Application of lactose-N-neotetraose in AKK bacterium culture, culture medium and method

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of lactose-N-neotetraose in AKK bacterium culture, a culture medium and a method. According to the invention, the lactose-N-neotetraose is used as a key nutrient source of an AKK bacterium culture medium, and the use concentration of the lactose-N-neotetraose is not lower than 0g / L, preferably 0.44-8.8 g / L, and more preferably 4-6.6 g / L. The media may be made based on a base formulation or an improved MRS formulation with addition of lactose-N-neotetraose. Experiments show that lactose-N-neotetraose can be efficiently utilized by AKK bacteria, has adaptability to strains such as AKKPROBIO, AKK ONE and the like, can remarkably improve the growth amount and metabolic activity of thalli in two culture media, has the effect superior to that of a conventional mucoprotein culture medium, is definite in component, controllable in cost and high in safety, can realize efficient, stable and large-scale culture of AKK bacteria, and has a wide application prospect. And the support is provided for the application in the fields of healthy food and clinic.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to application of lactose-N-neotetraose in culture of AKK bacteria, culture medium and method. BACKGROUND

[0002] In the human intestinal microecosystem, Akkermansia muciniphila is a gram-negative anaerobic bacterium closely related to host health. In recent years, it has become a research hotspot in the fields of microbiology, medicine and nutrition because of its important physiological activities such as degradation of mucus layer mucin, regulation of intestinal barrier function, improvement of metabolic disorders and inhibition of inflammatory response. A large number of studies have shown that the abundance change of the strain is significantly associated with the occurrence and development of obesity, type 2 diabetes, inflammatory bowel disease and other diseases. Its potential probiotic function shows broad application prospects in disease prevention and treatment, development of health food and other aspects. However, in vitro culture of Akkermansia muciniphila has been a key bottleneck restricting its research and application. The strain is a typical mucus-dependent bacterium, and its growth and reproduction is highly dependent on specific sugar chain structures in mucin as carbon source and energy source. In the traditional culture system, natural mucin such as pig stomach mucin is often used as substrate, but this kind of natural product has problems such as complex composition, large batch difference, low purity and containing unknown impurities, which not only interferes with the growth stability of the strain and the analysis of metabolic products, but also may limit its application in clinical and food fields due to carrying foreign antigens or pathogen-associated molecules. In addition, the extraction process of natural mucin is complicated and costly, which is difficult to meet the demand of large-scale culture, and seriously hinders the industrialization process of Akkermansia muciniphila.

[0003] The chemical structure of lactose-N-neotetraose is Galβ1-4GlcNAcβ1-3Galβ1-4Glc (2-(β-D-galactosyl)-(1→4)-N-acetyl-β-D-glucosaminyl-(1→3)-2-(β-D-galactosyl)-(1→4)-D-glucose), which contains a repeating disaccharide unit (Galβ1-4GlcNAc) similar to the mucin sugar chain, can be recognized and degraded by specific glycoside hydrolases of Akkermansia muciniphila, thereby providing sustained energy and material sources for the growth of the strain. Compared with natural mucin, lactose-N-neotetraose has the characteristics of single composition, high purity and clear structure, which can effectively avoid the interference of impurities in natural substrate on strain metabolism, and is conducive to the establishment of standardized culture system; at the same time, its clear chemical structure facilitates the study of the utilization mechanism of the strain to specific sugar chains, and provides an ideal tool for analyzing the interaction between Akkermansia muciniphila and host mucus layer.

[0004] At present, there is no report of a mucinophilic Akkermansia muciniphila special medium using lacto-N-neotetraose as a key raw material in the prior art, and the development of such a medium has important theoretical significance and practical application value. SUMMARY

[0005] In view of the above problems, the present application provides a culture medium for culturing mucinophilic Akkermansia muciniphila, which uses lacto-N-neotetraose as a key nutrient source, realizes efficient and stable culture of the strain, and solves the multiple technical pain points of the traditional culture scheme.

[0006] The technical scheme of the present application is as follows: On the one hand, the present application provides the use of lacto-N-neotetraose in AKK culture, and the lacto-N-neotetraose is used as a nutrient source in the culture medium.

[0007] Specifically, the use concentration of the lacto-N-neotetraose is not less than 0 g / L.

[0008] Preferably, the use concentration of the lacto-N-neotetraose is not less than 0.044 g / L.

[0009] Preferably, the use concentration of the lacto-N-neotetraose is 0.044-8.8 g / L, specifically 0.044 g / L, 0.44 g / L, 0.45 g / L, 0.46 g / L, 0.47 g / L, 048 g / L, 0.49 g / L, 0.5 g / L, 0.51 g / L, 0.52 g / L, 0.53 g / L, 0.54 g / L, 0.55 g / L, 0.56 g / L, 0.57 g / L, 0.58 g / L, 0.59 g / L, 0.6 g / L, 0.61 g / L, 0.62 g / L, 0.63 g / L, 0.64 g / L, 0.65 g / L, 0.66 g / L, 0.67 g / L, 0.68 g / L, 0.69 g / L, 0.7 g / L, 0.71 g / L, 0.72 g / L, 0.73 g / L, 0.74 g / L, 0.75 g / L, 0.76 g / L, 0.77 g / L, 0.78 g / L, 0.79 g / L, 0.8 g / L, 0.81 g / L, 0.82 g / L, 0.83 g / L, 0.84 g / L, 0.85 g / L, 0.86 g / L, 0.87 g / L, 0.88 g / L, 8.8 g / L, and any point value within the range, as well as a range value between any two points.

[0010] Preferably, the use concentration of the lacto-N-neotetraose is 2-8.8 g / L.

[0011] Preferably, the use concentration of the lacto-N-neotetraose is 2.5-8.8 g / L.

[0012] Preferably, the concentration of the lacto-N-neotetraose used is 3-8.8 g / L.

[0013] Preferably, the concentration of the lacto-N-neotetraose used is 3.5-8.8 g / L.

[0014] Preferably, the concentration of the lacto-N-neotetraose used is 4-8.8 g / L.

[0015] Preferably, the concentration of the lacto-N-neotetraose used is 4-8 g / L.

[0016] Preferably, the concentration of the lacto-N-neotetraose used is 4-7.5 g / L.

[0017] Preferably, the concentration of the lacto-N-neotetraose used is 4-7 g / L.

[0018] Preferably, the concentration of the lacto-N-neotetraose used is 4-6.6 g / L.

[0019] In another aspect, the present application provides a culture medium for culturing AKK bacteria, wherein the culture medium comprises lacto-N-neotetraose.

[0020] Specifically, the concentration of the lacto-N-neotetraose is not less than 0 g / L.

[0021] Preferably, the concentration of the lacto-N-neotetraose used is not less than 0.044 g / L.

[0022] Preferably, the concentration of the lacto-N-neotetraose used is 0.044-8.8 g / L, specifically 0.044 g / L, 0.44 g / L, 0.45 g / L, 0.46 g / L, 0.47 g / L, 048 g / L, 0.49 g / L, 0.5 g / L, 0.51 g / L, 0.52 g / L, 0.53 g / L, 0.54 g / L, 0.55 g / L, 0.56 g / L, 0.57 g / L, 0.58 g / L, 0.59 g / L, 0.6 g / L, 0.61 g / L, 0.62 g / L, 0.63 g / L, 0.64 g / L, 0.65 g / L, 0.66 g / L, 0.67 g / L, 0.68 g / L, 0.69 g / L, 0.7 g / L, 0.71 g / L, 0.72 g / L, 0.73 g / L, 0.74 g / L, 0.75 g / L, 0.76 g / L, 0.77 g / L, 0.78 g / L, 0.79 g / L, 0.8 g / L, 0.81 g / L, 0.82 g / L, 0.83 g / L, 0.84 g / L, 0.85 g / L, 0.86 g / L, 0.87 g / L, 0.88 g / L, 8.8 g / L and any point value within the range, and a range value between any two points.

[0023] Preferably, the concentration of the lacto-N-neotetraose used is 2-8.8 g / L.

[0024] Preferably, the concentration of the lacto-N-neotetraose used is 2.5-8.8 g / L.

[0025] Preferably, the concentration of the lacto-N-neotetraose used is 3-8.8 g / L.

[0026] Preferably, the concentration of the lacto-N-neotetraose used is 3.5-8.8 g / L.

[0027] Preferably, the concentration of the lacto-N-neotetraose used is 4-8.8 g / L.

[0028] Preferably, the concentration of the lacto-N-neotetraose used is 4-8 g / L.

[0029] Preferably, the concentration of the lacto-N-neotetraose used is 4-7.5 g / L.

[0030] Preferably, the concentration of the lacto-N-neotetraose used is 4-7 g / L.

[0031] Preferably, the concentration of the lacto-N-neotetraose used is 4-6.6 g / L.

[0032] In particular, the culture medium further comprises glucose, threonine, yeast extract, manganese sulfate, magnesium chloride hexahydrate, sodium chloride, sodium bicarbonate, sodium acetate and potassium dihydrogen phosphate.

[0033] Preferably, the culture medium further comprises glucose 5 g / L, threonine 4 g / L, yeast extract 30 g / L, manganese sulfate 0.02 g / L, magnesium chloride hexahydrate 0.2 g / L, sodium chloride 0.5 g / L, sodium bicarbonate 1 g / L, sodium acetate 2 g / L and potassium dihydrogen phosphate 0.4 g / L; or the culture medium further comprises peptone, beef infusion powder, yeast extract, glucose, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, Tween and biotin.

[0034] In another aspect, the present application provides a method for culturing AKK bacteria, comprising inoculating AKK bacteria into a culture medium containing lacto-N-neotetraose for culturing.

[0035] In particular, the concentration of the lacto-N-neotetraose used is not less than 0 g / L.

[0036] Preferably, the concentration of the lacto-N-neotetraose used is not less than 0.044 g / L.

[0037] Preferably, the concentration of the lacto-N-neotetraose used is 0.044-8.8 g / L, specifically 0.044 g / L, 0.44 g / L, 0.45 g / L, 0.46 g / L, 0.47 g / L, 0.48 g / L, 0.49 g / L, 0.5 g / L, 0.51 g / L, 0.52 g / L, 0.53 g / L, 0.54 g / L, 0.55 g / L, 0.56 g / L, 0.57 g / L, 0.58 g / L, 0.59 g / L, 0.6 g / L, 0.61 g / L, 0.62 g / L, 0.63 g / L, 0.64 g / L, 0.65 g / L, 0.66 g / L, 0.67 g / L, 0.68 g / L, 0.69 g / L, 0.7 g / L, 0.71 g / L, 0.72 g / L, 0.73 g / L, 0.74 g / L, 0.75 g / L, 0.76 g / L, 0.77 g / L, 0.78 g / L, 0.79 g / L, 0.8 g / L, 0.81 g / L, 0.82 g / L, 0.83 g / L, 0.84 g / L, 0.85 g / L, 0.86 g / L, 0.87 g / L, 0.88 g / L, 8.8 g / L, and any point value and range value within the range.

[0038] Preferably, the concentration of the lacto-N-neotetraose used is 2-8.8 g / L.

[0039] Preferably, the concentration of the lacto-N-neotetraose used is 2.5-8.8 g / L.

[0040] Preferably, the concentration of the lacto-N-neotetraose used is 3-8.8 g / L.

[0041] Preferably, the concentration of the lacto-N-neotetraose used is 3.5-8.8 g / L.

[0042] Preferably, the concentration of the lacto-N-neotetraose used is 4-8.8 g / L.

[0043] Preferably, the concentration of the lacto-N-neotetraose used is 4-8 g / L.

[0044] Preferably, the concentration of the lacto-N-neotetraose used is 4-7.5 g / L.

[0045] Preferably, the concentration of the lacto-N-neotetraose used is 4-7 g / L.

[0046] Preferably, the concentration of the lacto-N-neotetraose used is 4-6.6 g / L.

[0047] The present application has the beneficial effects of providing a natural source of nutrients (lacto-N-neotetraose) for the large-scale production of Akkermansia muciniphila (AKK), promoting clean production of AKK bacteria, and providing a feasible low-cost raw material for the large-scale production of AKK bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0048] Fig. 1 The bacterial morphology in the medium of Example 7 was observed under a professional microscope for microorganisms with an objective lens of 10 times and an oil lens (with cedar oil added) of 100 times.

[0049] Fig. 2 The bacterial morphology in the medium of the conventional control group was observed. DETAILED DESCRIPTION

[0050] The present application will be further clarified by the following examples, which are only a part of the present application and are not used to limit the present application, but only to illustrate the present application. The experimental methods used in the following examples are conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0051] EXAMPLE 1.1 Strain activation The frozen bacteria solution of Akkermansia muciniphila (strain numbers AKK PROBIO (CGMCC No. 20955, disclosed in CN116898883A) and AKK ONE (CGMCC 20954, disclosed in CN118286268B) from Shengenkang Company was inoculated into a conventional medium at a volume ratio of 1:10 under sterile operation conditions, and cultured at 37°C in an anaerobic environment for 48h until the bacterial solution was in the logarithmic growth phase, which was used as a seed solution for experiments.

[0052] 1.2 Medium preparation The base medium was prepared by accurately weighing each ingredient (glucose 5g / L, threonine 4g / L, yeast extract 30g / L, manganese sulfate 0.02g / L, magnesium chloride hexahydrate 0.2g / L, sodium chloride 0.5g / L, sodium bicarbonate 1g / L, sodium acetate 2g / L, potassium dihydrogen phosphate 0.4g / L) according to the formula, adding an appropriate amount of distilled water, and stirring until completely dissolved. Different concentrations of lacto-N-neotetraose (see Table 1: Examples 1-12) were added, the pH value of the medium was adjusted to 7.5-7.6, three replicates were set, and then high-pressure steam sterilization (115°C, 30min) was performed. After sterilization, the medium was cooled to room temperature for standby, and the AKK PROBIO strain was inoculated for culture study. Modified MRS medium: proteose peptone 10 g / L, beef infusion powder 8 g / L, yeast extract 8 g / L, glucose 20 g / L, potassium phosphate dibasic 2 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.05 g / L, Tween 80 1.0 g / L, biotin 0.5 g / L, add an appropriate amount of distilled water to constant volume and completely dissolve. Add different lacto-N-neotetraose (see Table 1: Examples 13-14) respectively, adjust the pH of the medium to 6.8-7.0, sterilize at 115°C for 30 minutes, cool to room temperature, and then inoculate AKK ONE strain for culture study.

[0053] Blank control group medium: basic medium formula (without lacto-N-neotetraose), prepare two, inoculate AKKPROBIO strain (blank control group-AKKPROBIO) and AKK ONE strain (blank control group-AKK ONE) respectively.

[0054] Conventional control group medium: modified BHI medium, specific composition: brain heart infusion 37 g / L + yeast extract 5 g / L + porcine gastric mucin 2.5 g / L + brilliant green 1 mg / L + L-cysteine 0.1 g / L; pH 7.5-7.6, sterilize at 121°C for 20 minutes, then prepare two, inoculate AKKPROBIO strain (conventional control group-AKKPROBIO) and AKK ONE strain (conventional control group-AKK ONE) respectively.

[0055] Culture conditions: all experimental groups and control groups are cultured under strict anaerobic conditions at 37°C.

[0056] Detection index: OD 600 , pH index to represent the influence on growth, respectively measure the OD 600 of each test carbon source group and control group at 20h of culture, the average value, Δ20h-OD 600 (i.e. the change amount of OD 600 in 20h), and Δ20h-pH (i.e. the change amount of pH in 20h).

[0057] Table 1 Concentration of lacto-N-neotetraose in examples

[0058] 1.3 Experimental results and analysis The experimental results are shown in Table 2: Table 2 Experimental results of examples

[0059] Table 2 Experimental results of the examples

[0060] As can be seen from Table 2, lacto-N-neotetraose as a core substance has an irreplaceable positive effect on the growth of the strain.

[0061] For the AKK PROBIO strain, in the concentration interval of 0.044-6.6 g / L, Δ20h-OD 600 increases linearly, from 0.50 (0.044 g / L) in Example 12 to 2.13 (6.6 g / L) in Example 9, with an increase of 326%, indicating that the higher the concentration of lacto-N-neotetraose in this interval, the more sufficient nutrients AKK strain can provide to promote cell reproduction. In the concentration interval of 6.6-8.8 g / L, Δ20h-OD 00 falls back, 2.01 in Example 10 (7.7 g / L) and 1.97 in Example 11 (8.8 g / L), it is speculated that there may be osmotic pressure inhibition at high concentrations, or the utilization of carbon source by the strain reaches saturation, so 6.6 g / L is a relatively optimal concentration node.

[0062] For the AKK ONE strain, in the lacto-N-neotetraose concentration of 2.2 g / L (Example 13) and 4.4 g / L (Example 14), Δ20h-OD 600 are 1.24 and 1.56 respectively, and Δ20h-pH are 1.15 and 1.30 respectively, showing the rule of "concentration increasing-growth increasing"; which are significantly higher than the self blank control group (Δ20h-OD 600 0.28) and the conventional control group (Δ20h-OD 600 1.06), among which the Δ20h-OD 600 of Example 14 is 1.47 times that of the conventional control group.

[0063] In addition, compared with the conventional medium, lacto-N-neotetraose is a scalable sugar substance (non-animal source), and there is an optimal concentration interval (4-6.6 g / L), which can not only ensure high growth (such as Δ20h-OD 600 1.71-2.13 in Example 7-Example 9) in this interval, but also avoid carbon source waste; at the same time, the basic ingredients of the medium (glucose, threonine, yeast extract, etc.) are conventional microbial culture raw materials, which are easy to purchase and cost controllable. The animal-derived mucin in the conventional medium may carry viral, bacterial residues or foreign proteins, which may cause cross-contamination risk and trigger immune response (such as foreign antigen sensitization), especially in clinical application.

[0064] In summary, the performance of lacto-N-neotetraose is better than that of the conventional control group in terms of key growth indicators such as 20h-OD600, Δ20h-OD600 and Δ20h-pH, and the morphology is good Figs. 1-2 , which fully proves that it can efficiently and stably support the growth of Akkermansia muciniphila and has excellent efficacy as a carbon source for the culture of the strain. Comparative Example With reference to the example, a comparative example is set up. The difference between the comparative example and Example 7 is shown in Table 3 (the test strain is AKKPROBIO). Two replicates are set up in the experiment, and the experimental results are shown in Table 4.

[0065] Table 3 Setting of the comparative example

[0066] Table 4 Experimental results of the comparative example

[0067] Continued Table 4 Experimental results of the comparative example

[0068] Continued Table 4 Experimental results of the comparative example

[0069] From the results in Table 4, it can be seen that the other test nutritional sources have a significant gap with the conventional control group in the above-mentioned core indicators, and cannot meet the demand of the strain growth on the nutritional source. Therefore, it can be determined that the lacto-N-neotetraose of the present application is the only nutritional source that can achieve and be superior to the conventional control medium in terms of equivalent culture effect, and its effectiveness and adaptability in supporting the growth of Akkermansia muciniphila are outstanding.

[0070] The above detailed description is a specific description of one feasible embodiment of the present application, and this embodiment is not used to limit the patent scope of the present application. It should be noted that any changes derived from the present application by biological professionals or any equivalent implementation or changes made without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of lacto-N-neotetraose in the cultivation of AKK bacteria, characterized in that, The lacto-N-neotetraose is used at a concentration of 4-8.8 g / L.

2. Use according to claim 1, characterized in that, The lacto-N-neotetraose is used at a concentration of 4-6.6 g / L.

3. A culture medium for culturing AKK bacteria, characterized by, The culture medium comprises lacto-N-neotetraose; the lacto-N-neotetraose is used at a concentration of 4-8.8 g / L.

4. The medium according to claim 3, characterized in that, The culture medium further comprises glucose, threonine, yeast extract, manganese sulfate, magnesium chloride hexahydrate, sodium chloride, sodium bicarbonate, sodium acetate and potassium dihydrogen phosphate; Or the culture medium further comprises peptone, beef infusion powder, yeast extract, glucose, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, Tween and biotin.

5. A method for culturing AKK bacteria, characterized by, The method comprises inoculating AKK bacteria into a culture medium containing lacto-N-neotetraose for culture; the lacto-N-neotetraose is used at a concentration of 4-8.8 g / L.

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