis i797 bacteria solution with 10 7 The bacteria solution is placed in a high-pressure sterilization pot, and is subjected to inactivation at 121°C for 10 min. The bacteria solution is inactivated according to the above two inactivation methods, and the obtained supernatant after centrifugation is inactivated cell-free supernatant; the obtained precipitate after centrifugation is washed with PBS buffer to obtain inactivated bacteria, which is resuspended in DMEM high-sugar culture medium to obtain a bacteria suspension with a concentration of 10 7 CFU / mL, which is recorded as dead bacteria, and is used. The inactivated cell-free supernatant is placed in an ultrasonic crusher, and is subjected to ultrasonic treatment for 10 min under ice bath conditions, with an ultrasonic power of 200 W and an interval of 2 s for each 2 s of ultrasonic treatment. The dead bacteria are placed in an ultrasonic crusher, and are subjected to ultrasonic treatment for 15 min under ice bath conditions, with an ultrasonic power of 600 W and an interval of 5 s for each 5 s of ultrasonic treatment; the obtained precipitate is collected by centrifugation at 4°C and 12,000 rpm for 30 min, and is resuspended in DMEM high-sugar culture medium without antibiotics to obtain a bacteria suspension with a concentration of 10 7 The lysate corresponding to the CFU bacteria is used.

[0060] 2. The method for determining the antioxidant capacity according to the present application, the free radical scavenging capacity, the antioxidant enzyme activity and the relative expression of antioxidant enzyme activity gene of live bacteria, live bacteria lysate, live bacteria cell-free supernatant, and two ways of inactivated dead bacteria, heat-inactivated dead bacteria lysate and heat-inactivated cell-free supernatant and MRS medium are determined.

[0061] The free radical scavenging capacity determination results of different groups are shown in Table 1, wherein Figure 1 A represents the determination results of hydroxyl radical scavenging capacity, Figure 1 B is the determination results of DPPH radical scavenging capacity, Figure 1 C is the determination results of superoxide anion radical scavenging capacity. The determination results of antioxidant enzyme activity of different groups are shown in Table 2, wherein Figure 2 A is the determination results of CAT enzyme activity, Figure 2 B is the determination results of GSH-Px enzyme activity, Figure 2 C is the determination results of SOD enzyme activity. The determination results of relative expression of antioxidant enzyme gene of different groups are shown in Table 3, wherein Figure 2 A is the determination results of SOD gene relative expression, Figure 3 B is the determination results of CAT gene relative expression, Figure 3 C is the determination results of GSH-Px gene relative expression. Among them, Figure 3 Different capital letters represent significant differences between DZ heat-inactivated groups (P < 0.05), different small letters represent significant differences between GW heat-inactivated groups (P < 0.05), and * represents significant differences between the two heat-inactivated groups (P < 0.05). The MRS medium group is the same medium as used for culturing the animal bifidobacterium, which is used as a substrate control. Figure 3 Figures 1-3 As can be seen from Table 1, Table 2 and Table 3, Figures 1-3 the free radical scavenging capacity of the inactivated bacteria, heat-inactivated dead bacteria lysate and heat-inactivated cell-free supernatant of animal bifidobacterium lactis i797 prepared by DZ method are stronger than the corresponding postbiotic components prepared by GW method. At the same time, compared with GW method, the heat-inactivated dead bacteria lysate of animal bifidobacterium lactis i797 prepared by DZ method also has strong antioxidant enzyme activity and relative expression level of its gene.

[0062] As can be seen from Table 1, Table 2 and Table 3, Figure 4 the free radical scavenging capacity of the inactivated bacteria, heat-inactivated dead bacteria lysate and heat-inactivated cell-free supernatant of animal bifidobacterium lactis i797 prepared by DZ method are stronger than the corresponding postbiotic components prepared by GW method. At the same time, compared with GW method, the heat-inactivated dead bacteria lysate of animal bifidobacterium lactis i797 prepared by DZ method also has strong antioxidant enzyme activity and relative expression level of its gene.

[0063] ​3. Following the method for determining anti-inflammatory capacity provided by this invention, the effects of live bacteria, live bacterial lysates, cell-free supernatant of live bacteria, and dead bacteria inactivated by two methods, heat-inactivated dead bacterial lysates, heat-inactivated cell-free supernatant, and MRS culture medium on the relative expression of inflammatory factors IL-10 and IL-1β genes were determined. A blank control group was used as a negative control, and the LPS group as a positive control. The results of the relative expression of inflammatory factor genes in different groups are shown below. Figure 4 As shown, where Figure 4 A represents the results of the relative expression of the IL-10 gene. Figure 4 B represents the results of the relative expression of the IL-1β gene. Figure 4 In the figure, different uppercase letters represent significant differences between the DZ heat inactivation groups (P<0.05), different lowercase letters represent significant differences between the GW heat inactivation groups (P<0.05), and * represents significant differences between the two heat inactivation groups (P<0.05).

[0064] Depend on Figure 5 The results showed that, compared with the LPS group, both inactivated Bifidobacterium animalis subsp. lactis i797 and heat-inactivated cell-free supernatant significantly upregulated the gene expression level of the anti-inflammatory factor IL-10, with the heat-inactivated cell-free supernatant showing a better effect. Compared with the LPS group, the heat-inactivated cell-free supernatant significantly downregulated the gene expression of the pro-inflammatory factor IL-1β.

[0065] Therefore, the present invention selects a low-temperature gradient inactivation method to inactivate live bacteria.

[0066] Example 2 This invention takes the effects of different treatment groups on DPPH free radical scavenging rate and SOD enzyme activity as examples, and examines the antioxidant capacity of the post-biotics prepared in Examples 1-4 and Comparative Examples 1-3. The results of the DPPH free radical scavenging rate determination of different groups are as follows: Figure 6 As shown in the figure, the results of SOD enzyme activity assays in different groups are as follows: Figures 5-6 As shown.

[0067] Depend on Figure 7The results showed that the DPPH radical scavenging rate of the post-biotic-heat-inactivated bacterial lysate obtained by ultrasonic disruption assisted by 0.05 mg / mL-0.5 mg / mL chlorophyllin sodium salt and 0.01 mg / mL-0.05 mg / mL riboflavin was higher than that of the PBS group. Furthermore, the SOD enzyme activity of the post-biotic-heat-inactivated bacterial lysate obtained by ultrasonic disruption assisted by 0.05 mg / mL-0.1 mg / mL chlorophyllin sodium salt and 0.01 mg / mL-0.05 mg / mL riboflavin was higher than that of the PBS group. In conclusion, ultrasonic disruption assisted by the concentrations of chlorophyllin sodium salt or riboflavin protected in this invention can enhance the antioxidant activity of the post-biotic in heat-inactivated bacterial lysate.

[0068] Example 3 This invention uses the relative expression of the IL-10 gene in different treatment groups as an example to examine the anti-inflammatory ability of the post-biotics prepared in Examples 3-8 and Comparative Examples 4-6. The results of the determination of the relative expression of the IL-10 gene in different groups are as follows: Figure 7 As shown.

[0069] Depend on ​ It was found that the relative expression level of the anti-inflammatory factor IL-10 gene in the heat-inactivated cell-free supernatant prepared by ultrasound with 0.05 mg / mL-0.1 mg / mL chlorophyll copper sodium salt or 0.01 mg / mL-0.05 mg / mL riboflavin was significantly higher than that in the ordinary ultrasound group. This indicates that ultrasound treatment with the concentration of chlorophyll copper sodium salt or riboflavin protected in the embodiments of the present invention can enhance the anti-inflammatory activity of the post-genetic agent in the heat-inactivated cell-free supernatant.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a Bifidobacterium postbiotic, characterized in that: The preparation method comprises the following steps: low-temperature gradient inactivation of a Bifidobacterium bacterial solution, solid-liquid separation to collect the precipitate and the solution respectively; and then at least one of the following treatments is performed: The first treatment: resuspension of the precipitate, addition of a sonosensitizer, ultrasonic disruption treatment, solid-liquid separation, collection of the precipitate, and obtaining of the Bifidobacterium postbiotic A; The second treatment: addition of a sonosensitizer to the solution, ultrasonic treatment, and obtaining of the Bifidobacterium postbiotic B; The low-temperature gradient inactivation is performed under the following conditions: sequentially at 48-52 DEG C, 58-62 DEG C, 68-72 DEG C, 78-82 DEG C and 98-100 DEG C, and the holding time at each temperature section is 8-12 min; The sonosensitizer comprises at least one of sodium copper chlorophyllin or riboflavin; The final concentration of the sodium copper chlorophyllin in the system is 0.05-0.1 mg / mL; The final concentration of the riboflavin in the system is 0.01-0.05 mg / mL.

2. The preparation method of the bifidobacterium postbiotic according to claim 1, characterized in that: The ultrasonic disruption treatment is performed under the following conditions: ultrasonic power of 550-650 W, 4-6 s of ultrasonic treatment and 4-6 s of intermittent treatment, and total treatment time of 12-18 min.

3. The preparation method of the bifidobacterium postbiotic according to claim 1, characterized in that: The ultrasonic treatment is performed under the following conditions: ultrasonic power of 180-220 W, 2-3 s of ultrasonic treatment and 2-3 s of intermittent treatment, and total treatment time of 8-12 min.

4. The preparation method of the bifidobacterium postbiotic according to claim 1, characterized in that: The preparation method of the Bifidobacterium bacterial solution comprises the following steps: anaerobic culture of Bifidobacterium at 36-38 DEG C for 12-20 h to obtain Bifidobacterium fermentation liquor; adjusting the viable count of the Bifidobacterium fermentation liquor to 10 6 CFU / mL-10 7 CFU / mL, to obtain the Bifidobacterium bacterial solution.

5. The method of producing a Bifidobacterium postbiotic according to any one of claims 1 to 4, characterized in that: The Bifidobacterium comprises animal Bifidobacterium lactis i797.

6. The preparation method of the bifidobacterium postbiotic according to claim 5, characterized in that: The preparation method specifically comprises the following steps: Inoculation of animal Bifidobacterium lactis i797 into MRS culture medium, anaerobic culture at 36-38 DEG C for 12-20 h, and obtaining of animal Bifidobacterium lactis i797 fermentation liquor; The bacterial concentration of the bifidobacterium fermentation broth was adjusted to 10 6 CFU / mL-10 7 CFU / mL, obtained from animal bifidobacterium lactis subspecies i797 broth; Low-temperature gradient inactivation of the animal Bifidobacterium lactis i797 bacterial solution, centrifugal separation to collect the precipitate and the solution, wherein the obtained precipitate is inactivated bacteria, and the obtained solution is inactivated cell-free supernatant; and then at least one of the following treatments is performed on the inactivated bacteria and the inactivated cell-free supernatant: The first treatment: resuspension of the inactivated bacteria, addition of a sonosensitizer, ultrasonic disruption treatment, centrifugal separation, and collection of the precipitate, and obtaining of animal Bifidobacterium lactis i797 postbiotic A; The second treatment: addition of a sonosensitizer to the inactivated cell-free supernatant, ultrasonic treatment, and obtaining of animal Bifidobacterium lactis i797 postbiotic B.

7. A Bifidobacterium postbiotic, characterized in that: Prepared by the preparation method of the Bifidobacterium postbiotic in any one of claims 1-6.

8. The Bifidobacterium postbiotic of claim 7, wherein: The Bifidobacterium postbiotic is animal Bifidobacterium lactis i797 postbiotic; and the animal Bifidobacterium lactis i797 postbiotic comprises at least one of the animal Bifidobacterium lactis i797 postbiotic A or the animal Bifidobacterium lactis i797 postbiotic B in claim 6.

9. Use of the Bifidobacterium postbiotic in claim 7 or 8 in the preparation of a product with antioxidant function and / or anti-inflammatory function.

10. The use of the bifidobacterium postbiotic according to claim 9, for the preparation of a product having antioxidant function and / or having anti-inflammatory function, characterized by: The product comprises fermented milk.

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