Ackerman bacterium powder and preparation method thereof

Abstract

The invention discloses Ackerman's bacteria powder and a preparation method thereof, the Ackerman's bacteria powder comprises Ackerman's bacteria and a fermentation medium of the Ackerman's bacteria, and the Ackerman's bacteria powder is prepared by inoculating the Ackerman's bacteria into the fermentation medium for culture and then directly freeze-drying; a nitrogen source of the fermentation culture medium is whey protein. Edible whey protein is adopted as a nitrogen source of a fermentation culture medium of Ackerman's bacteria, so that the fermentation culture medium of Ackerman's bacteria is an edible culture medium, Ackerman's bacteria are cultured in the culture medium and then are directly freeze-dried to prepare the Ackerman's bacteria powder, culture and thalli do not need to be separated, and the yield of the Ackerman's bacteria powder is improved. According to the method, the damage to the strain is effectively avoided, the survival rate of the thalli of the finally prepared bacterial powder is remarkably improved, the operation difficulty and bacterial contamination risk of bacterial powder preparation are reduced, and the method is suitable for industrial culture.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to Akkermansia powder and a preparation method thereof. Background Art

[0002] Akkermansia muciniphila, also known as AKK bacteria, is an oval Gram-negative bacterium isolated from human feces. It belongs to the phylum Verrucomicrobia and has unique morphology and ecological functions. In recent years, studies have found that Akkermansia has a significant role in promoting intestinal health and improving metabolic function. For example, Akkermansia can promote the proliferation of intestinal stem cells and relieve enteritis; in overweight or obese people, those with a higher abundance of Akkermansia in the intestine have a better effect on improving insulin resistance; Akkermansia can also promote mucus secretion, enhance the intestinal mucosal barrier, reduce the expression of blood lipopolysaccharide, and improve the metabolic function of mice fed a high-fat diet.

[0003] With the continuous advancement of science and technology, people's understanding of probiotics has gradually deepened, and intestinal health has received more and more attention, which has promoted the continuous expansion of the probiotic market. Probiotic products, whether used in clinical or market sales, face the important issue of storage. Only by ensuring sufficient probiotic dosage can it exert its beneficial effects on the host. Therefore, preparing probiotics into powder has become an ideal solution. This preparation method is not only convenient for storage and has high stability, but also reduces the storage volume, is easy to store and transport, and provides greater convenience for customers. Probiotic drying technology has been widely used as a simple and economical technology. The main purpose of this technology is to remove moisture from probiotic bacteria and their cultures.

[0004] The traditional preparation of probiotic powder requires centrifugation of the culture medium after bacterial culture, washing the culture medium with potential risks, and then adding a desiccant to dry and preserve it. For Akkermansia, the traditional powder preparation process will bring the risk of inactivating anaerobic bacteria, and the complicated operation is a great test for the cleanliness of the production process to avoid contamination. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an Akkermansia powder and a preparation method thereof, which can provide Akkermansia powder with a high number of viable bacteria.

[0006] The present invention provides an Akkermansia powder, which comprises Akkermansia and Akkermansia fermentation medium. The Akkermansia powder is obtained by inoculating Akkermansia into the fermentation medium for cultivation and then directly freeze-drying. The nitrogen source of the fermentation medium is whey protein.

[0007] The Akkermansia powder of the present invention adopts edible whey protein as the nitrogen source of the fermentation medium of Akkermansia, so that the fermentation medium of Akkermansia is an edible medium. After Akkermansia is cultured in the medium, the Akkermansia powder is directly freeze-dried, and the culture and the bacteria do not need to be separated, which effectively avoids the damage of the bacteria and significantly improves the survival rate of the bacteria of the finally prepared powder.

[0008] Optionally, the fermentation medium comprises: whey protein 75-250 g / L, cysteine ​​0.7-0.9 g / L, NaHCO3 3-5 g / L, KH2PO4 0.3-0.5 g / L, Na2HPO4·12H2O 1.3-1.4 g / L and glucose 10-25 g / L.

[0009] Optionally, the concentration of the whey protein is 222 g / L.

[0010] In another aspect, the present invention provides a method for preparing Akkermansia powder, which comprises:

[0011] (1) inoculating an Akkermansia strain into a seed culture medium, and after two resuscitations, inoculating the strain into the fermentation culture medium for cultivation to obtain an Akkermansia fermentation culture; the fermentation culture medium is a culture medium in a semi-solid state after sterilization;

[0012] (2) vacuum freeze-drying the Akkermansia fermentation culture to obtain the Akkermansia powder.

[0013] According to a method for preparing Akkermansia powder in an embodiment of the present invention, Akkermansia is inoculated into a semi-solid culture medium formed after sterilization for cultivation, and the obtained culture and bacterial bodies are directly subjected to vacuum freeze-drying to prepare bacterial powder. The survival rate of Akkermansia in the obtained bacterial powder is significantly improved compared with traditional separation and freeze-drying; and the operation difficulty and the risk of bacterial contamination are reduced, so that the powder is suitable for industrial cultivation.

[0014] Optionally, in step (1), the culture is anaerobic culture, the inoculation amount is 8-16%, the culture temperature is 35-38°C, the rotation speed is 90-110 rpm, and the initial pH is controlled at 7.5-7.9.

[0015] Optionally, in step (2), vacuum freeze drying is: pre-freezing the Akkermansia fermentation culture at -20°C for 3 hours, and then freeze drying it in a vacuum freeze dryer with a vacuum degree of 54 Pa for 24 hours.

[0016] Optionally, step (1) is: inoculating the Akkermansia strain into a BHI medium, culturing at 35-38° C. and 90-110 rpm for 15-24 h to obtain a primary seed solution, inoculating the primary seed solution into a BHI medium, culturing at 35-38° C. and 90-110 rpm for 15-24 h to obtain a secondary seed solution, and inoculating the secondary seed solution into the fermentation medium for culturing.

[0017] Optionally, the Akkermansia is ATCC BAA-835 Akkermansia muciniphila.

[0018] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of whey protein, whey powder and tryptone as nitrogen sources on the growth of Akkermansia.

[0020] Figure 2 The effect of different concentrations of whey protein and whey powder on the viable count of Akkermansia after culture;

[0021] Figure 3 To study the effects of freeze-drying and one-step freeze-drying on the survival rate of viable Akkermansia cells. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0023] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0024] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0025] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0026] Example 1 Cultivation of whey protein and trypsin as nitrogen sources

[0027] The culture medium with tryptone as the nitrogen source was: tryptone 37 g / L, NaHCO3 4 g / L, KH2PO4 0.4 g / L, Na2HPO4·12H2O 1.34 g / L, cysteine ​​0.4 g / L, and glucose 15 g / L.

[0028] The culture medium with whey protein or whey powder as the nitrogen source was: whey protein or whey powder 37 g / L, NaHCO3 4 g / L, KH2PO4 0.4 g / L, Na2HPO4·12H2O 1.34 g / L, cysteine ​​0.4 g / L, and glucose 15 g / L.

[0029] Akkermansia muciniphila (ATCC BAA-835) recovery medium uses BHI medium (brain heart infusion medium). The Akkermansia strain stored in cryopreserved glycerol was inoculated into a 10 mL, 37 g / L headspace bottle of BHI system at an inoculum of 1% (v / v). After culturing for 24 hours, it was inoculated into a 50 mL, 37 g / L serum bottle of BHI system at an inoculum of 4% and cultured until OD 600 =0.7, the secondary recovery seeds were obtained; 600 =0.7 of the secondary recovery seeds were inoculated into the above 50mL culture medium at an inoculum rate of 10%, and cultured anaerobically in 100mL serum bottles at 37°C and 100r / min. Samples were taken and diluted and spread for determination of viable bacterial count after 42h.

[0030] like Figure 1 As shown in Figure 2, the number of viable bacteria when tryptone was used as the nitrogen source was 1.73×10 9 CFU / mL, and the viable bacterial counts when whey protein was used as the nitrogen source were 1.36×10 9 CFU / mL, the number of viable bacteria when whey powder is used as nitrogen source is 1.42×10 9 CFU / mL.

[0031] Example 2 Optimization of concentration of whey protein and whey powder as nitrogen source

[0032] The culture medium with whey protein and whey powder as nitrogen sources, respectively, was as follows: whey protein or whey powder were optimized according to nitrogen source concentrations of 15 g / L, 37 g / L, 74 g / L, 148 g / L, and 222 g / L, NaHCO3 4 g / L, KH2PO4 0.4 g / L, Na2HPO4·12H2O 1.34 g / L, cysteine ​​0.4 g / L, and glucose 15 g / L.

[0033] The Akkermansia seed recovery procedure was the same as in Example 1. 600 =0.7 secondary recovery seeds were inoculated into 50mL culture medium of the above concentration at an inoculum size of 10%, and cultured anaerobically in 100mL shake flasks at 37°C, 100r / min, and the initial pH was controlled at 7.5-7.9. Samples were taken for dilution and smearing at 42h to determine the number of viable bacteria.

[0034] like Figure 2 As shown in the figure, when the whey powder was increased from 15 g / L to 74 g / L, the biomass of Akkermansia increased with the increase of nitrogen source concentration. When the nitrogen source concentration was higher than 74 g / L, there was no significant effect on the biomass of Akkermansia. When whey protein was used as the nitrogen source, the biomass of Akkermansia increased with the increase of nitrogen source concentration. The number of viable bacteria was the highest when the whey protein was used as the nitrogen source, reaching 2.90×10 9 CFU / mL, compared with 1.73×10 9 The CFU / mL increased by 67.6%.

[0035] Example 3 Freeze Drying Process Comparison

[0036] The bacterial solution after culture with whey protein at a concentration of 222 g / L in Example 2 was directly freeze-dried to obtain Akkermansia powder. Freeze-drying conditions: pre-freezing time 3 h, pre-freezing temperature -20°C, vacuum degree 54 Pa, freeze-drying 24 h.

[0037] Take 50 mL of Akkermansia cultured in the culture medium with tryptone as the nitrogen source in Example 1 and centrifuge at 8000 rpm for 3 min. After washing three times with 0.1 M sterilized PBS, take the bacterial sludge and mix it with edible whey protein with a concentration of 222 g / L, and freeze-dry it to obtain Akkermansia powder. Freeze-drying conditions: pre-freezing time 3 h, pre-freezing temperature -20 ° C, vacuum degree 54 pa, freeze-drying 24 h.

[0038] The survival rate of Akkermansia bacteria powder was determined by MTT method (survival rate = OD after freeze-drying). 490 / OD before freeze drying 490 × 100%). Figure 3As shown in the figure, the survival rate of Akkermansia can reach 52.81% by direct freeze drying without adding any protective agent, indicating that the whey protein culture medium has a good protective ability for Akkermansia; while the survival rate of Akkermansia cultured with tryptone, after separation and cleaning and then mixed with edible whey protein for freeze drying is only 22.75%, which is unacceptable in industry. The main reason for the low survival rate after separation and drying may be that the whey protein concentration is too high (222g / L), resulting in the inability of the bacteria to be fully mixed with the protein, most of the bacteria are not completely dispersed and exposed on the surface, and the protein does not play a protective role; after being cultured with whey protein, the bacteria have a strong adaptability to the whey protein culture medium, and direct drying plays a better protective role.

[0039] In summary, according to the embodiments of the present invention, the present application uses whey protein as a nitrogen source to culture Akkermansia, the ingredients are safe and edible, and the culture and the bacteria can be directly freeze-dried without separation, and the bacterial powder is wrapped in an edible culture medium, which greatly improves the survival rate of the bacterial powder compared to traditional separation freeze-drying.

[0040] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0041] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An Akkermansia powder, characterized in that: include: Akkermansia and fermentation medium of Akkermansia, the Akkermansia powder is obtained by inoculating the Akkermansia in the fermentation medium for cultivation and then directly freeze-drying; the nitrogen source of the fermentation medium is whey protein.

2. The Akkermansia powder according to claim 1, characterized in that The fermentation medium comprises: 75-250 g / L of whey protein, 0.7-0.9 g / L of cysteine, 3-5 g / L of NaHCO3, 0.3-0.5 g / L of KH2PO4, 1.3-1.4 g / L of Na2HPO4·12H2O and 10-25 g / L of glucose.

3. The Akkermansia powder according to claim 1 or 2, characterized in that The concentration of the whey protein is 222 g / L.

4. A method for preparing Akkermansia powder according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) inoculating an Akkermansia strain into a seed culture medium, and after two resuscitations, inoculating the strain into the fermentation culture medium for cultivation to obtain an Akkermansia fermentation culture; the fermentation culture medium is a culture medium in a semi-solid state after sterilization; (2) vacuum freeze-drying the Akkermansia fermentation culture to obtain the Akkermansia powder.

5. The preparation method according to claim 4, characterized in that: In step (1), the culture is anaerobic culture, the inoculation amount is 8-16%, the culture temperature is 35-38°C, the rotation speed is 90-110rpm, and the initial pH is controlled at 7.5-7.

9.

6. The preparation method according to claim 4, characterized in that: In step (2), vacuum freeze drying is as follows: pre-freezing the Akkermansia fermentation culture at -20°C for 3 hours, and then freeze drying it in a vacuum freeze dryer with a vacuum degree of 54 Pa for 24 hours.

7. The preparation method according to claim 4, characterized in that: Step (1) is: inoculating the Akkermansia strain into a BHI medium, culturing at 35-38° C. and 90-110 rpm for 15-24 hours to obtain a primary seed solution, taking the primary seed solution and inoculating it into a BHI medium, culturing at 35-38° C. and 90-110 rpm for 15-24 hours to obtain a secondary seed solution, and inoculating the secondary seed solution into the fermentation medium for culturing.

8. The preparation method according to claim 4, characterized in that: The Akkermansia is ATCC BAA-835 Akkermansia muciniphila.

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