Preparation method of ackermania powder

Abstract

The invention discloses a preparation method of Ackerman's bacterial powder, which comprises the following steps: inoculating Ackerman's bacterial strains into a seed culture medium, reviving, inoculating into a fermentation culture medium, and culturing to obtain a fermentation culture; centrifuging a fermentation culture, discarding supernatant, collecting thalli, washing with sterile PBS (Phosphate Buffer Solution), and resuspending; and mixing the thallus suspension with a composite freeze-drying protective additive, and performing vacuum freeze-drying to obtain ackermania powder. By optimizing a fermentation culture medium, a seed recovery process, a protective agent formula and freeze-drying conditions, the preparation method aims to improve the preparation efficiency of Ackerman's bacteria powder, and is suitable for development of probiotic preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for preparing Akkermansia powder. Background Art

[0002] Akkermansia muciniphila is a Gram-negative anaerobic bacterium belonging to the phylum Verrucomicrobia. It resides in the human intestine and utilizes intestinal mucin as its primary nutrient source. It accounts for 0.5% to 5% of the intestinal microbiota. Studies have shown that Akkermansia has the potential to contribute to maintaining intestinal health and improving metabolic function by promoting mucus secretion and enhancing intestinal mucosal barrier function. These properties make it an important candidate for the development of probiotic formulations.

[0003] In order to maintain the activity of probiotics during processing, transportation and storage, vacuum freeze-drying technology is usually used to prepare bacterial powder. This technology removes moisture through low-temperature freezing and vacuum sublimation, thereby extending the shelf life of the bacterial powder. However, the freezing and drying process may cause damage to the bacterial cell membrane or decrease metabolic activity, thereby reducing the survival rate. To alleviate this problem, the existing technology often adds protective agents such as sugars (sucrose, trehalose), amino acids (glutamic acid) or alcohols (mannitol) before freeze-drying to protect the bacteria from damage by freezing and dehydration stress. Different strains of bacteria respond differently to protective agents, and the effect of a single protective agent is limited. It is necessary to improve the survival rate through combination optimization.

[0004] There are few studies on the freeze-drying process for Akkermansia. Existing technologies usually use conventional anaerobic bacterial culture methods, such as using brain heart infusion medium (BHI) for seed recovery and fermentation, and freeze-drying with common protective agents (such as trehalose) after collecting the bacteria. These methods mostly draw on the experience of other probiotics (such as lactic acid bacteria) and lack targeted optimization of the anaerobic characteristics and cell structure of Akkermansia, resulting in a bacterial powder survival rate generally between 30% and 70%, which is difficult to meet the needs of large-scale production. In addition, the freeze-drying process parameters (such as pre-freezing and drying temperature) are mostly based on general conditions, and the special needs of Akkermansia are not fully considered, which affects the preparation efficiency and bacterial powder quality.

[0005] In industrial production, the survival rate of probiotic powder directly impacts the efficacy and production cost of the formulation. While some probiotics can achieve high survival rates under laboratory conditions, industrial production typically suffers from lower survival rates due to equipment and cost constraints. Akkermansia, as an emerging probiotic, faces challenges in preparation, including controlling culture conditions and scaling up the process. Optimized freeze-drying processes are urgently needed to improve the survival rate and stability of the powder to meet production and application requirements. Summary of the Invention

[0006] The present invention aims to overcome the defects of the prior art and provide a method for preparing Akkermansia powder.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing Akkermansia powder comprises the following steps:

[0009] (1) inoculating the Akkermansia strain into a seed culture medium for recovery, and then inoculating into a fermentation culture medium for fermentation culture to obtain an Akkermansia fermentation culture;

[0010] (2) centrifuging the Akkermansia fermentation culture obtained in step (1), discarding the supernatant, and collecting the bacterial cells;

[0011] (3) Wash the bacterial cells obtained in step (2) with sterile PBS and resuspend them with sterile PBS to obtain a bacterial suspension;

[0012] (4) mixing the bacterial suspension with a composite freeze-drying protective agent, and performing vacuum freeze-drying to obtain Akkermansia powder; wherein the composite freeze-drying protective agent includes sorbitol, sodium glutamate, and sucrose.

[0013] In a preferred embodiment of the present invention, the nitrogen source in the fermentation medium is tryptone.

[0014] Further preferably, the fermentation medium comprises: 37 g / L tryptone, 0.4 g / L L-cysteine, 1 g / L NaHCO3, 0.4 g / L KH2PO4, 1.34 g / L Na2HPO4 and 15 g / L glucose.

[0015] In a preferred embodiment of the present invention, the seed culture medium is BHI medium.

[0016] In a preferred embodiment of the present invention, in step (4), the composite protective agent consists of sorbitol, sodium glutamate and sucrose.

[0017] Further preferably, in the composite protective agent, the mass ratio of sorbitol, sodium glutamate and sucrose is 2:2:9.

[0018] More preferably, the amount of sorbitol added is 2 wt% of the bacterial suspension, the amount of sodium glutamate added is 2 wt% of the bacterial suspension, and the amount of sucrose added is 9 wt% of the bacterial suspension.

[0019] In a preferred embodiment of the present invention, in step (4), the vacuum freeze-drying comprises: pre-freezing the mixture of the bacterial suspension and the freeze-protectant at -20°C for 3 hours, and then freeze-drying at a vacuum degree of 54 Pa for 24 hours.

[0020] More preferably, the freeze-drying temperature is -50°C.

[0021] In a preferred embodiment of the present invention, the Akkermansia strain is ATCC BAA-835

[0022] The beneficial effects of the present invention are:

[0023] 1. Through optimized fermentation medium, seed recovery process, and freeze-drying process, the Akkermansia powder prepared in this invention achieves a survival rate of 93.4%, significantly higher than the 30% to 70% survival rate commonly seen in existing technologies and superior to the 19.2% achieved with conventional freeze-drying. This high survival rate ensures the activity of the powder during storage and application, providing a reliable foundation for the development of Akkermansia probiotic preparations.

[0024] 2. Through single-factor and orthogonal experiments, the present invention screened and optimized the formulation of a composite lyoprotectant, significantly improving the protective effect of the bacteria during the freeze-drying process. Compared with a single protectant alone, the survival rate of the composite protectant formulation increased from 73.3%-80.3% to 86.3%, effectively reducing the damage to Akkermansia cells caused by freezing and dehydration stress and enhancing the stability of the bacterial powder.

[0025] 3. Through experiments, the present invention determined that -50°C is the optimal freeze-drying temperature. Compared with the conventional -60°C to -80°C process, the survival rate further increased from 86.3% to 93.4%. This optimized temperature condition not only improves the survival rate of the strain but also has the potential to reduce energy consumption during the freeze-drying process, providing energy-saving potential for industrial production.

[0026] 4. Through a specific fermentation medium formulation, a two-stage seed recovery process, and a centrifugal washing step, this invention achieves high-density fermentation and efficient cell collection of Akkermansia, providing high-quality fermentation culture for subsequent freeze-drying. The coordinated optimization of each process step ensures the overall efficiency and quality stability of bacterial powder preparation.

[0027] 5. The preparation process provided by this invention achieves high survival rates and stable bacterial powder quality under laboratory conditions. The optimized process parameters have good potential for industrial scale-up. Compared with existing preparation methods that have lower survival rates and insufficient process stability, this invention provides technical support for the large-scale production of Akkermansia bacterial powder and the commercial application of probiotic preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is one of the experimental result diagrams of Example 2 of the present invention.

[0029] Figure 2This is the second diagram of the experimental results of Example 2 of the present invention.

[0030] Figure 3 This is the third figure of the experimental results of Example 2 of the present invention.

[0031] Figure 4 FIG4 is the experimental result diagram of Example 2 of the present invention.

[0032] Figure 5 This is one of the experimental result diagrams of Example 3 of the present invention.

[0033] Figure 6 This is the second diagram of the experimental results of Example 3 of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0035] Example 1 Preparation of Akkermansia cell suspension

[0036] In this embodiment, tryptone was used as the nitrogen source in the fermentation medium, and the specific formula was: tryptone 37 g / L, L-cysteine ​​0.4 g / L, NaHCO3 1 g / L, KH2PO4 0.4 g / L, Na2HPO4 1.34 g / L and glucose 15 g / L.

[0037] The recovery medium for Akkermansia ATCC BAA-835 is BHI medium (brain heart infusion medium). The Akkermansia strain stored in glycerol at low temperature was inoculated at a 1% (v / v) inoculum into a 10 mL, 37 g / L BHI system headspace bottle. After culturing for 24 hours, the inoculum was inoculated at a 4% inoculum into a 50 mL, 37 g / L BHI system serum bottle and cultured until the OD 600 =0.8-0.9, and obtain secondary recovery seeds; the secondary recovery seeds were inoculated into 50 mL of the above fermentation medium at an inoculum rate of 10%, and cultured anaerobically in 100 mL serum bottles at 37 ° C, 100 r / min, and cultured for 42 h (the number of viable Akkermansia bacteria after 42 h of fermentation was 4.18 × 10 9 After 100 CFU / mL), the suspension was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile PBS and then resuspended with 10 mL of sterile PBS to obtain an Akkermansia cell suspension.

[0038] Example 2 Optimization of Akkermansia Lyophilization Protectant

[0039] The Akkermansia cell suspension in Example 1 was mixed with different protective agents and freeze-dried to obtain Akkermansia powder. The freeze-drying conditions were as follows: pre-freezing time 3 hours, pre-freezing temperature -20°C, vacuum degree 54 Pa, and freeze-drying for 24 hours.

[0040] The survival rate of Akkermansia bacteria powder was determined by MTT method (survival rate = OD 490 / OD before freeze drying 490 Compared with the freeze-dried bacterial powder without protective agent, the survival rate of the freeze-dried bacterial powder with protective agent was significantly improved.

[0041] like Figure 1 As shown in the figure, the protective effect of sucrose with a sugar content of 6% was the best, and the survival rate of the freeze-dried bacterial powder reached 80.3%, which was 60.9% higher than that of the freeze-dried bacterial powder without the addition of protective agent.

[0042] like Figure 2 、 3 As shown in the figure, the amino acid protective agent with a 3% sodium glutamate addition had the best protective effect, and the survival rate of the freeze-dried bacterial powder reached 73.3%, which was 54.2% higher than that of the freeze-dried bacterial powder without the protective agent.

[0043] like Figure 4 As shown, the protective effect of 3% sorbitol added to the alcohol protective agent is the best, and the survival rate of the freeze-dried bacterial powder reaches 74.3%, which is 55.8% higher than that of the freeze-dried bacterial powder without protective agent.

[0044] To further explore the effect of a composite lyoprotectant on the survival rate of Akkermansia, this example used sucrose, sodium glutamate, and sorbitol, selected from the single-factor experiments as shown in Table 1, as test materials. An orthogonal experiment was performed to investigate the effects of different concentrations of the composite protectant on the survival rate of the strain. The results are shown in Table 2. The optimal composite protectant formula was 2% sorbitol, 2% sodium glutamate, and 9% sucrose, i.e., the optimal protectant combination was A2B2C2. The freeze-dried bacterial powder survival rate reached 86.3%, a result higher than that of any single protectant and a 67.1% increase compared to the control group. Recent studies have shown that by optimizing the protectant formula and freeze-drying process, the freeze-dried survival rate of Akkermansia can reach 70% to 90%, indicating that the composite lyoprotectant with this ratio has a very good protective effect on Akkermansia. An analysis of variance analysis of the orthogonal experiment results showed that all three protectants had a significant effect on the survival rate of freeze-dried Akkermansia powder (p < 0.05).

[0045] Table 1

[0046]

[0047] Table 2

[0048]

[0049] Example 3 Vacuum freeze-drying process optimization

[0050] In order to further improve the freeze-dried survival rate of Akkermansia, this example optimizes the freezing temperature. The Akkermansia suspension after fermentation and cleaning is mixed evenly with the optimal combination of protective agents, pre-frozen at -20°C for 3 hours, and then frozen at -50°C, -60°C, -70°C, and -80°C for 24 hours. The results are shown in Figure 2. Figure 5 As shown, freeze-drying at -50°C has the highest survival rate, reaching 93.4%, an 8.2% increase compared to conventional freeze-drying in Example 2. Currently, the survival rate of probiotics after freeze-drying varies significantly depending on the strain, preservative formulation, and process conditions. Some probiotics, such as Lactobacillus plantarum, can achieve a survival rate of 90%-95% under optimized conditions. However, due to production costs and process stability, it is generally difficult to achieve the optimal laboratory level on an industrial scale. The freeze-dried survival rate of most commercial probiotic preparations is between 30% and 70%.

[0051] The survival rates of the strains in three processes were compared and analyzed: conventional freeze drying without adding a protective agent (control group), conventional freeze drying with the addition of the composite protective agent obtained by optimization in Example 2, and optimized freeze temperature drying (protective agent + freezing temperature group) with the addition of the composite protective agent obtained by optimization in Example 2 (protective agent group). Figure 6 As shown, the survival rate of the control group was only 19.2%, while that of the protectant group increased by 67.1% compared with the control group, and finally increased to 93.4% after further temperature optimization. It can be seen that the present invention provides a method for preparing Akkermansia powder with high survival rate.

[0052] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing Akkermansia powder, characterized in that: The following steps are involved: (1) inoculating the Akkermansia strain into a seed culture medium for recovery, and then inoculating into a fermentation culture medium for fermentation culture to obtain an Akkermansia fermentation culture; (2) centrifuging the Akkermansia fermentation culture obtained in step (1), discarding the supernatant, and collecting the bacterial cells; (3) Wash the bacterial cells obtained in step (2) with sterile PBS and resuspend them with sterile PBS to obtain a bacterial suspension; (4) mixing the bacterial suspension with a composite freeze-drying protective agent, and performing vacuum freeze-drying to obtain Akkermansia powder; wherein the composite freeze-drying protective agent includes sorbitol, sodium glutamate, and sucrose.

2. The preparation method according to claim 1, wherein: The nitrogen source in the fermentation medium is tryptone.

3. The preparation method according to claim 2, wherein: The fermentation medium comprises 37 g / L tryptone, 0.4 g / L L-cysteine, 1 g / L NaHCO3, 0.4 g / L KH2PO4, 1.34 g / L Na2HPO4 and 15 g / L glucose.

4. The preparation method according to claim 1, wherein: The seed culture medium is BHI medium.

5. The preparation method according to claim 1, wherein: In the step (4), the composite protective agent consists of sorbitol, sodium glutamate and sucrose.

6. The preparation method according to claim 5, wherein: In the composite protective agent, the mass ratio of sorbitol, sodium glutamate and sucrose is 2:2:

9.

7. The preparation method according to claim 6, wherein: The amount of sorbitol added is 2 wt% of the bacterial suspension, the amount of sodium glutamate added is 2 wt% of the bacterial suspension, and the amount of sucrose added is 9 wt% of the bacterial suspension.

8. The preparation method according to claim 1, wherein: In step (4), the vacuum freeze drying comprises: pre-freezing the mixture of the bacterial suspension and the freeze-drying protectant at -20°C for 3 hours, and then freeze-drying at a vacuum degree of 54 Pa for 24 hours.

9. The preparation method according to claim 8, wherein: The freeze-drying temperature is -50°C.

10. The preparation method according to any one of claims 1 to 9, characterized in that: The Akkermansia strain is ATCC BAA-835.

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