Lactobacillus reuteri ccfm1528 that promotes growth of bifidobacteria by metabolism and survival upon freeze-drying

Abstract

The present application relates to a strain of promoting growth of Bifidobacterium by metabolism and freeze-drying survival of Lactobacillus reuteri CCFM1528, and belongs to the field of microbial fermentation engineering. The present application introduces Bifidobacterium into the fermentation supernatant of Lactobacillus reuteri for culture, so that the maximum biomass of Bifidobacterium CCFM1167 is increased by 90.05%, and the freeze-drying survival rate is increased from 12.52+0.37% to 17.52+1.69%; the maximum biomass of Bifidobacterium longum CCFM1510 is increased by 42.86%, and the freeze-drying survival rate is increased from 0.62+0.02% to 54.35+1.41%.

Description

Technical Field

[0001] This invention relates to a strain of *Lactobacillus reuteri* CCFM1528 that promotes the growth and freeze-dried survival of *Bifidobacterium* through metabolism, belonging to the field of microbial fermentation engineering. Background Technology

[0002] Bifidobacteria, as one of the main beneficial bacteria in the human gut, has had its probiotic functions extensively studied, showing great potential in maintaining gut microbiota balance, regulating immunity, and improving mental health. Given these significant probiotic functions, Bifidobacteria are increasingly being used in the food industry and in probiotic preparations.

[0003] Despite the significant probiotic value of Bifidobacterium, high-density cultivation in industrial production still faces severe challenges. Bifidobacterium is a strict anaerobe, requiring demanding cultivation environments. During cultivation, due to its sensitivity to osmotic pressure, controlling the substrate concentration in the culture medium is crucial: too low a concentration will not achieve optimal cultivation results, while too high a concentration will inhibit cell growth. Furthermore, due to species differences, there is no clear pattern to the nitrogen source utilization of Bifidobacterium. These factors collectively make it difficult to achieve high biomass of Bifidobacterium in traditional fermentation processes, resulting in low fermentation densities and hindering the upgrading of its industrial production.

[0004] After obtaining a fermentation broth with a high viable cell count, maintaining a high survival rate during freeze-drying remains a major technical bottleneck. Vacuum freeze-drying is a common method for preparing Bifidobacterium powder, but the freeze-drying process causes various types of damage to the bacteria, including mechanical damage, membrane damage, protein denaturation, and DNA damage, leading to a significant decrease in viable cell count. To address this issue, freeze-drying protectants are usually added; however, Bifidobacterium generally has poor resistance to stress, and existing freeze-drying protectants are often ineffective, resulting in a still low survival rate. Therefore, there is an urgent need in industrial production to develop new methods that can synergistically improve the growth density and freeze-drying survival rate of Bifidobacterium to overcome the shortcomings of existing technologies and meet industrialization needs.

[0005] Bifidobacterium longum CCFM1510 is a strain that the inventors previously discovered can improve colonic autophagy and delay colonic aging. Bifidobacterium bifidum CCFM1167 is a publicly disclosed functional strain that can relieve constipation. How to improve the growth efficiency of Bifidobacterium longum CCFM1510 and Bifidobacterium bifidum CCFM1167, while improving the freeze-drying survival rate, is a technical problem that urgently needs to be solved in the existing technology. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides the application of Lactobacillus reuteri CCFM1528 and its fermentation supernatant in promoting the growth and freeze-dried survival of Bifidobacteria.

[0007] This invention provides Lactobacillus reuteri CCFM1528, which was deposited on December 26, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67538, and the deposit address is Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0008] In one embodiment, the *Lactobacillus reuteri* CCFM1528 has the following characteristics: In one embodiment, the *Lactobacillus reuteri* CCFM1528 cells appear under a microscope as slightly irregular, round-ended, non-motile curved bacteria. After inoculation on MRS solid medium and cultured for 48 hours, the colonies are generally slightly transparent at the edges, milky white overall, smooth and raised, and round with a diameter of 1-2 mm.

[0009] In one embodiment, the *Lactobacillus reuteri* CCFM1528 is a Gram-positive, facultatively anaerobic, thermophilic bacterium with an optimal growth temperature of 35-40°C and an optimal growth pH of 6.0-7.0.

[0010] The present invention also provides a method for preparing fermentation supernatant of Lactobacillus reuteri, wherein Lactobacillus reuteri CCFM1528 is cultured at 35~39℃ for 24h, the supernatant is obtained by centrifugation, sugar is added to 15-20g / L, the pH is adjusted to 6.0, and the supernatant is filtered for sterilization.

[0011] Furthermore, the culture medium for *Lactobacillus reuteri* CCFM1528 consists of: 5-10 g / L beef extract, 5-10 g / L peptone, 5-10 g / L yeast extract, 20-30 g / L glucose, 2-5 g / L sodium acetate, 2-5 g / L diammonium citrate, 2-5 g / L K₂HPO₄·3H₂O, 0-0.5 g / L MgSO₄·7H₂O, 0-0.5 g / L MnSO₄·H₂O, 1-2 mL / L between 80, 1-2 g / L cysteine, and 1000 g / L distilled water, adjusted to pH 6.0.

[0012] The present invention also provides the application of the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 in promoting the growth of *Bifidobacterium*. When *Bifidobacterium* is inoculated at a 2% inoculum, the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 can promote the growth of *Bifidobacterium*.

[0013] In one embodiment, the bifidobacteria include Bifidobacterium bifidum CCFM1167 or Bifidobacterium longum CCFM1510.

[0014] The present invention also provides a method for preparing Bifidobacterium powder, the method comprising the following steps: (1) Use the fermentation supernatant of the above-mentioned Lactobacillus reuteri to culture Bifidobacterium to the stationary phase; (2) Collect the Bifidobacterium sludge from step (1), mix it with the protectant at a mass ratio of (1:2) to (1:4), and then freeze-dry it to obtain Bifidobacterium powder.

[0015] In one embodiment, the freeze-drying protectant is an aqueous solution containing 50 g / L trehalose, 50 g / L sucrose, and 100 g / L skim milk powder.

[0016] This invention provides the application of the above-mentioned fermentation supernatant of *Lactobacillus reuteri* CCFM1528 or the above-mentioned method of promoting the growth and freeze-drying survival of *Lactobacillus reuteri* CCFM1528 through metabolism in promoting the growth of *Bifidobacterium* and improving the freeze-drying resistance of *Bifidobacterium*.

[0017] Beneficial effects: This invention provides a strain of *Lactobacillus reuteri* CCFM1528, the fermentation supernatant of which can promote the growth and freeze-dried survival of *Bifidobacterium*, as detailed below: Compared with culture in MRS medium, culturing in the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 increased the maximum biomass of *Bifidobacterium bifidum* CCFM1167 by 90.05% and the maximum biomass of *Bifidobacterium longum* CCFM1510 by 42.86%.

[0018] Compared with lyophilization after culturing in MRS medium, lyophilization after culturing in the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 increased the lyophilization survival rate of *Bifidobacterium bifidum* CCFM1167 from 12.52±0.37% to 17.52±1.69%, and the lyophilization survival rate of *Bifidobacterium longum* CCFM1510 from 0.62±0.02% to 54.35±1.41%.

[0019] Preservation of biological materials Lactobacillus reuteri ( Limosilactobacillus reuteri (CCFM1528, categorized as follows) Limosilactobacillus reuteri It was deposited on December 26, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67538, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0020] Bifidobacterium longum ( Bifidobacterium longum subsp. longum (CCFM1510, categorized as follows) Bifidobacterium longum subsp. longumIt was deposited on July 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66746, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0021] Lactobacillus reuteri ( Limosilactobacillus reuteri CCFM1502, categorized as follows: Limosilactobacillus reuteri It was deposited on July 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66740, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description Figure 1 Growth curves of Bifidobacterium in fermentation supernatant and MRS of Lactobacillus reuteri CCFM1528: A. Bifidobacterium bifidum CCFM1167; B. Bifidobacterium longum CCFM1510.

[0022] Figure 2 Growth curves of Bifidobacterium in fermentation supernatants of Lactobacillus reuteri CCFM1528 and other Lactobacillus reuteri: A. Bifidobacterium bifidum CCFM1167; B. Bifidobacterium longum CCFM1510.

[0023] Figure 3 This represents the maximum biomass of Bifidobacterium cultured in the fermentation supernatant of Lactobacillus reuteri CCFM1528 and MRS.

[0024] Figure 4 This represents the maximum biomass of Bifidobacterium cultured in the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 and other *Lactobacillus reuteri* species.

[0025] Figure 5 A. Generation time; B. Exponential growth rate of carbon source bacteria per unit mass during the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 and MRS.

[0026] Figure 6 A. Generation time; B. Exponential growth rate of carbon source bacteria per unit mass during the fermentation supernatant of Bifidobacterium reuteri CCFM1528 and other Lactobacillus reuteri.

[0027] Figure 7 The lyophilization survival rate of Bifidobacterium in the fermentation supernatant and MRS of Lactobacillus reuteri.

[0028] Figure 8 The lyophilized survival rate of Bifidobacterium in fermentation supernatants of Lactobacillus reuteri CCFM1528 and other Lactobacillus reuteri is given. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments.

[0030] The following examples involve Bifidobacterium bifidum CCFM1167, which is disclosed in patent CN112940980B, with accession number GDMCC No: 61482; Bifidobacterium longum CCFM1510, with accession number GDMCC No: 66746; Lactobacillus reuteri CCFM1502, with accession number GDMCC No: 66740; and Lactobacillus reuteri FYNLJ83L8.

[0031] The culture media involved in the following examples are as follows: MRS medium: 10 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 2 g / L sodium acetate, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.05 g / L LnSO4·H2O, 1 mL / L Tween 80, 1 g / L cysteine, 1000 g / L distilled water, adjusted to pH 6.0.

[0032] MRS solid culture medium: 10 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 2 g / L sodium acetate, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.05 g / L LnSO4·H2O, 1 mL / L Tween 80, 1 g / L cysteine, 20 g / L agar, 1000 g / L distilled water, adjusted to pH 6.0.

[0033] The detection methods involved in the following embodiments are as follows: ; Where G represents the generation time of the strain, and A0 represents the OD of the strain when it enters the logarithmic growth phase. 600 A t Indicates the OD of the strain at time t 600 .

[0034] The method for detecting the total viable count of Bifidobacteria was as follows: The national standard GB 4789.35-2016, "National Food Safety Standard for Microbiological Testing of Food - Lactic Acid Bacteria Detection", was adopted.

[0035] Glucose consumption was detected using a glucose test kit (Nanjing Jiancheng Bioengineering Institute).

[0036] The formula for calculating the proliferation of carbon source bacteria per unit mass during the exponential phase is: ; The formula for calculating the freeze-dried product survival rate is: .

[0037] Example 1: Screening and identification of Lactobacillus reuteri CCFM1528 The isolation method for *Lactobacillus reuteri* is as follows: fecal samples were serially diluted with 0.9% physiological saline (10... -1 -10 -5 Afterwards, 100 μL of the sample suspension was spread onto an MRS solid plate and anaerobically cultured at 37°C for 48 h. Based on colony morphology, single colonies with different morphologies were picked, streaked onto new MRS solid plates for purification, and anaerobically cultured at 37°C for 48 h. A single colony was then picked and cultured in MRS liquid medium for 24 h, followed by subculturing at a 2% inoculum. 0.5 mL of the bacterial culture was transferred to a 2 mL culture tube, 0.5 mL of 60% glycerol was added, vortexed, and stored at -80°C. Further 16S sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The nucleic acid sequence of *Lactobacillus reuteri* was identified as 100% similar to that of *Lactobacillus reuteri*, and it was named *Lactobacillus reuteri*. Limosilactobacillus reuteri (CCFM1528)

[0038] Example 2: Effect of fermentation supernatant of *Lactobacillus reuteri* CCFM1528 on the growth of *Bifidobacterium*. 1. Preparation of fermentation supernatant of *Lactobacillus reuteri*: *Lactobacillus reuteri* CCFM1528 was cultured in MRS liquid medium for 24 h until the viable count reached 1×10⁻⁶. 9 CFU / mL, centrifuge to collect supernatant, add glucose to 20 g / L, adjust pH to 6.0, and filter sterilize using a 0.22 μm filter membrane.

[0039] 2. Activation of *Bifidobacterium bifidum* CCFM1167 and *Bifidobacterium longum* CCFM1510: After thawing the culture tubes, shake well, and use a sterile inoculation loop to pick up an appropriate amount of bacterial suspension and streak it onto an MRS solid plate for isolation. Incubate anaerobically for 48 h. Pick single colonies and inoculate them into MRS liquid medium, incubate anaerobically for 24 h, and subculture at an inoculation rate of 2% (v / v). After three stable subcultures, a viable count higher than 10⁻⁶ was obtained. 8 Seed culture of CFU / mL.

[0040] Growth curve plotting: Activated Bifidobacterium was inoculated into the fermentation supernatant of *Lactobacillus reuteri* at a 2% inoculum and fermented. The fermentation group inoculated into MRS medium served as a control. OD was measured every 2 hours. 600(See Figure 1 ).

[0041] Result: From Figure 1 It can be seen that the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 has a significant promoting effect on *Bifidobacterium bifidum* CCFM1167 and *Bifidobacterium longum* CCFM1510, thus increasing the OD of the fermentation broth during the stationary phase of both strains. 600 The values ​​increased significantly, from 0.623±0.016 and 1.588±0.028 to 0.957±0.009 and 1.873±0.034, respectively.

[0042] Comparative Example 1: Effect of *Lactobacillus reuteri* CCFM1528 fermentation supernatant on Bifidobacterium growth compared to other *Lactobacillus reuteri* fermentation supernatants. 1. Preparation of fermentation supernatant of Lactobacillus reuteri: Fermentation supernatant of Lactobacillus reuteri CCFM1502 and FYNLJ83L8 was prepared according to the method in Example 2.

[0043] 2. Growth curve plotting: Following the method in Example 1, activated Bifidobacterium was inoculated into the fermentation supernatant of Lactobacillus reuteri at an inoculum size of 2%, and OD was measured every 2 hours. 600 (See Figure 2 ).

[0044] Result: From Figure 2 It was found that the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 significantly promoted the growth of *Bifidobacterium bifidum* CCFM1167 and *Bifidobacterium longum* CCFM1510, which was significantly better than that of *Lactobacillus reuteri* CCFM1502 (0.813±0.014 and 1.650±0.023) and *Lactobacillus reuteri* FYNLJ83L8 (0.794±0.008 and 1.552±0.025).

[0045] Example 3: Effect of fermentation supernatant of *Lactobacillus reuteri* CCFM1528 on the maximum biomass of *Bifidobacterium* Determination of maximum biomass: Following the method described in Example 1, *Bifidobacterium bifidum* CCFM1167 and *Bifidobacterium longum* CCFM1510 were cultured in MRS medium and fermentation supernatant of *Lactobacillus reuteri* CCFM1528, respectively. *Bifidobacterium bifidum* was cultured for 14 h and 16 h, and *Bifidobacterium longum* for 12 h and 14 h. Maximum biomass was then determined (see Example 1). Figure 3 ).

[0046] Result: From Figure 3It was found that, compared with MRS medium, the maximum biomass of Bifidobacterium bifidum CCFM1167 and Bifidobacterium longum CCFM1510 in the fermentation supernatant of Lactobacillus reuteri CCFM1528 was significantly increased, from 7.03±0.58×10⁻⁶ in MRS medium. 8 CFU / mL and 1.12±0.05×10 9 CFU / mL increased to 1.34 ± 0.05 × 10⁻⁶. 9 CFU / mL and 1.60±0.09×10 9 CFU / mL.

[0047] Comparative Example 2: Effect of *Lactobacillus reuteri* CCFM1528 fermentation supernatant on the maximum biomass of *Bifidobacterium* compared to other *Lactobacillus reuteri* fermentation supernatants. Determination of maximum biomass: The maximum biomass of Bifidobacterium was determined in the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 and other *Lactobacillus reuteri* according to the detection method described in Example 3 (see Example 3). Figure 4 ).

[0048] Result: From Figure 4 It can be seen that the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 significantly promoted the maximum biomass of *Bifidobacterium bifidum* CCFM1167 and *Bifidobacterium longum* CCFM1510, which was significantly better than the promotion of the maximum biomass of *Lactobacillus reuteri* CCFM1502 (8.37 ± 0.23 × 10⁻⁶). 8 CFU / mL and 1.16±0.04×10 9 CFU / mL, and 7.80±0.27×10⁻⁶ CFU / mL for *Lactobacillus reuteri* FYNLJ83L8. 8 CFU / mL and 1.24±0.10×10 9 CFU / mL.

[0049] Example 4: Effect of fermentation supernatant of *Lactobacillus reuteri* CCFM1528 on the proliferation of Bifidobacterium per unit mass of carbon source bacteria during generation time and exponential phase. 1. Determination of generation time: Following the method described in Example 2, activated Bifidobacterium was inoculated into MRS and Lactobacillus reuteri CCFM1528 fermentation supernatant at a 2% inoculum and cultured for 4 h. Then, the generation time of the strains was determined (see Example 2). Figure 5 A).

[0050] 2. Determination of carbon source bacteria proliferation per unit mass during the exponential phase: Following the method described in Example 2, activated Bifidobacterium was inoculated at a 2% inoculum into the fermentation supernatant of MRS and Lactobacillus reuteri CCFM1528, and cultured separately. The proliferation of carbon source bacteria per unit mass during the exponential phase of the strains was determined (see Example 2). Figure 5 B).

[0051] Result: From Figure 5 As shown in A, the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 significantly shortened the generation time of *Bifidobacterium bifidum* CCFM1167, from 2.00±0.02 h to 1.90±0.01 h. Figure 5 B indicates that the fermentation supernatant of *Lactobacillus reuteri* CCFM1528 significantly increased the proliferation of carbon source bacteria per unit mass of *Bifidobacterium bifidum* CCFM1167 during the exponential phase, from 6.06 ± 0.19 × 10⁻⁶. 11 CFU / g increased to 7.49±0.44×10 11 CFU / g.

[0052] Comparative Example 3: Effect of *Lactobacillus reuteri* CCFM1528 fermentation supernatant on the proliferation of Bifidobacterium per unit mass of carbon source bacteria during generation time and exponential phase compared to other *Lactobacillus reuteri* fermentation supernatants. 1. Determination of generation time: The generation time of Bifidobacterium cultured in MRS and Lactobacillus reuteri CCFM1528 fermentation supernatant was determined according to the method described in Example 4 (see Example 4). Figure 6 A).

[0053] 2. Determination of the proliferation rate of carbon source bacteria per unit mass during the exponential phase: The proliferation rate of Bifidobacterium per unit mass in the fermentation supernatant of MRS and Lactobacillus reuteri CCFM1528 during the exponential phase was determined according to the method described in Example 4 (see Example 4). Figure 6 B).

[0054] Result: From Figure 6 As shown in A, the intermediate fermentation time of *Bifidobacterium bifidum* CCFM1167 in the supernatant of *Lactobacillus reuteri* CCFM1528 was significantly shorter than that of *Lactobacillus reuteri* FYNLJ83L8 (2.24 ± 0.04 h); the intermediate fermentation time of *Bifidobacterium longum* CCFM1510 in the supernatant of *Lactobacillus reuteri* CCFM1528 was significantly shorter than that of *Lactobacillus reuteri* CCFM1502 (1.47 ± 0.01 h) and *Lactobacillus reuteri* FYNLJ83L8 (1.66 ± 0.01 h). Figure 6 As shown in B, the proliferation rate of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus reuteri CCFM1528 was significantly higher than that of Lactobacillus reuteri FYNLJ83L8 (4.54 ± 0.35 × 10⁻⁶). 11 CFU / g.

[0055] Example 5: Effect of fermentation supernatant of *Lactobacillus reuteri* CCFM1528 on the survival of freeze-dried *Bifidobacterium* 1. Determination of freeze-drying survival rate: Bifidobacterium was cultured in MRS medium and fermentation supernatant of *Lactobacillus reuteri* CCFM1528, respectively, according to the method described in Example 2. After culturing, the bacterial cells were collected by centrifugation and mixed evenly with the freeze-drying protectant. The freeze-drying survival rate was calculated by measuring the number of viable bacteria in the bacterial suspension before and after freeze-drying (see Example 2). Figure 7 The freeze-drying protectant consists of 50 g / L trehalose, 50 g / L sucrose, and 100 g / L skim milk powder; the freeze-drying protectant is mixed with the bacterial cells at a ratio of 2:1 v / m and then freeze-dried.

[0056] 2. Freeze-drying process parameter settings: Pre-freeze, control the laminate temperature to drop from room temperature to -50℃ within 1 hour and maintain it for 4 hours; First drying, adjust the lamination temperature to -30℃ within 1.3 hours and maintain it at a vacuum of 0.2 μbar for 30 hours to remove free water; Second drying, control the laminate temperature to rise to 25℃ within 1 hour at a vacuum of 20 μbar and maintain it for 20 hours.

[0057] Result: From Figure 7 It can be seen that, compared with MRS medium, the freeze-dried survival rates of Bifidobacterium bifidum CCFM1167 and Bifidobacterium longum CCFM1510 in the fermentation supernatant of Lactobacillus reuteri CCFM1528 were significantly increased, from 12.52±0.37% and 0.62±0.02% in MRS medium to 17.52±1.69% and 54.35±1.41% in the fermentation supernatant of Lactobacillus reuteri CCFM1528, respectively.

[0058] Comparative Example 4: Effect of *Lactobacillus reuteri* CCFM1528 fermentation supernatant on the survival of *Bifidobacterium* lyophilized compared to other *Lactobacillus reuteri* fermentation supernatants. Determination of freeze-dried survival rate: The freeze-dried survival rate of Bifidobacterium cultured in fermentation supernatants of Lactobacillus reuteri CCFM1528 and other Lactobacillus reuteri was determined according to the method described in Example 5 (see Example 5). Figure 8 ).

[0059] Result: From Figure 8 It was found that the freeze-drying survival rate of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus reuteri CCFM1528 was significantly higher than that of Lactobacillus reuteri CCFM1502 (12.36±0.98%) and Lactobacillus reuteri FYNLJ83L8 (13.36±0.56%). Similarly, the freeze-drying survival rate of Bifidobacterium longum CCFM1510 in the fermentation supernatant of Lactobacillus reuteri CCFM1528 was significantly higher than that of Lactobacillus reuteri CCFM1502 (46.25±3.79%).

[0060] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1528, the Lactobacillus reuteri CCFM1528, has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67538.

2. The fermentation supernatant of *Lactobacillus reuteri* CCFM1528 according to claim 1, characterized in that, The fermentation supernatant is obtained by fermenting the Lactobacillus reuteri CCFM1528 of claim 1, removing the bacterial cells, and adding glucose to a final concentration of 15-20 g / L.

3. The method according to claim 2, characterized in that, The fermentation conditions are as follows: cultured at 35~39℃ for 24-30 hours.

4. The method according to claim 3, characterized in that, The fermentation medium contains 5-10 g / L beef extract, 5-10 g / L peptone, 5-10 g / L yeast extract, 20-30 g / L glucose, 2-5 g / L sodium acetate, 2-5 g / L diammonium citrate, 2-5 g / L K2HPO4·3H2O, 0-0.5 g / L MgSO4·7H2O, 0-0.5 g / L MnSO4·H2O, 1-2 mL / L between 80, and 1-2 g / L cysteine.

5. A method for promoting the growth of Bifidobacteria, characterized in that, Bifidobacteria are inoculated into the fermentation supernatant according to any one of claims 2 to 4 for fermentation.

6. The method according to claim 5, characterized in that, Cultured anaerobically at 35~39℃ until the early stage of stable growth.

7. The method according to claim 5, characterized in that, The bifidobacteria include Bifidobacterium bifidum CCFM1167 or Bifidobacterium longum CCFM1510.

8. A method for promoting the survival of freeze-dried Bifidobacteria, characterized in that, After inoculating Bifidobacterium into the fermentation supernatant of any one of claims 2 to 4 for fermentation, a freeze-drying protectant is added for freeze-drying.

9. The method according to claim 8, characterized in that, The fermentation conditions are 35-39℃, anaerobic culture.

10. The method according to claim 9, characterized in that, The freeze-drying protectant contains 40-60 g / L trehalose, 50-80 g / L sucrose, and 100-120 g / L skim milk powder; the freeze-drying protectant is mixed with bacterial cells at a ratio of (2:1) to (4:1) v / m and then freeze-dried.

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