Lactobacillus reuteri imau12801 having probiotic properties and use thereof

Abstract

This invention provides a strain of *Lactobacillus reuteri* IMAU12801 with probiotic properties and its applications, belonging to the field of food microbiology technology. The *Lactobacillus reuteri* IMAU12801 with probiotic properties is listed under the preservation number CGMCC No. 31226. The *Lactobacillus reuteri* IMAU12801 provided by this invention exhibits good tolerance to gastrointestinal fluids and possesses various probiotic properties, such as antibacterial potential, DPPH and hydroxyl radical scavenging ability, and antioxidant activity. When *Lactobacillus reuteri* IMAU12801 is combined with a basic starter culture for fermentation of cow's milk, the stability of the fermented milk can be improved under storage conditions at 4°C. This invention provides a novel approach for the utilization and development of *Lactobacillus reuteri* IMAU12801.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology technology, specifically relating to a strain of Lactobacillus reuteri IMAU12801 with probiotic properties and its applications. Background Technology

[0002] Lactic acid bacteria are widely used in fermented dairy products. Commercially available fermented milk typically uses *Lactobacillus bulgaricus* and *Streptococcus thermophilus* strains for fermentation. These two active strains continue to ferment and grow in the fermented milk, often disrupting its quality stability and leading to a deterioration in taste and texture. Currently, to improve the stability of fermented milk, various stabilizers are usually added. However, the addition of stabilizers often adversely affects the flavor of the fermented milk and contradicts consumers' desire for healthy foods without any additives. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the purpose of this invention is to provide a strain of *Lactobacillus reuteri* with probiotic properties. This *Lactobacillus reuteri* can be used together with commercial starter cultures *Lactobacillus bulgaricus* and *Streptococcus thermophilus* as starter cultures to improve the stability of fermented milk. Moreover, the *Lactobacillus reuteri* has probiotic properties, which can meet consumers' demand for healthy food without any additives.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] This invention provides a strain of *Limosilactobacillus reuteri* IMAU12801 with probiotic properties, the preservation number of which is CGMCC No. 31226.

[0006] This invention provides a probiotic preparation comprising the fermentation broth of *Lactobacillus reuteri* IMAU12801 described in the above technical solution.

[0007] Preferably, the viability of *Lactobacillus reuteri* IMAU12801 in the fermentation broth is ≥10⁻⁶. 6 CFU / mL.

[0008] This invention provides a method for preparing the probiotic preparation described in the above technical solution, comprising:

[0009] Lactobacillus reuteri IMAU12801 was cultured in a culture medium to obtain a probiotic preparation containing the fermentation broth of Lactobacillus reuteri IMAU12801.

[0010] This invention provides a fermentation agent comprising Lactobacillus reuteri IMAU12801 as described in the above technical solution.

[0011] Preferably, the fermenting agent also includes a commercial fermenting agent.

[0012] This invention provides the application of *Lactobacillus reuteri* IMAU12801 described in the above-mentioned technical solutions, the probiotic preparation described in the above-mentioned technical solutions, the probiotic preparation prepared by the preparation method described in the above-mentioned technical solutions, or the starter culture described in the above-mentioned technical solutions in any one or more of the following (1) to (4):

[0013] (1) Prepare products that improve gut microbiota;

[0014] (2) Preparation of antioxidant products;

[0015] (3) Prepare antibacterial products;

[0016] (4) Preparation of anti-antibiotic products; the antibiotics include any one or more of amikacin, gentamicin, norfloxacin, ciprofloxacin, compound sulfamethoxazole, chloramphenicol, neomycin, oxacillin, kanamycin, tetracycline and doxycycline.

[0017] Preferably, the product includes one or more of pharmaceuticals, food, and health products; the food includes fermented milk.

[0018] Preferably, the pathogens include any one or more of Escherichia coli, Staphylococcus aureus, and Salmonella.

[0019] This invention provides a method for preparing fermented milk using *Lactobacillus reuteri* IMAU12801, comprising:

[0020] Milk and sugar are mixed, and a starter culture is added for fermentation to obtain fermented milk.

[0021] The fermentation agent includes Lactobacillus reuteri IMAU12801.

[0022] The beneficial effects of this invention are:

[0023] This invention provides a strain of *Limosilactobacillus reuteri* IMAU12801 with probiotic properties, whose preservation number is CGMCC No. 31226. The *Limosilactobacillus reuteri* IMAU12801 provided by this invention exhibits good tolerance to gastrointestinal fluids and possesses various probiotic properties, such as antibacterial potential, DPPH and hydroxyl radical scavenging ability, antioxidant activity, high self-polymerization ability, and good cell surface hydrophobicity. Furthermore, the *Limosilactobacillus reuteri* IMAU12801 is resistant to multiple antibiotics, including amikacin, gentamicin, norfloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, neomycin, oxacillin, kanamycin, tetracycline, and doxycycline. Corresponding antibiotic-resistant probiotic preparations can be prepared from the *Limosilactobacillus reuteri* IMAU12801. Furthermore, the *Lactobacillus reuteri* IMAU12801, when combined with a commercial starter culture for fermentation of cow's milk, can improve the stability of fermented milk under storage conditions of 4°C. This invention provides a novel approach for the utilization and development of *Lactobacillus reuteri* IMAU12801.

[0024] Biological Preservation Information

[0025] Limosilactobacillus reuteri IMAU12801 was deposited on July 8, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 31226. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The acid production rate curve of Lactobacillus reuteri IMAU12801;

[0028] Figure 2 Growth curve of Lactobacillus reuteri IMAU12801;

[0029] Figure 3 A graph showing the sensory evaluation scores of fermented milk during storage;

[0030] Figure 4This is a graph showing the changes in viable bacterial count during storage;

[0031] Figure 5 This is a graph showing the pH changes of fermented milk during storage.

[0032] Figure 6 This is a graph showing the change in titration acidity of fermented milk during storage;

[0033] Figure 7 This is a graph showing the change in viscosity of fermented milk samples during storage;

[0034] Figure 8 This is a graph showing the changes in the water-holding capacity of fermented milk during storage. Detailed Implementation

[0035] This invention provides a strain of *Limosilactobacillus reuteri* IMAU12801 with probiotic properties, the preservation number of which is CGMCC No. 31226.

[0036] In this invention, *Lactobacillus reuteri* IMAU12801 is a strain with potential probiotic properties screened from 85 lactic acid bacteria isolated from naturally fermented milk in Yunnan Province in 2022, and named strain IMAU12801. This invention identifies strain IMAU12801 as *Lactobacillus reuteri* by performing whole-genome sequencing and classification using 16S rDNA. *Lactobacillus reuteri* IMAU12801 was deposited on July 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 31226.

[0037] In this invention, *Lactobacillus reuteri* IMAU12801 is a Gram-positive bacterium, morphologically a slightly irregular, round-ended curved bacillus, arranged in pairs or chains; the colonies of *Lactobacillus reuteri* IMAU12801 are milky white, opaque, round, with a smooth surface, and relatively small. In this invention, *Lactobacillus reuteri* IMAU12801 can be cultured on MRS and TPY media; *Lactobacillus reuteri* IMAU12801 can be cultured anaerobically; the culture temperature for *Lactobacillus reuteri* IMAU12801 can be 37°C. In this invention, the viable count of *Lactobacillus reuteri* IMAU12801 after entering the stationary phase can reach as high as 1.55 × 10⁻⁶. 9CFU / mL; The Lactobacillus reuteri IMAU12801, after fermentation in MRS medium for 24 h, tended to stabilize at pH 4.03, exhibiting good acid production performance.

[0038] In this invention, the fermentation supernatant and cell suspension of *Lactobacillus reuteri* IMAU12801 possess antioxidant activity. The fermentation supernatant of *Lactobacillus reuteri* IMAU12801 exhibits scavenging rates of 35.41% and 85.38% against DPPH free radicals and hydroxyl free radicals, respectively; the cell suspension of *Lactobacillus reuteri* IMAU12801 exhibits scavenging rates of 28.62% and 54.14% against DPPH free radicals and hydroxyl free radicals, respectively.

[0039] In this invention, the *Lactobacillus reuteri* IMAU12801 exhibits good tolerance to gastrointestinal digestive fluids, enabling it to enter the human intestine in a live state and exert health benefits. The results of the embodiments of this invention show that the viable count of *Lactobacillus reuteri* IMAU12801 decreased after 3 hours in simulated gastric juice (pH 2.5), with a survival rate of 97.80% at 3 hours. After being kept in simulated gastric juice at pH 2.5 at 37°C for 3 hours, and then transferred to simulated intestinal juice at pH 8.0 for 8 hours, its survival rate was 96.37%.

[0040] In this invention, the *Lactobacillus reuteri* IMAU12801 exhibits antibacterial effects, showing good antibacterial activity against *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*. The fermentation broth of *Lactobacillus reuteri* IMAU12801 described in this invention can significantly inhibit the growth of *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*, producing a good antibacterial effect.

[0041] In this invention, the *Lactobacillus reuteri* IMAU12801 has a high self-polymerization ability, good cell surface hydrophobicity, and has the potential to be used as a probiotic.

[0042] In this invention, *Lactobacillus reuteri* IMAU12801 is sensitive to 12 antibiotics, including penicillin, ampicillin, piperacillin, carbenicillin, minocycline, cefoperazone, ceftazidime, cefuroxime, cefradine, ceftriaxone, cefalexin, and cefazolin; and resistant to 11 antibiotics, including amikacin, gentamicin, norfloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, neomycin, oxacillin, kanamycin, tetracycline, and doxycycline.

[0043] In this invention, the *Lactobacillus reuteri* IMAU12801 can utilize D-fructose (FRU), L-arabinose (LARA), D-ribose (RIB), D-galactose (GAL), D-glucose (GLU), ferric citrate of aesculin (ESC), D-maltose (MAL), D-lactose (LAC), D-maltodextrose (MEL), D-sucrose (SAC), and D-raffinose (RAF). However, the *Lactobacillus reuteri* IMAU12801 cannot utilize arbutin (ARB), salicin (SAL), and D-trehalose (TRE).

[0044] In this invention, *Lactobacillus reuteri* IMAU12801 can be combined with a commercial starter culture to ferment dairy products, thereby improving the textural properties of the fermented milk. In this invention, the combined fermentation of *Lactobacillus reuteri* IMAU12801 with a commercial starter culture (Mild1.0) achieved a minimum fermentation endpoint (pH 4.5) of 6.20 hours. The storage stability (hardness, consistency, and cohesiveness) of the Mild1.0 + IMAU12801 fermented milk at 4°C was higher than that of the Mild1.0 group.

[0045] This invention provides a probiotic preparation comprising the fermentation broth of *Lactobacillus reuteri* IMAU12801 described in the above-mentioned technical solution. In this invention, the probiotic preparation comprising the fermentation broth of *Lactobacillus reuteri* IMAU12801 preferably has a live bacterial count of ≥10⁻⁶. 6 CFU / mL. In this invention, the fermentation broth can be directly used in probiotic preparations. In this invention, the fermentation broth can also be further separated to obtain a cell suspension and a fermentation supernatant; the cell suspension and fermentation supernatant can be used as probiotic preparations, respectively.

[0046] This invention provides a method for preparing the probiotic preparation described in the above technical solution, comprising:

[0047] Lactobacillus reuteri IMAU12801 was cultured in a culture medium to obtain a probiotic preparation containing the fermentation broth of Lactobacillus reuteri IMAU12801.

[0048] In this invention, the culture medium can be MRS medium or TPY medium; the culture temperature can be 37℃; the culture time for one generation can be 12h-48h or 24h; the culture can be carried out continuously for 2-3 generations. After the culture is completed, the present invention obtains the fermentation broth of *Lactobacillus reuteri* IMAU12801. In this invention, the viable count of the fermentation broth of *Lactobacillus reuteri* IMAU12801 after entering the stationary phase reaches a maximum of 1.55 × 10⁻⁶. 9CFU / mL. In this invention, the fermentation broth can be used directly as a probiotic preparation, or the fermentation broth can be separated to obtain a fermentation supernatant and a cell suspension; the fermentation supernatant and cell suspension are then used as probiotic preparations, respectively. This invention does not specifically limit the separation method; any conventional separation method in the art can be used. In this invention, the separation method preferably includes centrifugation and filtration; the centrifugation speed can be 6000×g or 5000 r / min; the centrifugation time can be 5–10 min; the filtration preferably uses a 0.22 μm filter membrane. This invention preferably obtains the fermentation supernatant and *Lactobacillus reuteri* IMAU12801 cells through filtration. After obtaining the *Lactobacillus reuteri* IMAU12801 cells, this invention preferably washes the cells with PBS buffer and resuspends them to obtain a cell suspension; the washing can be performed three times.

[0049] This invention provides a starter culture, comprising *Lactobacillus reuteri* IMAU12801 as described in the above-mentioned technical solution. This invention allows for the direct fermentation of dairy products using *Lactobacillus reuteri* IMAU12801 to obtain fermented milk. When fermenting cow's milk using *Lactobacillus reuteri* IMAU12801, fermentation takes 15.34 hours, reaching a pH of 4.5, to obtain fermented milk. The fermented milk prepared using *Lactobacillus reuteri* IMAU12801 exhibits superior texture parameters (hardness, consistency, and cohesion) compared to fermented milk prepared with commercial starter cultures during storage for 14–21 days.

[0050] In this invention, the preferred method for preparing the fermenting agent includes: activating and culturing *Lactobacillus reuteri* IMAU12801, followed by three subcultures to obtain *Lactobacillus reuteri* IMAU12801 fermentation broth; separating the bacterial cells from the *Lactobacillus reuteri* IMAU12801 fermentation broth, washing, and freeze-drying to obtain *Lactobacillus reuteri* IMAU12801 fermenting agent. In this invention, the culture medium for activation and subculture includes MRS medium; the activation temperature can be 37°C, and the activation time can be 24 hours; the temperature for each subculture can be 37°C, and the subculture time can be 24 hours. After three subcultures, this invention obtains *Lactobacillus reuteri* IMAU12801 fermentation broth. After obtaining the *Lactobacillus reuteri* IMAU12801 fermentation broth, this invention preferably obtains *Lactobacillus reuteri* IMAU12801 bacterial cells by centrifugation. In this invention, the centrifugation speed is preferably 5000 r / min; the centrifugation time can be 10 min. After centrifugation, the precipitate is collected to obtain *Lactobacillus reuteri* IMAU12801 cells. After obtaining *Lactobacillus reuteri* IMAU12801 cells, this invention preferably washes the *Lactobacillus reuteri* IMAU12801 cells. The washing is preferably performed three times; the washing is preferably done with sterile physiological saline with a mass concentration of 0.85%. After washing, this invention preferably adds a lyophilization protectant to the obtained cells and then freeze-dries them. This invention does not have a specific limitation on the freeze-drying method; any conventional freeze-drying method in the art can be used. After freeze-drying, this invention preferably further includes grinding the freeze-dried material into powder to obtain a fermentation agent containing *Lactobacillus reuteri* IMAU12801. This invention preferably stores the fermentation agent in an aluminum foil bag under vacuum sealing.

[0051] In this invention, the starter culture may further include a commercial starter culture; the commercial starter culture may include Chr. Hansen Mild 1.0. In this invention, the *Lactobacillus reuteri* IMAU12801 is combined with the commercial starter culture Chr. Hansen Mild 1.0 for fermentation. The shortest time to reach the fermentation endpoint (pH 4.5) is 6.20 hours. The storage stability (hardness, consistency, and cohesiveness) of the Mild 1.0 + IMAU12801 fermented milk at 4°C is higher than that of the Mild 1.0 group.

[0052] This invention provides the application of *Lactobacillus reuteri* IMAU12801 described in the above-mentioned technical solutions, the probiotic preparation described in the above-mentioned technical solutions, the probiotic preparation prepared by the preparation method described in the above-mentioned technical solutions, or the starter culture described in the above-mentioned technical solutions in any one or more of the following (1) to (4):

[0053] (1) Prepare products that improve gut microbiota;

[0054] (2) Preparation of antioxidant products;

[0055] (3) Prepare antibacterial products;

[0056] (4) Preparation of anti-antibiotic products; the antibiotics include any one or more of amikacin, gentamicin, norfloxacin, ciprofloxacin, compound sulfamethoxazole, chloramphenicol, neomycin, oxacillin, kanamycin, tetracycline and doxycycline.

[0057] In this invention, the product may include one or more of the following: pharmaceuticals, food, and health products. In this invention, the food may include fermented milk. In this invention, the pathogens include one or more of the following: Escherichia coli, Staphylococcus aureus, and Salmonella.

[0058] This invention provides a method for preparing fermented milk using *Lactobacillus reuteri* IMAU12801, comprising:

[0059] Milk and sugar are mixed, and a starter culture is added for fermentation to obtain fermented milk.

[0060] The fermentation agent includes Lactobacillus reuteri IMAU12801.

[0061] The preparation method of the fermenting agent in this invention has been described above and will not be repeated here. Preferably, the fermenting agent in this invention may also include other commercial fermenting agents.

[0062] In this invention, when mixing milk and sugar, there are no special limitations on the amount of milk and sugar used or the mixing method; any conventional mixing method in the art can be used. In a specific embodiment of this invention, 93.5% milk and 6.5% granulated sugar are uniformly stirred in a mass percentage ratio to obtain a milk-sugar mixture; the sugar may include granulated sugar. To ensure complete dissolution of the sugar, the milk and sugar mixture can be heated to a temperature of 60°C for 15 minutes. After heating, a milk-sugar mixture is obtained. After obtaining the milk-sugar mixture, it can be homogenized and sterilized before adding a starter culture for fermentation. In a specific embodiment of this invention, homogenization can be carried out at 15-20 MPa; after homogenization, sterilization is performed at a temperature of 95°C for 15 minutes. After sterilization, the sterilized milk-sugar mixture is rapidly cooled to 42°C before adding the starter culture. If the starter culture contains only *Lactobacillus reuteri* IMAU12801, the inoculum size can achieve a *Lactobacillus reuteri* IMAU12801 viability of 1 × 10⁻⁶ cells / year in the mixed system. 7 CFU / mL. In this invention, if the starter culture includes *Lactobacillus reuteri* IMAU12801 and other commercial starter cultures, the inoculum amount of the starter culture can make the viability of *Lactobacillus reuteri* IMAU12801 in the mixed system 1×10⁻⁶. 7 CFU / mL; the commercial starter culture can be added according to conventional methods. In a specific embodiment of the present invention, when the commercial starter culture is Chr. Hansen Mild 1.0, the amount of Chr. Hansen Mild 1.0 added is 0.003% of the mass of the lactose mixture. The present invention does not specifically limit the fermentation method; any conventional fermentation method in the art can be used. In a specific embodiment of the present invention, the fermentation temperature can be 37°C; the fermentation ends when the pH reaches 4.5, yielding fermented milk. After obtaining the fermented milk, the present invention can refrigerate the fermented milk at 4°C.

[0063] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] Isolation and identification of Lactobacillus reuteri IMAU12801

[0066] 1. *Lactobacillus reuteri* IMAU12801 is a strain of *Lactobacillus reuteri* with potential probiotic properties, screened from 85 lactic acid bacteria isolated from fermented milk samples in Yunnan Province in 2022. This strain was mainly isolated from fermented milk in Yunnan Province through acid and bile salt tolerance and probiotic property evaluation experiments. The specific isolation process is as follows:

[0067] The bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. It was then passaged three times to restore strain viability. The bacterial culture was serially diluted to obtain 10-1... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Dilution gradient. Take 200 μL of each of the 10... -4 10 -5 10 -6 10 -7 Serially diluted isolates were evenly spread onto MRS solid medium plates and anaerobically incubated at 37°C for 48–72 h. Single clones of different morphology, size, and color were picked and inoculated into liquid medium and incubated at 37°C for 24 h. The test strains were obtained. After the strains had grown well, Gram staining and microscopic examination were performed. The isolates were preserved, and genomic DNA was extracted for subsequent analysis.

[0068] The MRS medium consists of: 10g soybean peptone, 5g beef extract, 5g yeast powder, 20g glucose, 1mL Tween 80, 2g sodium dihydrogen phosphate, 5g anhydrous sodium acetate, 2g triamine citrate, 0.02g manganese sulfate, 0.1g magnesium sulfate, 1L distilled water, pH adjusted to approximately 6.2, 15g agar, and sterilized at 121℃ for 15 minutes.

[0069] 2. Molecular biological identification

[0070] (1) The cryopreserved test strain was inoculated into MRS enrichment liquid medium and cultured at 37℃ for 24 h. After 3 generations of MRS subculture, 2 mL of the bacterial culture at the end of the logarithmic growth phase was placed in a sterile EP tube and centrifuged at 8000×g for 3 min (4℃). The bacterial cells were collected, the supernatant was discarded, and the cells were resuspended in PBS buffer and centrifuged again. The resuspension and centrifugation were repeated until the culture medium in the bacterial cells was removed. Then, whole genomic DNA was extracted. The whole genomic DNA extraction method was based on that of Zhong Zhi et al. (Zhong Zhi, Sun Tiansong, Chen Yongfu. Genomic analysis reveals the molecular mechanism of extracellular polysaccharide enrichment in Streptococcus thermophilus ND-07 [J]. China Dairy Industry, 2018, 9-11+21). The extracted DNA was tested for integrity and purity by 1% agarose gel electrophoresis. The high-quality DNA that met the requirements was used to build a 10kb library according to the Pac-Bio SMRT whole genome DNA library construction process. After library construction, whole genome sequencing was performed according to the Pac-Bio SMRT RSII sequencing platform.

[0071] The whole genome of strain IMAU12801 was sequenced using the PacBio SMRT RSII third-generation sequencing platform, yielding a genome sequence length of 1,897,512 bp, a GC content of 38.69%, and containing 1,902 protein-coding regions and 61 RNA genes. After evaluation and assembly of the sequencing data, the strain was identified as *Limosilactobacillus reuteri* and named *Limosilactobacillus reuteri* IMAU12801.

[0072] (2) DNA samples of the isolated strain IMAU12801 were extracted and identified by Shanghai Paisennong Biotechnology Co., Ltd. The 16S rDNA sequence of strain IMAU12801 is shown in SEQ ID NO.1. The molecular sequence was compared with Limosilactobacillus reuteri by Blast in the NCBI database and the similarity was 99.72%. Therefore, strain IMAU12801 can also be identified as Limosilactobacillus reuteri.

[0073] SEQ ID NO.1:

[0074]

[0075] 3. Morphological characteristics of *Lactobacillus reuteri* IMAU12801

[0076] Lactobacillus reuteri IMAU12801 has the following morphological characteristics: it is a Gram-positive bacterium with a slightly irregular, rounded end, and is arranged in pairs or chains.

[0077] 4. Colony morphology characteristics of *Lactobacillus reuteri* IMAU12801

[0078] When Lactobacillus reuteri IMAU12801 grows on MRS medium, the colonies are milky white, opaque, round, with a smooth surface, and the colonies are relatively small.

[0079] 5. Detection of viable count and acid production of *Lactobacillus reuteri* IMAU12801 after it enters the stationary phase.

[0080] Lactobacillus reuteri IMAU12801 was inoculated into MRS medium and cultured at 37°C. During culture, the viable cell count and pH of the culture system were measured every 2 hours to determine the viable cell count and acid-producing capacity of Lactobacillus reuteri IMAU12801 after entering the stationary phase. The results are as follows: Figures 1-2 As shown. Among them. Figure 1 The acid production rate curve of Lactobacillus reuteri IMAU12801; Figure 2 The growth curve of *Lactobacillus reuteri* IMAU12801.

[0081] Depend on Figure 1 and Figure 2 It can be seen that the viable count of *Lactobacillus reuteri* IMAU12801 after entering the stationary phase can be as high as 1.55 × 10⁻⁶. 9 CFU / mL; the Lactobacillus reuteri IMAU12801 fermented for 24 h tended to be stable (pH 4.03), and had good acid production performance.

[0082] Example 2

[0083] Gastrointestinal fluid tolerance of Lactobacillus reuteri IMAU12801

[0084] After sterilizing PBS, add 3.0 mg / mL pepsin, adjust the pH to 2.5 with 1 mol / L HCl, and filter sterilize using a 0.22 μm microporous membrane to prepare simulated gastric fluid; after sterilizing PBS, add 0.1% trypsin and 1.8% ox bile salt, adjust the pH to 8.0 with 0.1 mol / L NaOH, and filter sterilize using a 0.22 μm microporous membrane to prepare simulated intestinal fluid.

[0085] Lactobacillus reuteri IMAU12801 was anaerobically cultured in MRS medium at 37°C for 24 h, and then cultured for another 24 h at 37°C using the MRS medium for two consecutive generations. After culturing, the bacterial culture was centrifuged at 6000×g for 5 min, the supernatant was discarded, and the bacteria were washed with sterile PBS. This process was repeated 2-3 times, followed by centrifugation and discarding of PBS to collect the bacterial cells. 5 mL of sterile PBS was added to the bacterial cells, and the mixture was shaken to prepare a stock solution. 0.5 mL of the stock solution was added to 4.5 mL of pH 2.5 simulated gastric fluid and digested at 37°C for 3 h. Viable bacteria were counted using the MRS agar pour method at 0 h and 3 h after digestion. Then, 0.5 mL of the digested 3-h simulated gastric fluid containing bacteria was added to 4.5 mL of simulated intestinal fluid and cultured in a 37°C water bath. Viable bacteria were counted using the MRS agar pour method at 4 h and 8 h after the addition of the intestinal fluid. The growth of *Lactobacillus reuteri* IMAU12801 in artificial simulated gastrointestinal fluid is shown in Table 1.

[0086] Table 1. Growth of *Lactobacillus reuteri* IMAU12801 in simulated gastrointestinal fluid.

[0087]

[0088] Table 1 shows that the viable count of *Lactobacillus reuteri* IMAU12801 decreased after 3 hours in simulated gastric juice (pH 2.5), with a survival rate of 97.80%. After being kept in simulated gastric juice at 37°C for 3 hours, and then transferred to simulated intestinal juice at pH 8.0 for 8 hours, the survival rate of *Lactobacillus reuteri* IMAU12801 in the intestinal juice was 96.37%, demonstrating good tolerance to gastrointestinal digestive fluids and the ability to enter the human intestine in a live state to exert health benefits. Due to its good tolerance to gastrointestinal digestive fluids, *Lactobacillus reuteri* IMAU12801, as a probiotic, can resist the adverse effects of the digestive tract environment and adhere and colonize well in the intestine. It also has the functions of regulating intestinal flora, improving immunity, lowering cholesterol, and reducing cardiovascular disease.

[0089] Example 3

[0090] Antioxidant activity of Lactobacillus reuteri IMAU12801

[0091] Using the DPPH free radical scavenging ability kit from Nanjing Jiancheng Bioengineering Institute, the scavenging levels of DPPH free radicals and hydroxyl free radicals in the fermentation supernatant and bacterial suspension of the strain were determined according to the instructions.

[0092] Lactobacillus reuteri IMAU12801 strain was cultured in MRS liquid medium for 12 h, followed by centrifugation (5000 r / min, 10 min) to collect the supernatant. The supernatant was obtained by filtration through a 0.22 μm filter membrane. The Lactobacillus reuteri IMAU12801 cell pellet was washed three times with PBS buffer and resuspended to adjust the cell concentration to 102. 9 CFU / mL was used as the cell suspension.

[0093] Meanwhile, the model strain of *Lactobacillus reuteri* DSM 20016 was also tested. T The antioxidant activity of fermentation supernatant and cell suspension of *Lactobacillus reuteri* strain DSM 20016 was detected by (Vogel, RF, et al., Identification of lactobacilli from sourdough and description of *Lactobacillus pontis* sp. nov. 1994. 44(2): p. 223-229). T The preparation methods for fermentation supernatant and cell suspension are the same as those for Lactobacillus reuteri IMAU12801.

[0094] The obtained *Lactobacillus reuteri* IMAU12801 and *Lactobacillus reuteri* type strain DSM 20016 were analyzed. T The antioxidant activity of fermentation supernatant and cell suspension was determined using a DPPH free radical scavenging assay kit. This assay was applied to *Lactobacillus reuteri* IMAU12801 and the *Lactobacillus reuteri* model strain DSM 20016. T The results of the scavenging capacity tests for DPPH free radicals and hydroxyl free radicals are shown in Tables 2 and 3.

[0095] Table 2. Scavenging ability of *Lactobacillus reuteri* IMAU12801 against DPPH free radicals.

[0096] IMAU12801 <![CDATA[35.41±0.33 a ]]> <![CDATA[28.62±0.23 a ]]> <![CDATA[DSM20016 T ]]> <![CDATA[31.72±0.51 b ]]> <![CDATA[25.17±0.23 b ]]>

[0097] Note: Data in the table are mean (D) ± standard deviation (SD), where the mean is the average of the three groups. Significance analysis results are those within the same column. The same applies below.

[0098] Table 3. Scavenging ability of Lactobacillus reuteri IMAU12801 against hydroxyl radicals.

[0099]

[0100] As shown in Tables 2 and 3, the scavenging capacity of *Lactobacillus reuteri* IMAU12801 for DPPH and hydroxyl radicals was higher than that of the model strain DSM 20016. T This indicates that *Lactobacillus reuteri* IMAU12801 is a relative variant of the type strain DSM 20016. T It has higher antioxidant activity.

[0101] Example 4

[0102] Determination of the antibacterial activity of Lactobacillus reuteri IMAU12801

[0103] The inhibition diameter of the strains against Escherichia coli, Salmonella and Staphylococcus aureus was determined using the Oxford cup method described by Wang et al. (Wang, X., et al., Probiotic potential and wide-spectrum antimicrobial activity of lactic acid bacteria isolated from infant feces.[J]. Probiotics and antimicrobial proteins 2021.13:p.90-101).

[0104] After culturing the strain in MRS liquid medium for 12 h, the supernatant was collected by centrifugation (5000 r / min, 10 min). The supernatant was obtained by filtration through a 0.22 μm filter membrane. The concentration of the indicator bacteria (Escherichia coli, Salmonella, and Staphylococcus aureus) suspension was adjusted to 10. 6 CFU / mL. Indicator bacterial suspension was added to NA medium at a 1% inoculum volume, mixed, and 20 mL was transferred to a sterile agar plate. After the agar plate solidified, a sterile Oxford cup was placed on the NA medium and pressed down. 100 μL of fermentation supernatant was added to each well, and the mixture was incubated at 37°C for 18 h. The diameter of the inhibition zone was measured, and each group was tested in triplicate.

[0105] The antibacterial activity of *Lactobacillus reuteri* IMAU12801 against *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus* is shown in Table 4.

[0106] Table 4. Antibacterial activity of IMAU12801 against Escherichia coli, Salmonella, and Staphylococcus aureus.

[0107]

[0108] As shown in Table 4, the inhibition diameter of *Lactobacillus reuteri* IMAU12801 against *Escherichia coli* and *Salmonella* is higher than that of the model strain DSM 20016T.

[0109] Example 5

[0110] Determination of the self-aggregation ability and hydrophobicity of *Lactobacillus reuteri* IMAU12801

[0111] (1) After overnight culture of the strain in MRS liquid medium, the bacterial sludge was collected by centrifugation (5000 r / min, 10 min). The bacterial sludge was washed twice with PBS buffer and resuspended to adjust the OD of the bacterial suspension. 600nm The value was 0.6 ± 0.05. The bacterial suspension was added to MRS medium and incubated at 37°C. The OD values ​​of the upper layer of the bacterial suspension were measured at 600 nm using a microplate reader at different time points (0 h, 2 h, 4 h, 24 h). 600nm The formula for the self-aggregation rate is:

[0112]

[0113] in:

[0114] A0 represents the absorbance value at 0h;

[0115] At represents the absorbance value of th.

[0116] The self-aggregation ability of Lactobacillus reuteri IMAU12801 and the model strain DSM 20016T was determined using the above method. Three parallel experiments were conducted for each strain, and the results are shown in Table 5.

[0117] (2) After overnight culture in MRS liquid medium, the bacterial strain was centrifuged (5000 r / min, 10 min) to collect the bacterial sludge. The bacterial sludge was washed twice with PBS buffer and resuspended, and the OD was adjusted. 600nm The value was 0.6 ± 0.05 (A0). 1 mL of xylene was added to 3 mL of the prepared bacterial cell suspension, mixed, and incubated for 10 min. The mixture was then vortexed for 2 min and incubated for another 30 min at room temperature to separate the layers. The upper aqueous phase was collected, and the OD value was measured. 600nm Value (A). The hydrophobicity of bacteria is calculated using the following formula:

[0118]

[0119] in:

[0120] A0 represents the initial absorbance value;

[0121] A represents the absorbance value after processing.

[0122] The above methods were used to determine the levels of *Lactobacillus reuteri* IMAU12801 and the model strain DSM 20016, respectively. T The hydrophobicity of each strain was assessed by conducting three parallel experiments, and the results are shown in Table 6.

[0123] Table 5 Evaluation of the self-aggregation ability of *Lactobacillus reuteri*

[0124]

[0125] Table 5 shows that the self-aggregation ability under static conditions continuously increases with the increase of bacterial cell count. IMAU12801 exhibited a significantly higher self-aggregation rate at 24 hours compared to DSM 20016. T (P < 0.05).

[0126] Table 6 Evaluation of the hydrophobicity of *Lactobacillus reuteri*

[0127] IMAU12801 <![CDATA[56.57±0.30 a ]]> <![CDATA[DSM20016 T ]]> <![CDATA[39.77±1.19 c ]]>

[0128] Table 6 shows the hydrophobicity of the two lactic acid bacteria strains, IMAU12801 and DSM 20016. T All are classified as mesohydrophobic. Among them, IMAU12801 has the highest hydrophobicity (56.57%), which is significantly higher than DSM 20016T (P<0.05). IMAU12801 exhibits good cell surface hydrophobicity and has the potential to be used as a probiotic.

[0129] Example 6

[0130] Antibiotic sensitivity of Lactobacillus reuteri IMAU12801

[0131] The susceptibility of lactic acid bacteria to 24 antibiotics was determined using the disk diffusion method (KB method).

[0132] Take 0.1 mL of lactic acid bacteria fermentation broth and spread it on MRS medium. Use sterile forceps to attach a drug sensitivity paper strip with a certain amount of antibiotic to the plate after spreading the bacterial broth. Incubate at 37℃ for 48 h and observe the size of the inhibition diameter.

[0133] The method for preparing lactic acid bacteria fermentation broth is as follows: lactic acid bacteria are cultured on MRS medium at 37℃ for 24 hours to obtain lactic acid bacteria fermentation broth.

[0134] Lactobacillus reuteri IMAU12801 and the model strain DSM 20016 were prepared using the above method. T The fermentation broth was then analyzed, and its sensitivity to different antibiotics was determined. The results are shown in Table 7.

[0135] Table 7. Antibiotic susceptibility studies of *Lactobacillus reuteri*.

[0136]

[0137] Note: Criteria for determining the diameter of the inhibition zone: Sensitive (S), Intermediate (I), Resistant (-)

[0138] The results are shown in Table 7. *Lactobacillus reuteri* IMAU12801 was sensitive to 12 antibiotics: penicillin, ampicillin, piperacillin, carbenicillin, minocycline, cefoperazone, ceftazidime, cefuroxime, cefradine, ceftriaxone, cefalexin, and cefazolin. It showed varying degrees of resistance to the remaining antibiotics. Compared to DSM 20016... T In contrast, Lactobacillus reuteri IMAU12801 has developed resistance to chloramphenicol and neomycin, indicating that this strain is not easily inhibited by the corresponding antibiotics during fermentation, which is beneficial for its stable application in fermented foods and can improve the flavor and quality of the products.

[0139] Example 7

[0140] Carbon source utilization of Lactobacillus reuteri IMAU12801

[0141] API50 CHL is a simple culture medium composed of 49 fermentable carbohydrates used for the identification of Lactobacillus and related bacteria. Carbohydrates are the main energy source for bacterial growth, and the ability of lactic acid bacteria to utilize carbohydrates affects their growth and metabolism. Following the bioMérieux API50 CHL kit instructions, API50 CHL was used to identify IMAU12801 and the type strain DSM 20016. T The carbohydrate utilization capacity of *Lactobacillus reuteri* IMAU12801 was determined. Table 8 shows the utilization of the corresponding carbon sources by *Lactobacillus reuteri* IMAU12801 after culturing for 24 h and 48 h.

[0142] Table 8 Carbon source utilization of *Lactobacillus reuteri* IMAU12801

[0143]

[0144]

[0145] Note: + indicates a positive reaction (reacts with the tested enzyme); - indicates a negative reaction (does not react with the tested enzyme); D indicates a delayed reaction.

[0146] The results are shown in Table 8. The comparison revealed that IMAU12801 and the model strain DSM20016 TAll strains utilized L-arabinose (LARA), D-ribose (RIB), D-galactose (GAL), D-glucose (GLU), ferric citrate of aesculin (ESC), D-maltose (MAL), D-lactose (LAC), D-maltodextrose (MEL), D-sucrose (SAC), and D-raffinose (RAF). IMAU12801 did not utilize arbutin (ARB), salicin (SAL), and D-trehalose (TRE), and only IMAU12801 could utilize D-fructose (FRU). The fact that *Lactobacillus reuteri* IMAU12801 can utilize fructose indicates that this strain has a stronger fermentation capacity and may produce more beneficial intestinal products.

[0147] Example 8

[0148] Fermentation of Lactobacillus reuteri IMAU12801 with basic starter culture

[0149] (1) Preparation of Lactobacillus reuteri IMAU12801 fermentation broth used in the experiment

[0150] Lactobacillus reuteri IMAU12801 was activated by culturing in MRS at 37°C for 24 hours. After activation, it was passaged three times in MRS liquid medium. The first passage involved inoculating the activated culture into MRS liquid medium and culturing at 37°C for 24 hours. The second passage involved inoculating the culture into fresh MRS liquid medium and culturing at 37°C for 24 hours. Finally, the second passage culture was inoculated into fresh MRS liquid medium and cultured at 37°C for 24 hours to complete the third passage, yielding the fermentation broth of Lactobacillus reuteri IMAU12801.

[0151] After three subcultures, the obtained *Lactobacillus reuteri* IMAU12801 fermentation broth was centrifuged (5000 r / min, 10 min), and the precipitate was collected. The resulting bacterial cells were washed three times with sterile 0.85% physiological saline, and the bacterial sludge was weighed. A freeze-drying protectant was added. The sample was pre-frozen at -80℃ for 24 h, followed by freeze-drying for 48 h. The freeze-dried sample was ground into powder to obtain *Lactobacillus reuteri* IMAU12801 fermentation primordial. This primordial primordial IMAU12801 fermentation primordial broth can be vacuum-sealed in aluminum foil bags for storage. The viable count of the obtained *Lactobacillus reuteri* IMAU12801 fermentation primordial broth was 3 × 10⁻⁶. 7 CFU / g.

[0152] (2) Mix 93.5% of commercially available pure milk and 6.5% of white sugar in a uniform ratio according to mass percentage, ensuring the sugar is completely dissolved. Heat to 60℃ for 15 minutes, then homogenize at 15-20 MPa. After homogenization, sterilize at 95℃ for 15 minutes and rapidly cool to 42℃. Divide the sterilized milk-sugar mixture into three experimental groups: Experiment 1, Experiment 2, and Experiment 3. Experiment 1 was inoculated with commercial starter culture Mild1.0 and designated as the Mild1.0 group; Experiment 2 was inoculated with commercial starter culture Mild1.0 and *Lactobacillus reuteri* IMAU12801 and designated as the Mild1.0+IMAU12801 group; Experiment 3 was inoculated with *Lactobacillus reuteri* IMAU12801 and designated as the IMAU12801 group. The specific inoculation amounts of starter culture were as shown in Table 9. Each group was tested in triplicate. After inoculation, stir and dispense the mixture, place it in a 37°C water bath for 10 minutes to warm up, and then place it at 37°C to ferment until the pH reaches 4.5, at which point fermentation is complete. Cool and store the mixture.

[0153] The fermentation time to pH 4.5 for each experimental group was statistically analyzed, and the results are shown in Table 10. After obtaining the fermented milk, the fermented milk from different experimental groups was stored at 4℃, 15℃, and 25℃, respectively. The texture parameters of the fermented milk were tested at 1, 7, 14, and 21 days of storage, and the test results are shown in Tables 13-16. Sensory evaluation of the fermented milk was conducted at 1, 7, 14, and 21 days of storage. The sensory evaluation method was based on the national standard GB 19302-2010 "Fermented Milk," and the sensory quality scoring criteria for fermented milk were evaluated from four aspects: color, texture, taste and aroma, and mouthfeel. The sensory evaluation results are shown in Tables 12 and 13. Figure 3 As shown.

[0154] The viable cell count of the fermented milk was tested at 1, 7, 14, and 21 days of storage. The method involved accurately weighing 25.0 g of fermented milk sample, diluting it tenfold, shaking it at low temperature for 15 min, and then serially diluting 1 mL of the diluted sample using the same method. Plate counting was performed using an appropriate gradient. The sample was incubated at 37℃ for 72 h, and the colony count was then recorded. The viable cell count results are shown in Table 17. Figure 4 As shown in the figures. The pH and titratable acidity of the fermented milk were measured at 1, 7, 14, and 21 days of storage, and the results are shown in Tables 18-19. Figures 5-6As shown. The water-holding capacity and viscosity of the fermented milk were tested at 1, 7, 14, and 21 days of storage. The method involved accurately weighing 20.0 g of fermented milk sample, placing it in a funnel lined with filter paper, allowing it to stand at room temperature for 2 hours, collecting the filtrate, and weighing it. The calculation formula was as follows: Water-holding capacity (%) = 1 - filtrate weight (g) / sample weight (g) × 100%. Under room temperature conditions, the viscosity of the fermented milk sample was measured using a BROOKFIELD DV-1VISCOMETER viscometer. A #4 rotor was selected, the rotation speed was 100 rpm, the torque was 10-100%, and the measurement time was 30 s. The results of the water-holding capacity and viscosity tests are shown in Tables 20-21 and 21, respectively. Figures 7-8 As shown.

[0155] The commercial fermentation agent Mild1.0 strain is named Chr. Hansen Mild1.0, with strain type Lactobacillus delbrueckii subsp. Bulgaricus, Streptococcus thermophilus, and viable count of 4.3 × 10⁻⁶. 7 CFU / g.

[0156] Table 9. Composition and inoculum size of fermentation agents in different experimental groups

[0157] Mild1.0 group 0.3g / L - Mild1.0 + IMAU12801 group 0.3g / L <![CDATA[1×10 7 ]]> IMAU12801 group - <![CDATA[1×10 7 ]]>

[0158] Table 10 Fermentation time of different experimental groups

[0159] Mild1.0 group <![CDATA[7.10±0.03 b ]]> Mild1.0 + IMAU12801 group <![CDATA[6.20±0.02 a ]]> IMAU12801 group <![CDATA[15.34±0.06 c ]]>

[0160] Note: Different letters in the superscript indicate significant differences.

[0161] Table 10 shows the time required for the fermented milk samples in the three experimental groups to reach a pH of 4.5. The fermentation time of the single strain IMAU12801 group was the longest, at 15.34±0.06 h, while the fermentation time of the Mild1.0 group was 7.10±0.03 h. The fermentation time of the Mild1.0+IMAU12801 group was the shortest, at 6.20±0.02 h, which was significantly less than the fermentation time of the Mild1.0 group and the IMAU12801 group (P<0.05).

[0162] Table 11 Sensory Quality Scoring Criteria for Fermented Milk

[0163]

[0164] Table 12 Sensory evaluation results of fermented milk

[0165]

[0166]

[0167] Table 13 Analysis of textural parameters of fermented milk from different experimental groups after 1 day of storage.

[0168]

[0169] Table 14 Analysis of textural parameters of fermented milk from different experimental groups after 7 days of storage.

[0170] Mild1.0 4℃ <![CDATA[55.91±1.87 b ]]> <![CDATA[440.07±10.98 de ]]> <![CDATA[-51.43±6.01 b <!-- 16 -->]]> Mild1.0+IMAU12801 4℃ <![CDATA[68.44±0.57 a ]]> <![CDATA[502.57±8.51 a ]]> <![CDATA[-61.17±4.93 c ]]> IMAU12801 4℃ <![CDATA[48.21±3.48 c ]]> <![CDATA[401.16±11.31 e ]]> <![CDATA[-39.92±4.53 a ]]> Mild1.0 15℃ <![CDATA[56.15±1.97 b ]]> <![CDATA[426.06±10.32 d ]]> <![CDATA[-51.36±4.43 b ]]> Mild1.0+IMAU12801 15℃ <![CDATA[53.50±7.39 b ]]> <![CDATA[427.61±17.34 d ]]> <![CDATA[-46.90±3.19 ab ]]> IMAU12801 15℃ <![CDATA[56.71±2.18 b ]]> <![CDATA[448.18±9.81 c ]]> <![CDATA[-52.12±6.08 b ]]> Mild1.0 25℃ <![CDATA[47.00±1.82 c ]]> <![CDATA[359.39±5.06 f ]]> <![CDATA[-45.07±1.94 ab ]]> Mild1.0+IMAU12801 25℃ <![CDATA[54.08±4.09 b ]]> <![CDATA[423.96±5.38 de ]]> <![CDATA[-49.55±3.45 b ]]> IMAU12801 25℃ <![CDATA[57.23±2.59 b ]]> <![CDATA[467.23±6.18 b ]]> <![CDATA[-52.16±7.29 b ]]>

[0171] Table 15 Analysis of textural parameters of fermented milk from different experimental groups after 14 days of storage.

[0172] Mild1.0 4℃ <![CDATA[58.69±1.43 bc ]]> <![CDATA[491.44±5.24 bcd ]]> <![CDATA[-55.54±2.72 cd ]]> Mild1.0+IMAU12801 4℃ <![CDATA[75.24±2.37 a ]]> <![CDATA[565.44±20.58 a ]]> <![CDATA[-71.24±3.47 f ]]> IMAU12801 4℃ <![CDATA[63.72±2.39 b ]]> <![CDATA[501.14±11.40 bc ]]> <![CDATA[-66.15±1.74 e ]]> Mild1.0 15℃ <![CDATA[60.12±6.14 b ]]> <![CDATA[469.31±40.33 cde ]]> <![CDATA[-60.34±3.90 d ]]> Mild1.0+IMAU12801 15℃ <![CDATA[60.92±1.97 b ]]> <![CDATA[506.99±7.56 b ]]> <![CDATA[-57.97±2.20 d ]]> IMAU12801 15℃ <![CDATA[54.43±1.73 c ]]> <![CDATA[459.32±10.05 de ]]> <![CDATA[-50.97±1.77 bc ]]> Mild1.0 25℃ <![CDATA[44.38±1.47 e ]]> <![CDATA[446.58±3.60 e ]]> <![CDATA[-45.50±3.67 a ]]> Mild1.0+IMAU12801 25℃ <![CDATA[49.55±1.56 d ]]> <![CDATA[481.23±14.85 bcd ]]> <![CDATA[-46.45±2.62 ab ]]> IMAU12801 25℃ <![CDATA[42.70±2.18 e ]]> <![CDATA[402.79±12.79 f ]]> <![CDATA[-43.58±1.97 a ]]>

[0173] Table 16 Analysis of textural parameters of fermented milk from different experimental groups after 21 days of storage.

[0174] Mild1.0 4℃ <![CDATA[48.56±1.58 c ]]> <![CDATA[393.00±12.19 c ]]> <![CDATA[-45.99±1.43 cd ]]> Mild1.0+IMAU12801 4℃ <![CDATA[68.39±1.00 a ]]> <![CDATA[529.52±5.29 a ]]> <![CDATA[-63.31±2.77 e ]]> IMAU12801 4℃ <![CDATA[53.19±2.62 b ]]> <![CDATA[460.96±15.54 b ]]> <![CDATA[-47.25±5.98 d ]]> Mild1.0 15℃ <![CDATA[46.89±1.88 c ]]> <![CDATA[385.84±15.35 cd ]]> <![CDATA[-43.46±3.55b cd ]]> Mild1.0+IMAU12801 15℃ <![CDATA[46.88±1.89 c ]]> <![CDATA[367.32±10.32 d ]]> <![CDATA[-42.16±2.54 bcd ]]> IMAU12801 15℃ <![CDATA[50.03±2.37 bc ]]> <![CDATA[406.83±8.33 c ]]> <![CDATA[-45.47±3.76 cd ]]> Mild1.0 25℃ <![CDATA[37.41±4.32 e ]]> <![CDATA[323.80±24.83 e ]]> <![CDATA[-35.87±2.40 a ]]> Mild1.0+IMAU12801 25℃ <![CDATA[45.97±1.34 c ]]> <![CDATA[405.14±6.72 c ]]> <![CDATA[-40.52±1.91 abc ]]> IMAU12801 25℃ <![CDATA[41.29±0.96 d ]]> <![CDATA[392.33±7.68 c ]]> <![CDATA[-39.14±1.39 ab ]]>

[0175] As shown in Tables 12-16, the textural properties of fermented milk exhibited a trend of first increasing and then decreasing during storage. After 7 days of storage, the fermented milk in the Mild1.0 group, Mild1.0+IMAU12801 group, and IMAU12801 group showed an increase in hardness and consistency due to the decrease in acidity, which improved the structural strength of the fermented milk. The cohesiveness of the fermented milk also increased accordingly. The Mild1.0 + IMAU12801 group of fermented milk stored at 4℃ for 14 days exhibited the highest hardness, consistency, and cohesiveness, at 75.24 g, 565.44 g·s, and -71.24 g·s, respectively. This was followed by the Mild1.0 group, with values ​​of 58.69 g, 491.44 g·s, and -55.54 g·s, respectively. The IMAU12801 group showed values ​​of 63.72 g, 501.14 g·s, and -66.15 g·s, respectively. The internal gel system of the fermented milk was disrupted, leading to a continuous decline in quality over time. After storage, the Mild1.0 + IMAU12801 group of fermented milk stored at 4℃ showed higher hardness than the group stored for 1 day, and the fermented milk with added IMAU12801 also had a higher hardness than the Mild1.0 group, consistent with the sensory evaluation findings. Fermented milk stored at 15℃ and 25℃ showed a decreasing trend in hardness.

[0176] Table 17 Viable bacteria counts in fermented milk from different experimental groups at different storage times

[0177]

[0178] The results of the viable bacteria count determination are shown in Table 17 and Figure 4 As shown, after 21 days of storage, the viable bacterial count of each group of fermented milk remained at 10. 6 CFU / mL or higher. Figure 4As shown, the viable cell count decreased during storage. After storage at 4℃, the viable cell counts of the Mild1.0+IMAU12801 group and the IMAU12801 group of fermented milk were (6.9±0.04)×10⁻⁶, respectively. 8 CFU / mL and (7.7±0.05)×10 8 CFU / mL. The viable counts of fermented milk from Mild1.0+IMAU12801 group and IMAU12801 group stored at 15℃ were (7.4±0.1)×10⁻⁶ CFU / mL. 8 CFU / mL and (7.6±0.06)×10 8 The viable cell counts (CFU / mL) of fermented milk from the Mild1.0+IMAU12801 group and the IMAU12801 group stored at 25℃ were (4.6±0.08)×10⁻¹⁰. 8 CFU / mL and (3.0±0.1)×10 8 The overall live bacteria count (CFU / mL) of fermented milk stored at 4℃ and 15℃ was higher than that stored at 25℃. This may be because the higher ambient temperature leads to increased acid production by lactic acid bacteria, resulting in a decrease in the number of lactic acid bacteria.

[0179] Table 18 pH values ​​of fermented milk from different experimental groups at different storage times

[0180]

[0181]

[0182] Table 19 Titration acidity of fermented milk from different experimental groups at different storage times

[0183]

[0184]

[0185] Changes in acidity effectively reflect the growth of lactic acid bacteria in fermented milk, significantly impacting the taste and aroma of fermented dairy products. pH value and titratable acidity are important indicators for assessing the degree of acidification after fermentation. Acidity changes during storage are shown in Tables 18-19 and... Figures 5-6As shown, at the end of storage, the pH values ​​of the Mild1.0 group, Mild1.0+IMAU12801 group, and IMAU12801 group stored at 4℃ were 4.24, 4.19, and 4.18, respectively; the pH values ​​of the Mild1.0 group, Mild1.0+IMAU12801 group, and IMAU12801 group stored at 15℃ were 4.02, 4.07, and 4.1, respectively; and the pH values ​​of the Mild1.0 group, Mild1.0+IMAU12801 group, and IMAU12801 group stored at 25℃ were 4.01, 3.96, and 3.92, respectively. There were no significant differences among the groups (P>0.05). The pH value of the fermented milk at the three storage temperatures showed an increasing trend with increasing temperature. This may be because higher temperatures promote the symbiosis of lactic acid bacteria, accelerating metabolism and increasing acid production. During storage at 4℃ and 15℃, the pH value of the fermented milk was within the optimal range for consumption.

[0186] The titratable acidity of the nine fermented milk samples showed an increasing trend during storage, mainly due to the continuous decomposition of lactose by lactic acid bacteria, leading to a continuous increase in acidity. During storage at 4℃, the titratable acidity of the Mild1.0 group increased the slowest, reaching 93°T after 21 days. In contrast, the titratable acidity values ​​of the Mild1.0+IMAU12801 and IMAU12801 groups were 100°T and 105°T, respectively, at the end of storage. The differences among the groups during storage were significant (P<0.05). The titratable acidity values ​​of Mild1.0, Mild1.0+IMAU12801, and IMAU12801 groups stored at 15℃ were 109°T, 108°T, and 117°T, respectively. The titratable acidity values ​​of these groups stored at 25℃ were 113°T, 123°T, and 130°T, respectively. The titratable acidity value of the Mild1.0+IMAU12801 group was significantly lower than that of the IMAU12801 group (P < 0.05). Donkor et al. {Donkor, 2006#18} found that fermented milk with a titratable acidity value between 70-110°T had the best quality and was most popular with consumers. Zhang Yong's research (Zhang Yong, 2018#19) found that fermented milk is not suitable for drinking when the acidity exceeds 120°T because a large amount of whey is separated, which reduces the overall quality.

[0187] Table 20 Viscosities of Fermented Milk from Different Experimental Groups at Different Storage Times

[0188]

[0189] Table 21 Water-holding capacity of fermented milk from different experimental groups at different storage times

[0190]

[0191]

[0192] Viscosity is an important indicator for evaluating yogurt quality. As the acidity of fermented milk decreases, casein coagulates, and bacteria release various extracellular polysaccharides, causing viscosity to increase. Viscosity changes are shown in Table 20 and... Figure 7 As shown, the viscosity of fermented milk stored at 4℃ increased continuously from 1 to 14 days, resulting in a smoother and creamier texture. In the later stages of storage, the viscosity decreased. After storage at 4℃, the viscosity values ​​of the Mild1.0 group, Mild1.0+IMAU12801 group, and IMAU12801 group were 1867 cp, 2538 cp, and 1651 cp, respectively, showing significant differences among the groups (P<0.05). The viscosity of fermented milk products stored at 15℃ and 25℃ decreased slightly, but the changes were small. In conclusion, the viscosity value of the Mild1.0+IMAU12801 group was higher than that of the IMAU12801 group, with the Mild1.0+IMAU12801 group maintaining a higher viscosity value at 4℃.

[0193] Water-holding capacity is an important indicator of the quality of fermented dairy products, and the three-dimensional network structure of fermented milk proteins determines its water-holding capacity. Fermented dairy products with strong water-holding capacity and a well-structured protein network can prevent whey separation and maintain the quality of the fermented milk. Changes in water-holding capacity are shown in Table 21 and... Figure 8 As shown, the water-holding capacity of fermented milk stored at 4℃ showed an increasing trend from 1 to 14 days, and decreased after the storage period ended, as the water-holding capacity continuously decreased with the decrease in pH value. At 14 days of storage, the water-holding capacity of fermented milk in the Mild1.0 group stored at 4℃ was 73.5%, while the water-holding capacities of the Mild1.0+IMAU12801 group and the IMAU12801 group were 75.7% and 65.05%, respectively. The water-holding capacity of the Mild1.0+IMAU12801 group was higher than that of the Mild1.0 group and the IMAU12801 group. Among them, the water-holding capacity of the Mild1.0+IMAU12801 group stored at 4℃ remained the highest at 14 days, and then showed a decreasing trend after 21 days of storage.

[0194] In summary, the *Lactobacillus reuteri* IMAU12801 provided by this invention exhibits good tolerance to gastrointestinal fluids and possesses various probiotic properties, such as antibacterial potential, DPPH and hydroxyl radical scavenging ability, and antioxidant activity. The *Lactobacillus reuteri* IMAU12801, when combined with a basic starter culture, can increase the stability of fermented milk under 4°C storage conditions. This invention provides a novel approach for the utilization and development of *Lactobacillus reuteri* IMAU12801.

[0195] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus (L. rhamnosus) having probiotic properties, L. rhamnosus IMAU 12801, characterized by, Limosilactobacillus reuteri )IMAU12801, characterized by, The preservation number of *Lactobacillus reuteri* IMAU12801 is CGMCC No. 31226.

2. A probiotic preparation, characterized in that, Includes the fermentation broth of *Lactobacillus reuteri* IMAU12801 as described in claim 1.

3. The probiotic preparation according to claim 2, characterized in that, the viable count of L. reuteri IMAU12801 in the probiotic preparation is ≥ 10 6 CFU / mL.

4. A method for preparing the probiotic preparation according to claim 2 or 3, characterized in that, include: Lactobacillus reuteri IMAU12801 was cultured in a culture medium to obtain a probiotic preparation containing the fermentation broth of Lactobacillus reuteri IMAU12801.

5. A fermenting agent, characterized in that, Includes *Lactobacillus reuteri* IMAU12801 as described in claim 1.

6. The fermenting agent according to claim 5, characterized in that, The fermenting agent also includes commercial fermenting agents.

7. The use of *Lactobacillus reuteri* IMAU12801 as described in claim 1 or the use of *Lactobacillus reuteri* IMAU12801 as described in claim 1 and commercial starter culture Mild1.0 in the preparation of fermented milk; The applications include shortening fermentation time and / or improving the storage quality of fermented milk; The improvement of the storage quality of fermented milk includes at least one of the following: (1) Optimize the textural parameters during the storage of fermented milk; the textural parameters include any one or more of hardness, consistency and cohesiveness; (2) Increase the number of viable bacteria during the storage of fermented milk; (3) Improve the acidity of fermented milk during storage; (4) Increase the viscosity of fermented milk during storage; (5) Improve the water-holding capacity of fermented milk during storage.

8. A method for preparing fermented milk using *Lactobacillus reuteri* IMAU12801, characterized in that, include: Milk and sugar are mixed, and a starter culture is added for fermentation to obtain fermented milk. The starter culture comprises Lactobacillus reuteri IMAU12801 as described in claim 1 or Lactobacillus reuteri IMAU12801 as described in claim 1 and the commercial starter culture Mild1.0.

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