Lactobacillus paracasei CCFM1529 for promoting growth and freeze-drying survival of bifidobacterium bifidum through metabolism

Abstract

The invention discloses a lactobacillus paracasei CCFM1529 for promoting growth and freeze-drying survival of bifidobacterium bifidum through metabolism, and belongs to the field of microbial fermentation engineering. According to the invention, the bifidobacterium bifidum is inoculated into the fermentation supernatant of the casei paracasei for culturing, so that the maximum biomass of the bifidobacterium bifidum CCFM1167 is increased by 115.17%, and the freeze-drying survival rate is increased by 20.18%.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus paracasei CCFM1529 that promotes the growth and freeze-dried survival of Bifidobacterium bifidum through metabolism, belonging to the field of microbial fermentation engineering. Background Technology

[0002] Bifidobacterium bifidum, as one of the core probiotics in the human gut, plays a crucial role in early colonization and the construction of a healthy gut microbiota in infants. It not only efficiently degrades various dietary fibers to produce short-chain fatty acids and regulates intestinal pH, but also interacts with the host immune system through its unique surface molecules, reducing the risk of allergies and inflammatory bowel disease. Furthermore, the application value of its strains in metabolic syndrome, mental health, and other fields is becoming a research hotspot, highlighting the importance of its in-depth development.

[0003] However, the industrial-scale high-density culture of Bifidobacterium bifidum faces significant biological limitations. As a strictly anaerobic heterotrophic microorganism, its growth is heavily dependent on complex nutrient sources and it is extremely sensitive to the redox potential of the culture medium; even trace amounts of oxygen can irreversibly inhibit its growth. During high-density fermentation, the bacterial cells rapidly metabolize to produce organic acids, leading to a rapid increase in the osmotic pressure of the culture system. Due to the relatively weak cell structure of Bifidobacterium bifidum, it is sensitive to changes in osmotic pressure, severely limiting its final biomass. Furthermore, it lacks a complete anabolic metabolic pathway and is dependent on various growth factors such as amino acids, vitamins, and nucleotides. These complex nutritional requirements and stringent anaerobic environmental control make it difficult to achieve economically feasible industrial-scale high-density culture through conventional process optimization.

[0004] The subsequent freeze-drying process also poses a significant challenge to maintaining the activity of Bifidobacterium bifidum. Physicochemical stresses such as ice crystal formation, drastic changes in osmotic pressure, and membrane lipid phase transitions during freeze-drying directly damage the integrity of the bacterial cell wall and cell membrane, leading to leakage of intracellular substances and ultimately loss of biological activity. Although freeze-drying protectants such as trehalose and skim milk are widely used, conventional protectant formulations are often limited in their effectiveness for environmentally sensitive strains like Bifidobacterium bifidum, resulting in a significant decrease in the survival rate and stability of live bacteria after freeze-drying. Therefore, developing a biotechnological method that can optimize the fermentation process to obtain high biomass and effectively enhance the strain's resistance to freeze-drying stress without relying on complex equipment has become an urgent need to overcome formulation bottlenecks and improve product competitiveness.

[0005] Bifidobacterium bifidum CCFM1167 is a publicly known functional strain with constipation-relieving effects. However, in practical industrial applications, it faces challenges such as slow growth and low freeze-drying survival rate. Improving the growth efficiency of Bifidobacterium bifidum CCFM1167 while simultaneously increasing its freeze-drying survival rate is a pressing technical problem that needs to be solved in the current technology. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides the application of Lactobacillus paracasei CCFM1529 and its fermentation supernatant in promoting the growth and freeze-dried survival of Bifidobacterium bifidum.

[0007] This invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei The Lactobacillus paracasei CCFM1529 was deposited on December 26, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67539, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In one embodiment, the *Lactobacillus paracasei* CCFM1529 has the following characteristics: In one embodiment, the *Lactobacillus paracasei* CCFM1529 cells appear as slightly irregular, round-ended, non-motile curved bacteria under a microscope. After inoculation on MRS solid medium and cultured for 48 h, the colonies are generally milky white, smooth, raised, and round with a diameter of 1-2 mm.

[0009] In one embodiment, the *Lactobacillus paracasei* CCFM1529 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 paracasei, wherein Lactobacillus paracasei CCFM1529 is cultured at 35~39℃ for 24 h, the supernatant is obtained by centrifugation, sugar is added to 20 g / L, the pH is adjusted to 6.0, and the supernatant is filtered to remove bacteria.

[0011] Furthermore, the culture medium for *Lactobacillus paracasei* CCFM1529 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 above-mentioned Lactobacillus paracasei fermentation supernatant in promoting the growth of Bifidobacterium bifidum. Bifidobacterium bifidum is inoculated at a 2% inoculum, and the growth of Bifidobacterium bifidum is promoted when the above-mentioned Lactobacillus paracasei fermentation supernatant is used to culture Bifidobacterium bifidum.

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

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

[0015] In one embodiment, the freeze-drying protectant is an aqueous solution containing 30-50 g / L trehalose, 30-50 g / L sucrose, and 100-200 g / L skim milk powder.

[0016] This invention provides the application of the above-mentioned Lactobacillus paracasei CCFM1529 fermentation supernatant in improving the survival rate of freeze-dried Bifidobacterium bifidum.

[0017] Beneficial effects: (1) This invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei Using the fermentation supernatant of *Lactobacillus paracasei* CCFM1529 to culture *Bifidobacterium bifidum* can promote the growth of *Bifidobacterium bifidum* and improve freeze-drying resistance, specifically: Compared with culture in MRS medium, culturing in the fermentation supernatant of Lactobacillus paracasei CCFM1529 increased the maximum biomass of Bifidobacterium bifidum CCFM1167 by 115.17%.

[0018] Compared with lyophilization after culturing in MRS medium, lyophilization after culturing in the fermentation supernatant of Lactobacillus paracasei CCFM1529 can increase the lyophilization survival rate of Bifidobacterium bifidum CCFM1167 by 20.18%.

[0019] Preservation of biological materials Lactobacillus paracasei CCFM1529, classified and named Lacticaseibacillus paracasei It was deposited on December 26, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67539, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached image description: Figure 1 The growth curves of Bifidobacterium bifidum CCFM1167 in fermentation supernatant and MRS of Lactobacillus paracasei CCFM1529 are shown.

[0020] Figure 2 The growth curves of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus paracasei CCFM1529 and other Lactobacillus paracasei are shown.

[0021] Figure 3 The maximum biomass of Bifidobacterium bifidum CCFM1167 cultured in fermentation supernatant and MRS of Lactobacillus paracasei CCFM1529 is given.

[0022] Figure 4 The maximum biomass of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus paracasei CCFM1529 and other Lactobacillus paracasei is given.

[0023] Figure 5 The lyophilized survival rate of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant and MRS of Lactobacillus paracasei CCFM1529 is given.

[0024] Figure 6 The freeze-dried survival rate of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus paracasei CCFM1529 and other Lactobacillus paracasei. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments.

[0026] 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.

[0027] 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 LmnSO4·H2O, 1 mL / L Tween 80, 1 g / L cysteine, 20 g / L agar, 1000 g / L distilled water, adjusted to pH 6.0.

[0028] The detection methods involved in the following embodiments are as follows: The method for detecting the total viable count of Bifidobacterium bifidum was as follows: The national standard GB 4789.35-2016, "National Food Safety Standard for Microbiology of Food - Detection of Lactic Acid Bacteria", was adopted.

[0029] The formula for calculating freeze-dried product survival rate is: Freeze-dried product survival rate (%)

[0030] Example 1: Screening and identification of Lactobacillus paracasei CCFM1529 The isolation method for *Lactobacillus paracasei* is as follows: Kimchi brine samples were serially diluted with 0.9% physiological saline (10... -1 -10 -5 Afterwards, 100 μL of 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 bacterial culture was transferred to a 2 mL culture preservation 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 paracasei* was identified as 100% similar, and it was named *Lactobacillus paracasei*. Lacticaseibacillus paracasei (CCFM1529)

[0031] Example 2: Effect of fermentation supernatant of *Lactobacillus paracasei* CCFM1529 on the growth of *Bifidobacterium bifidum*. 1. Preparation of fermentation supernatant of Lactobacillus paracasei: Lactobacillus paracasei CCFM1529 was cultured in MRS liquid medium for 24 h until the viable count reached 8 × 10⁻⁶. 8 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.

[0032] 2. Activation of Bifidobacterium bifidum CCFM1167: After thawing the preservation 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 upside down for 48 h. Pick a single colony and inoculate it 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.

[0033] 3. Growth curve plotting: Activated Bifidobacterium bifidum CCFM1167 was inoculated into the fermentation supernatant of Lactobacillus paracasei CCFM1529 at a 2% inoculum. Groups inoculated into MRS medium for fermentation served as controls. OD was measured every 2 hours. 600 (See Figure 1 ).

[0034] Result: From Figure 1 It can be seen that the fermentation supernatant of *Lactobacillus paracasei* CCFM1529 has a significant promoting effect on *Bifidobacterium bifidum* CCFM1167, increasing the OD during the stable phase of the fermentation broth. 600 It increased significantly, from 0.623±0.016 to 0.657±0.005.

[0035] Comparative Example 1: Effect of Lactobacillus paracasei fermentation supernatant on the growth of Bifidobacterium bifidum compared to other Lactobacillus paracasei fermentation supernatants 1. Preparation of fermentation supernatant of Lactobacillus paracasei: The fermentation supernatant of Lactobacillus paracasei was prepared according to the method described in Example 2.

[0036] 2. Growth curve plotting: Activated Bifidobacterium bifidum CCFM1167 was inoculated into the fermentation supernatant of Lactobacillus paracasei at a 2% inoculum. OD was measured every 2 hours. 600 (See Figure 2 ).

[0037] Result: From Figure 2 It can be seen that the fermentation supernatant of Lactobacillus paracasei CCFM1529 significantly promoted the growth of Bifidobacterium bifidum CCFM1167, which was significantly better than that of Lactobacillus paracasei DJXSRYG2L10 (0.618±0.008) and Lactobacillus paracasei CCFM1224 (0.531±0.006).

[0038] Example 3: Effect of fermentation supernatant of Lactobacillus paracasei CCFM1529 on the maximum biomass of Bifidobacterium bifidum Determination of maximum biomass: Bifidobacterium bifidum was cultured in MRS medium and Lactobacillus paracasei fermentation supernatant for 14 h and 18 h, respectively, according to the method described in Example 2, and the maximum biomass of Bifidobacterium bifidum was determined (see Example 2). Figure 3 ).

[0039] Result: From Figure 3 It was found that, compared with MRS medium, the maximum biomass of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus paracasei CCFM1529 was significantly increased, from 7.03 ± 0.58 × 10⁻⁶. 8 CFU / mL increased to 1.51 ± 0.09 × 10⁻⁶ 9 CFU / mL.

[0040] Comparative Example 2: Effect of Lactobacillus paracasei fermentation supernatant on the maximum biomass of Bifidobacterium bifidum compared to other Lactobacillus paracasei fermentation supernatants Determination of maximum biomass: The maximum biomass of Bifidobacterium bifidum in fermentation supernatants of Lactobacillus paracasei CCFM1529 and other Lactobacillus paracasei was determined according to the detection method described in Example 3 (see Example 3). Figure 4 ).

[0041] Result: From Figure 4 It was found that the fermentation supernatant of *Lactobacillus paracasei* CCFM1529 significantly promoted the maximum biomass of *Bifidobacterium bifidum* CCFM1167, which was significantly better than that of *Lactobacillus paracasei* DJXSRYG2L10 (6.57 ± 0.15 × 10⁻⁶). 8 CFU / mL and Lactobacillus paracasei CCFM1224 were 7.33 ± 0.47 × 10⁻⁶. 8 CFU / mL.

[0042] Example 4: Effect of Lactobacillus paracasei fermentation supernatant on the survival of freeze-dried Bifidobacterium bifidum. Determination of freeze-drying survival rate: Following the method described in Example 3, *Bifidobacterium bifidum* was cultured in MRS medium and *Lactobacillus paracasei* CCFM1529 fermentation supernatant, respectively. After culturing, the bacterial cells were collected by centrifugation and mixed thoroughly with the freeze-drying protectant. The freeze-drying survival rate was calculated by measuring the viable bacterial count in the bacterial suspension before and after freeze-drying (see Example 3). Figure 5 The freeze-drying protectant was an aqueous solution consisting of 50 g / L trehalose, 50 g / L sucrose, and 100 g / L skim milk powder. The freeze-drying protectant was mixed with the bacterial cells at a ratio of 2:1 v / m and then freeze-dried.

[0043] Freeze-drying process parameter settings: Pre-freezing, controlling the laminate temperature to drop from room temperature to -50℃ within 1 h and maintaining it for 4 h; First drying, adjusting the lamination temperature to -30℃ within 1.3 h and maintaining it under a vacuum of 0.2 μbar for 30 h to remove free water; Second drying, controlling the laminate temperature to rise to 25℃ within 1 h under a vacuum of 20 μbar and maintaining it for 20 h.

[0044] Result: From Figure 5 It can be seen that, compared with MRS medium, the survival rate of Bifidobacterium bifidum CCFM1167 in the supernatant of Lactobacillus paracasei CCFM1529 fermentation was significantly increased, from 12.52±0.37% to 15.05±0.90%.

[0045] Comparative Example 3: Effect of Lactobacillus paracasei fermentation supernatant CCFM1529 on the survival of Bifidobacterium bifidum freeze-dried compared to other Lactobacillus paracasei fermentation supernatants Determination of freeze-dried survival rate: The freeze-dried survival rate of Bifidobacterium cultured in fermentation supernatants of Lactobacillus paracasei CCFM1529 and other Lactobacillus paracasei was determined according to the method described in Example 4 (see Example 4). Figure 6 ).

[0046] Result: From Figure 6 It can be seen that the freeze-dried survival rate of Bifidobacterium bifidum CCFM1167 in the fermentation supernatant of Lactobacillus paracasei CCFM1529 was significantly higher than that of Lactobacillus paracasei DJXSRYG2L10 (11.19±0.48%) and Lactobacillus paracasei CCFM1224 (12.97±0.35%).

[0047] 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 paracasei ( Lacticaseibacillus paracasei The Lactobacillus paracasei CCFM1529 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67539.

2. The metabiotic prepared from *Lactobacillus paracasei* CCFM1529 according to claim 1, characterized in that, The metabiotic is obtained by fermenting the Lactobacillus paracasei CCFM1529 and collecting the fermentation supernatant.

3. The epigenetic agent according to claim 2, characterized in that, The fermentation conditions are 35~39℃, and the culture time is 20-30h.

4. The epigenetic agent according to claim 3, characterized in that, The metagener also contains glucose at a final concentration of 20-30 g / L; the pH of the metagener is 6.0-6.

1.

5. The epigenetic agent according to claim 4, characterized in that, The fermentation medium consisted 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 Tween 80, 1-2 g / L cysteine, with the remainder being water.

6. A method for promoting the growth of Bifidobacterium bifidum, characterized in that, Bifidobacterium bifidum is inoculated into the metagenerium as described in any one of claims 2 to 5 for fermentation.

7. The method according to claim 6, characterized in that, The Bifidobacterium bifidum includes Bifidobacterium bifidum CCFM1167.

8. The method according to claim 7, characterized in that, The fermentation conditions are 35~39℃, anaerobic culture.

9. A method for promoting the survival of freeze-dried Bifidobacterium bifidum, characterized in that, Bifidobacterium bifidum is inoculated into the postgenetic agent according to any one of claims 2 to 5 for fermentation, and the bacterial cells are collected and freeze-dried after fermentation; the Bifidobacterium bifidum includes Bifidobacterium bifidum CCFM1167.

10. The method according to claim 9, characterized in that, After collecting the bacterial cells, the freeze-drying protectant solution was mixed with the bacterial cells at a ratio of (2:1) to (4:1) v / m; the freeze-drying protectant solution contained 30-50 g / L trehalose, 30-50 g / L sucrose, and 100-200 g / L skim milk powder.

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