Co-culture method of Ackerman's bacteria and animal bifidobacterium lactis

Abstract

The invention discloses a co-culture method of Ackerman's bacteria and animal bifidobacterium lactis. The co-culture method comprises the following steps: (1) carrying out recovery culture on Ackerman's bacteria twice by using a BHI culture medium; (2) resuscitating the animal bifidobacterium lactis BB-12 twice by using an MRS culture medium to obtain an animal bifidobacterium lactis culture; and (3) respectively and sequentially centrifuging and washing cultures obtained in the step (1) and the step (2) with sterile PBS, and simultaneously inoculating the cultures into a whey protein hydrolysate culture medium for anaerobic culture. According to the method disclosed by the invention, ackermania and bifidobacterium lactis are co-cultured, so that lactose in a culture medium is fully utilized, and meanwhile, the product has the functions of two strains.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and particularly relates to a co-culture method of Akkermansia and animal lactic bifidobacterium. Background Art

[0002] Akkermansia muciniphila, or Akk bacteria for short, is an intestinal bacterium belonging to the phylum Verrucomicrobia. It is an oval Gram-negative bacterium isolated from human feces. Akkermansia can colonize in the intestinal mucosa, feed on mucin, and account for 0.5-5% of the intestinal flora. Although it was originally considered to be an ordinary commensal bacterium, recent studies have found that Akkermansia has a variety of health benefits, especially in promoting intestinal health and improving metabolic function.

[0003] For example, Akkermansia can promote the proliferation of intestinal stem cells, thereby alleviating enteritis. In overweight or obese people, those with a higher abundance of Akkermansia in the intestines tend to have a better effect in improving insulin resistance. In addition, Akkermansia can promote mucus secretion, enhance the intestinal mucosal barrier, and reduce the level of lipopolysaccharide in the blood. Its reduction may disrupt the intestinal barrier function, leading to increased endotoxin levels, which in turn triggers low-grade inflammation and metabolic disorders.

[0004] The functions of fermentation products of a single strain of Akkermansia are relatively simple, mainly focusing on improving intestinal health and metabolic function, and cannot meet people's demand for diversified functions of probiotic products. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art and provide a method for co-culturing Akkermansia and Bifidobacterium lactis.

[0006] The technical solution of the present invention is as follows:

[0007] A method for co-culturing Akkermansia and Bifidobacterium lactis, comprising the following steps:

[0008] (1) resuscitating Akkermansia twice using BHI medium to obtain Akkermansia culture;

[0009] (2) resuscitating animal lactic acid Bifidobacterium twice with MRS culture medium to obtain animal lactic acid Bifidobacterium culture;

[0010] (3) the Akkermansia culture obtained in step (1) and the Bifidobacterium animalis culture obtained in step (2) were centrifuged and washed with sterile PBS, respectively, and then inoculated into a whey protein hydrolyzate culture medium, and anaerobically cultured at a temperature of 37° C. and a rotation speed of 100 r / min;

[0011] The formula of the hydrolyzed whey protein medium is as follows: whey protein 222 g / L, L-cysteine 0.4 g / L, NaHCO 3 4 g / L, KH 2 PO 4 0.4 g / L, Na 2 HPO 4 1.34 g / L, and glucose 15 g / L.

[0012] In a preferred embodiment of the present invention, step (1) includes: inoculating Akkermansia muciniphila strains cryopreserved with glycerol into 10 mL of 37 g / L BHI medium at an inoculation amount of 1% (v / v), culturing for 15 - 17 h, and then inoculating into 50 mL of 37 g / L BHI medium at an inoculation amount of 4%, and culturing until OD600 = 0.8 - 0.9 to obtain secondary resuscitation seeds, namely the Akkermansia muciniphila culture.

[0013] In a preferred embodiment of the present invention, step (2) includes: inoculating Bifidobacterium animalis subsp. lactis strains cryopreserved with glycerol into 10 mL of MRS medium at an inoculation amount of 1% (v / v), culturing for 15 - 17 h, and then inoculating into 50 mL of MRS medium at an inoculation amount of 4%, and culturing until OD 600 = 0.8 - 0.9 to obtain secondary resuscitation seeds, namely the Bifidobacterium animalis subsp. lactis culture.

[0014] In a preferred embodiment of the present invention, in step (3), the centrifugation speed is 8000 r / min and the time is 4 min.

[0015] In a preferred embodiment of the present invention, in step (3), the volume ratio of the Akkermansia muciniphila culture / the Bifidobacterium animalis subsp. lactis culture to the hydrolyzed whey protein medium is 4:50.

[0016] In a preferred embodiment of the present invention, the Akkermansia muciniphila is Akkermansia muciniphila ATCC BAA - 835, and the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis BB - 12.

[0017] Use of the hydrolyzed whey protein medium in the co - culture of Akkermansia muciniphila and Bifidobacterium animalis subsp. lactis. The formula of the hydrolyzed whey protein medium is as follows: whey protein 222 g / L, L - cysteine 0.4 g / L, NaHCO 3 4 g / L, KH 2 PO 4 0.4 g / L, Na 2 HPO 4 1.34 g / L, and glucose 15 g / L.

[0018] In a preferred embodiment of the present invention, the Akkermansia is Akkermansia muciniphila ATCC BAA-835.

[0019] In a preferred embodiment of the present invention, the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis BB-12.

[0020] In a preferred embodiment of the present invention, the Akkermansia is Akkermansia muciniphila ATCC BAA-835, and the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis BB-12.

[0021] The beneficial effects of the present invention are as follows: The present invention uses a co-culture method of Akkermansia and Bifidobacterium animalis subsp. lactis, enabling the full utilization of lactose in the culture medium and endowing the product with the functions of both strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Showing the curve of the viable count of Akkermansia muciniphila cultured alone in the hydrolyzed whey protein medium in Example 1 of the present invention over time, as well as the curves of the glucose and lactose content changes.

[0023] Figure 2 Showing the curve of the viable count of Bifidobacterium animalis subsp. lactis BB-12 cultured alone in the hydrolyzed whey protein medium in Example 2 of the present invention over time, as well as the curves of the glucose and lactose content changes.

[0024] Figure 3 Showing the curve of the viable count of Akkermansia muciniphila and Bifidobacterium animalis subsp. lactis BB-12 co-cultured in the hydrolyzed whey protein medium in Example 3 of the present invention over time, as well as the curves of the glucose and lactose content changes.

[0025] Figure 4 Showing the organic acid content and the inhibitory effect on the growth of Escherichia coli when the hydrolyzed whey protein medium in Example 4 of the present invention is used to culture Akkermansia muciniphila, Bifidobacterium animalis subsp. lactis BB-12, and co-culture Akkermansia muciniphila and Bifidobacterium animalis subsp. lactis BB-12. DETAILED DESCRIPTION OF THE INVENTION

[0026] The technical solutions of the present invention will be further described and illustrated below through specific embodiments in conjunction with the drawings.

[0027] The formula of the hydrolyzed whey protein medium in the following examples is: whey protein 222 g / L, L-cysteine 0.4 g / L, NaHCO 3 4 g / L, KH 2 PO 4 0.4 g / L, Na 2 HPO 41.34 g / L and 15 g / L of glucose.

[0028] Example 1: Akkermansia muciniphila was cultured alone in a hydrolyzed whey protein medium

[0029] The resuscitation medium for Akkermansia muciniphila (ATCC BAA - 835) was BHI medium (brain heart infusion medium). According to an inoculation amount of 1% (v / v), the Akkermansia muciniphila strain preserved at low temperature with glycerol was inoculated into a headspace bottle containing 10 mL of 37 g / L BHI. After culturing for 15 - 17 h, it was then inoculated into a serum bottle containing 50 mL of 37 g / L BHI according to an inoculation amount of 4%. After culturing until OD 600 = 0.8 - 0.9, a secondary resuscitation seed was obtained. Take 4 mL of the secondary resuscitation seed with OD 600 = 0.8 - 0.9, centrifuge at 8000 r / min for 4 min, discard the supernatant, wash the bacterial cells twice with sterile PBS, and then inoculate them into the above 50 mL hydrolyzed whey protein medium for anaerobic culture in a 100 mL serum bottle. The culture conditions were 37 °C and 100 r / min. Samples were taken regularly for dilution coating to determine the viable cell count, and high - performance liquid chromatography was used to measure the consumption of glucose and lactose.

[0030] As Figure 1 shown, the curve of the viable cell count of Akkermansia muciniphila cultured alone in the hydrolyzed whey protein medium and the curves of the changes in glucose and lactose contents showed that the viable cell count of Akkermansia muciniphila reached 3.25×10 9 CFU / mL at 42 h, lactose was almost completely consumed at 28 h, and glucose was almost completely consumed at 36 h.

[0031] Example 2: Bifidobacterium animalis subsp. lactis BB - 12 was cultured alone in a hydrolyzed whey protein medium

[0032] The resuscitation medium for Bifidobacterium animalis subsp. lactis was MRS medium. According to an inoculation amount of 1% (v / v), Bifidobacterium animalis subsp. lactis BB - 12 preserved at low temperature with glycerol was inoculated into a headspace bottle containing 10 mL of MRS. After culturing for 15 - 17 h, it was then inoculated into a serum bottle containing 50 mL of MRS according to an inoculation amount of 4%. After culturing until OD 600 = 0.8 - 0.9, a secondary resuscitation seed was obtained. Take 4 mL of OD 600Secondary resuscitation seeds with an OD of 0.8 - 0.9 were centrifuged at 8000 r / min for 4 min, the supernatant was discarded, and the cells were washed twice with sterile PBS and then inoculated into the above-mentioned 50 mL hydrolyzed whey protein medium. Anaerobic culture was carried out in a 100 mL serum bottle at 37 °C and 100 r / min. Samples were taken regularly for dilution plating to determine the viable cell count, and high-performance liquid chromatography was used to measure the consumption of glucose and lactose.

[0033] As Figure 2 shown, the curve of the viable cell count of Bifidobacterium animalis subsp. lactis BB - 12 and the curves of the changes in glucose and lactose contents during the separate culture of Bifidobacterium animalis subsp. lactis BB - 12 in the hydrolyzed whey protein medium showed that the viable cell count of Bifidobacterium animalis subsp. lactis BB - 12 reached 2.40×10 9 CFU / mL at 16 h, lactose was almost completely consumed at 16 h, and glucose was almost completely consumed at 36 h.

[0034] Example 3 Co - culture of Akkermansia muciniphila and Bifidobacterium animalis subsp. lactis BB - 12 in the hydrolyzed whey protein medium

[0035] Akkermansia muciniphila (ATCC BAA - 835) and Bifidobacterium animalis subsp. lactis BB - 12 were each resuscitated twice and cultured until OD 600 = 0.8 - 0.9 to obtain secondary resuscitation seeds (refer to Example 1 and Example 2). 4 mL of the secondary resuscitation seeds of Akkermansia muciniphila and Bifidobacterium animalis subsp. lactis BB - 12 were taken respectively, centrifuged at 8000 r / min for 4 min, the supernatant was discarded, and after washing twice with sterile PBS, they were simultaneously inoculated into the above-mentioned 50 mL hydrolyzed whey protein medium. Anaerobic culture was carried out in a 100 mL serum bottle at 37 °C and 100 r / min. Samples were taken regularly for dilution plating to determine the viable cell count, and high-performance liquid chromatography was used to measure the consumption of glucose and lactose.

[0036] As Figure 3 shown, the curve of the viable cell count of the co - culture of Akkermansia muciniphila and Bifidobacterium animalis subsp. lactis BB - 12 and the curves of the changes in glucose and lactose contents in the hydrolyzed whey protein medium showed that the total viable cell count was 4.20×10 9 CFU / mL at 16 h, of which the viable cell count of Akkermansia muciniphila was 2.00×10 9 CFU / mL, and the viable cell count of Bifidobacterium animalis subsp. lactis was 2.20×10 9 CFU / mL. Lactose was almost completely consumed at 12 h, and glucose was almost completely consumed at 36 h.

[0037] Example 4 Inhibitory effect of the supernatant of the fermentation broth obtained in Examples 1 to 3 on Escherichia coli

[0038] Collect the fermentation broth obtained from Example 1, Example 2, and Example 3 after 50 h of fermentation. Add three times the volume of sterile PBS, centrifuge at 10,000 r / min for 10 min, take the supernatant, filter and sterilize it with a sterile filter membrane to obtain a sterile supernatant. Then, take 10 mL of the sterile supernatant corresponding to the fermentation broth of Examples 1 to 3 into a shake flask, add Escherichia coli cultured overnight, and dilute the Escherichia coli with sterile PBS to OD 600 = 0.8 before adding, and measure the growth of Escherichia coli every 2 h.

[0039] As Figure 4 shown, the sterile supernatants of the fermentation broths of the two strains cultured separately (Example 1 and 2, corresponding to the diluted Akk fermentation broth and the BB-12 fermentation broth in Figure 4 respectively) and co-cultured (Example 3, corresponding to the co-cultured fermentation broth in Figure 4 ) all have an inhibitory effect on Escherichia coli, and the inhibitory effect is higher than that of the blank medium supernatant containing 0.1% potassium sorbate (corresponding to the blank medium + 0.1% potassium sorbate in Figure 4 ). Among them, the sterile supernatant of the fermentation broth of Example 3 has the best inhibitory effect.

[0040] The above is only a preferred embodiment of the present invention, and thus the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A method for co-culturing Akkermansia and Bifidobacterium lactis, characterized in that: The steps include: (1) resuscitating Akkermansia twice using BHI medium to obtain Akkermansia culture; (2) resuscitating animal lactic acid Bifidobacterium twice with MRS culture medium to obtain animal lactic acid Bifidobacterium culture; (3) the Akkermansia culture obtained in step (1) and the Bifidobacterium animalis culture obtained in step (2) were centrifuged and washed with sterile PBS, respectively, and then inoculated into a whey protein hydrolyzate culture medium, and anaerobically cultured at a temperature of 37° C. and a rotation speed of 100 r / min; The formula of the hydrolyzed whey protein culture medium is: whey protein 222g / L, L-cysteine ​​0.4g / L, NaHCO3 4g / L, KH2PO4 0.4g / L, Na2HPO4 1.34g / L and glucose 15g / L.

2. The co-cultivation method according to claim 1, characterized in that: The step (1) comprises: inoculating the Akkermansia strain stored in cryopreserved glycerol at a 1% (v / v) inoculum into 10 mL 37 g / L BHI medium, culturing for 15-17 hours, and then inoculating into 50 mL 37 g / L BHI medium at a 4% inoculum, and culturing until the OD 600 =0.8-0.9, the secondary recovery seed, i.e. the Akkermansia culture, is obtained.

3. The co-cultivation method according to claim 1, characterized in that: The step (2) comprises: inoculating the animal lactobacillus preserved in glycerol cryogenic temperature into 10 mL MRS medium at an inoculation volume of 1% (v / v), culturing for 15-17 hours, and then inoculating into 50 mL MRS medium at an inoculation volume of 4%, and culturing until OD 600 =0.8-0.9, the secondary recovery seed is obtained, that is, the animal lactic Bifidobacterium culture.

4. The co-cultivation method according to claim 1, characterized in that: In the step (3), the centrifugal speed is 8000 r / min and the time is 4 min.

5. The co-cultivation method according to claim 1, characterized in that: In the step (3), the volume ratio of the Akkermansia culture / the Bifidobacterium lactis culture and the whey protein hydrolyzate culture medium is 4:

50.

6. The co-cultivation method according to any one of claims 1 to 5, characterized in that: The Akkermansia is Akkermansia ATCC BAA-835, and the Bifidobacterium lactis is Bifidobacterium lactis BB-12.

7. Use of a whey protein hydrolyzate culture medium in co-culturing Akkermansia and Bifidobacterium of animal lactis, characterized in that: The formula of the hydrolyzed whey protein culture medium is: whey protein 222g / L, L-cysteine ​​0.4g / L, NaHCO3 4g / L, KH2PO4 0.4g / L, Na2HPO4 1.34g / L and glucose 15g / L.

8. The use according to claim 7, characterized in that: The Akkermansia is Akkermansia ATCC BAA-835.

9. The use according to claim 7, characterized in that: The animal lactic acid Bifidobacterium is animal lactic acid Bifidobacterium BB-12.

10. The use according to claim 7, characterized in that: The Akkermansia is Akkermansia ATCC BAA-835, and the Bifidobacterium lactis is Bifidobacterium lactis BB-12.

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