Chemically defined medium for supporting growth of bifidobacteria and use thereof

Abstract

The application relates to the technical field of microbial culture medium, and discloses a chemical component limited culture medium for supporting growth of bifidobacterium and application thereof. The CDM comprises a carbon source, salts, amino acids, vitamins and water; the carbon source is selected from at least one of glucose, fructose, lactose, maltose and galactooligosaccharide, the salts comprise sodium acetate, potassium hydrogen phosphate, ammonium acetate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium chloride and manganese sulfate monohydrate, the amino acids comprise alanine, cystine, cysteine, tryptophan and asparagine, and the vitamins comprise pyridoxine hydrochloride, nicotinic acid, calcium pantothenate, thiamine hydrochloride, riboflavin, p-aminobenzoic acid, folic acid and biotin. The CDM of the application is a liquid culture medium, only comprises 21 definite components, is simple in components, convenient in preparation, and is high in universality, and can effectively support growth of bifidobacterium.

Description

Technical Field

[0001] This application relates to the technical field of microbial culture media, and in particular to a chemically defined culture medium for supporting the growth of Bifidobacteria and its application. Background Technology

[0002] Bifidobacteria ( Bifidobacterium Bifidobacterium carbohydrate metabolism is highly complex, exhibiting a significant preference for carbohydrate utilization. This is primarily due to the extensive genetic diversity among different species and strains in terms of carbohydrate transporters and glycoside hydrolases. Furthermore, cross-feeding interactions are prevalent among different Bifidobacterium species, enabling them to synergistically utilize various polysaccharides, further complicating the study of their carbohydrate metabolism mechanisms. However, most current research still relies on enrichment or semi-limited media containing undefined components, hindering the precise elucidation of metabolic phenotypes and regulatory mechanisms. Therefore, chemically defined medium (CDM) is of significant value in Bifidobacterium research, as it provides a stable, reproducible, and easily elucidated experimental background for understanding metabolic mechanisms.

[0003] To date, the number of CDMs specifically developed for Bifidobacteria is very limited, and most CDMs used in Bifidobacteria experiments are modified from the same type of lactobacillus (Lactobacillus). Lactobacillus The applicability of the CDM formulation (Petry et al., 2000) across different Bifidobacterium species has not been fully validated. Although some studies have successfully developed fully defined culture media containing 46 chemical components using genome-scale modeling (Schöpping et al., 2021), which can support Bifidobacterium growth with effects comparable to enrichment media, this strategy relies on genome annotation and metabolic network reconstruction for specific strains, making the analysis process complex. Furthermore, it has only tested two Bifidobacterium species, making it difficult to widely apply in routine experiments. In addition, reported CDM formulations typically contain about 40 components and require the addition of various trace elements, resulting in cumbersome and time-consuming preparation processes (Ferrario et al., 2015; Schöpping et al., 2021), severely limiting their large-scale application and daily use in research and industrial settings. In addition, the minimal medium for Bifidobacterium (BMM) reported by Sakaguchi et al. contains 22 substances (Sakaguchi et al., 2013). Although the composition is simple, it can only be used for growth on solid media and cannot be applied to liquid culture.

[0004] In summary, providing a chemically defined culture medium that is versatile, simple in composition, easy to prepare, and can efficiently support the growth of Bifidobacteria is a technical problem that urgently needs to be solved by those skilled in the art.

[0005] References: Document 1 (Petry et al., 2000): PÉTRY S, FURLAN S, CRÉPEAU MJ, et al. Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. Bulgarian grown in a chemically defined medium[J]. Applied andEnvironmental Microbiology, 2000, 66(8): 3427-3431. Document 2 (Schöpping et al., 2021): SCHÖPPING M, GASPAR P, NEVES AR, et al.Identifying the essential nutritional requirements of the probioticbacteria Bifidobacterium animalum and Bifidobacterium longum through genome-scale modeling[J]. NPJ Systems Biology and Applications, 2021, 7(1): 47. Document 3 (Ferrario et al., 2015): FERRARIO C, DURANTI S, MILANI C, et al. Exploring Amino Acid Auxotrophy in Bifidobacterium bifidum PRL2010[J].Frontiers in Microbiology, 2015, 6. Document 4 (Sakaguchi et al., 2013): SAKAGUCHI K, HE X, YASUI H, et al. The pyre Gene as a Bidirectional Selection Marker in Bifidobacterium longum 105-A[J].Biosci Microbiota Food Health, 2013, 32(2):59-68. Summary of the Invention The purpose of this invention is to solve the above-mentioned technical problems and provide a chemically defined culture medium (CDM) for supporting the growth of Bifidobacteria and its application. This CDM consists of only 21 components, is simple in composition, easy to prepare, and highly versatile, and can efficiently support the growth of Bifidobacteria.

[0006] In a first aspect, the present invention provides a chemically defined culture medium for supporting the growth of Bifidobacteria, employing the following technical solution: A chemically defined culture medium for supporting the growth of Bifidobacteria includes a carbon source, salts, amino acids, vitamins, and water; wherein the carbon source is selected from at least one of glucose, fructose, lactose, maltose, and galactooligosaccharides; the salts include sodium acetate, potassium hydrogen phosphate, ammonium acetate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium chloride, and manganese sulfate monohydrate; the amino acids include alanine, cystine, cysteine, tryptophan, and asparagine; and the vitamins include pyridoxine hydrochloride, niacin, calcium pantothenate, thiamine hydrochloride, riboflavin, para-aminobenzoic acid, folic acid, and biotin.

[0007] Optionally, per 1L, the composition and content of each raw material used in the preparation of the chemically defined culture medium are as follows: carbon source 5-20g, sodium acetate 15-35g, dipotassium hydrogen phosphate 1.5-4.0g, ammonium acetate 1.0-3.5g, magnesium sulfate heptahydrate 100-400mg, ferrous sulfate heptahydrate 5-20mg, sodium chloride 5-20mg, manganese sulfate monohydrate 3-12mg, alanine 100-400mg, cystine 10 0-400mg, cysteine ​​100-400mg, tryptophan 100-400mg, asparagine 50-200mg, pyridoxine hydrochloride 600-2000μg, nicotinic acid 300-1000μg, calcium pantothenate 200-700μg, thiamine hydrochloride 100-400μg, riboflavin 100-400μg, para-aminobenzoic acid 5-20μg, folic acid 5-20μg, biotin 2-8μg, balance water.

[0008] Optionally, per 1L, the composition and content of each raw material used in the preparation of the chemically defined culture medium are as follows: carbon source 10g, sodium acetate 25g, dipotassium hydrogen phosphate 2.5g, ammonium acetate 2.0g, magnesium sulfate heptahydrate 200mg, ferrous sulfate heptahydrate 10mg, sodium chloride 10mg, manganese sulfate monohydrate 6.75mg, alanine 200mg, cystine 200mg, cysteine ​​200mg, tryptophan 200mg, asparagine 100mg, pyridoxine hydrochloride 1200μg, nicotinic acid 600μg, calcium pantothenate 400μg, thiamine hydrochloride 200μg, riboflavin 200μg, para-aminobenzoic acid 10μg, folic acid 10μg, biotin 4μg, with the remainder being water.

[0009] Optionally, the chemical composition defines the pH of the culture medium as 6.7-7.2.

[0010] In a second aspect, the present invention provides the application of a chemically defined culture medium for supporting the growth of Bifidobacteria in the culture of Bifidobacteria.

[0011] Optionally, the Bifidobacterium is selected from at least one of the following: Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium short Bifidobacterium longum ( Bifidobacterium longum Bifidobacterium densiflorum ( Bifidobacterium teeth Bifidobacterium pseudosporidis ( ), Bifidobacterium pseudosporidis ( ) Bifidobacterium pseudocatenulatum Bifidobacterium adolescentis ( ) Bifidobacterium adolescentis Bifidobacterium bifidum ( Bifidobacterium bifidum ).

[0012] Optionally, the Bifidobacterium is selected from: B. short GZX43 B. dentium LFYP24 B. pseudo-chained LFYP29 B. adolescent LFYP80 B. short LFYP81, B. longum subsp. long LFYP82, B. longum subsp. child ATCC 15697 or B. bifidum JCM 1254.

[0013] Optionally, specific steps include: activating Bifidobacteria and then adjusting OD. 600 Up to 1.0, inoculate at a volume ratio of 1:100 into a chemically defined medium for supporting the growth of Bifidobacteria and culture anaerobically.

[0014] Optionally, the culture temperature is controlled at 35-42℃.

[0015] This invention includes at least one of the following beneficial technical effects: 1. The CDM of the present invention contains only 21 clearly defined ingredients and does not contain any animal-derived, plant-derived extracts or undefined ingredients. It has a simple composition, is easy to prepare, and has a low cost. 2. The CDM of the present invention can support the liquid culture of Bifidobacteria, and is suitable for both large and small volumes (e.g., 200 μL); 3. The CDM of the present invention can support the normal growth of Bifidobacteria and is superior to existing chemically defined culture media; specifically, the 6 species (8 strains) tested with the present CDM all showed good growth status, and after being replaced with a single carbon source suitable for specific strains, their growth status was even comparable to enrichment medium (LYHBHI).

[0016] In summary, this invention provides a culture medium with a clearly defined chemical composition, strong versatility, and the ability to efficiently support the growth of different Bifidobacteria, providing a stable and reproducible experimental basis for the study of the metabolic mechanisms of Bifidobacteria and their industrial applications. Attached Figure Description

[0017] Figure 1 This is a comparative analysis of the growth curves of eight Bifidobacterium strains in chemically defined media and enrichment media (LYHBHI) supplemented with different single carbon sources; among them, Figure 1 The strain in A is B. short GZX43 Figure 1 The strain in B is B. teeth LFYP24, Figure 1 The strain in C is B. pseudocatenulatum LFYP29, Figure 1 The strain in D is B. of a young man LFYP80, Figure 1 The strain in E is B. short LFYP81, Figure 1 The strain in F is B. long subsp. long LFYP82, Figure 1 The strain in G is B. bifidum JCM 1254, Figure 1 The strain in H is B. long subsp. child ATCC 15697.

[0018] Figure 2 These are growth curves comparing six Bifidobacterium strains in BMM and LYHBHI; among them, Figure 2 The strain in A is B. short GZX43 Figure 2 The strain in B is B. dentium LFYP24, Figure 2 The strain in C is B. pseudocatenulatum LFYP29, Figure 2 The strain in D is B. adolescent LFYP80, Figure 2 The strain in E is B. short LFYP81, Figure 2 The strain in F is B. longum subsp. long LFYP82. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments of the present invention are intended to fully demonstrate the technical solution and effects of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, improvements, etc., made to the embodiments of the present invention based on the technical essence and general principles of the present invention without creative effort should be within the scope of protection of the present invention.

[0020] Unless otherwise specified, the materials and equipment used in the various embodiments of the present invention are all commercially available products in the art.

[0021] The strain information used in this embodiment is as follows: B. bifidum JCM 1254, strain number: CICC 6170, purchased from China Industrial Microbial Culture Collection Center (CICC). B. longum subsp. child ATCC 15697, strain number: CICC 6069, purchased from China Industrial Microbial Culture Collection Center (CICC). The following six strains are all deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of March 5, 2026: Strain name GZX43, Bifidobacterium breve Bifidobacterium breve Collection number: CGMCC NO.37843; Strain name LFYP24, Bifidobacterium denticum Bifidobacterium dentium Collection number: CGMCCNO.37846; Strain name LFYP29, Bifidobacterium pseudosporidioides Bifidobacterium pseudocatenulatum Collection number: CGMCC NO.37845; Strain name LFYP80, Bifidobacterium adolescentis Bifidobacterium adolescentis Collection number: CGMCCNO.37844; Strain name LFYP81, Bifidobacterium breve Bifidobacterium breve Collection number: CGMCC NO.37841; Strain name LFYP82, Bifidobacterium longum subsp. longum Bifidobacterium longum subsp. long Collection number: CGMCC NO.37842.

[0022] Examples 1-3: Preparation of Chemically Defined Culture Media (CDM) A CDM used to support the growth of Bifidobacteria, the components and contents of the raw materials used in its preparation per 1L are shown in Table 1 below: Table 1. Components and contents of each CDM raw material in Examples 1-3

[0023] The carbon sources mentioned above can also be other carbon sources that can support the growth of Bifidobacteria, such as fructose, lactose, maltose, or galactooligosaccharides.

[0024] The above-mentioned method for preparing CDM to support the growth of Bifidobacteria specifically includes the following steps: S1: Magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium chloride and manganese sulfate monohydrate are dissolved in sterile water in sequence to prepare 100× salt mother liquor. S2: Dissolve pyridoxine hydrochloride, nicotinic acid, calcium pantothenate, thiamine hydrochloride and riboflavin in sterile water in sequence to prepare 100× vitamin stock solution A. An appropriate amount of NaOH can be added to promote the dissolution of vitamin stock solution A. S3: Dissolve para-aminobenzoic acid, folic acid and biotin in sterile water in sequence to prepare 10000× vitamin stock solution B. An appropriate amount of NaOH can be added to promote the dissolution of vitamin stock solution B. S4: Dissolve sodium acetate, dipotassium hydrogen phosphate, ammonium acetate, 100× salt stock solution, 100× vitamin stock solution A, and 10000× vitamin stock solution B in sterile water in sequence, stir and mix well to obtain mixture I; S5: Add alanine, cystine, cysteine, tryptophan and asparagine to mixture I in sequence, stir and mix well to obtain mixture II; S6: Add glucose to mixture II and stir until completely dissolved. Then add sterile water to make up to 1L and adjust the pH to 6.8 with NaOH or HCl to obtain crude CDM for supporting the growth of Bifidobacteria. S7: Use a 0.22µm microporous membrane to sterilize the crude CDM by aseptic filtration, avoiding high temperature damage to heat-sensitive components, to obtain CDM that supports the growth of Bifidobacteria.

[0025] The final CDM contains 21 precisely quantified components, contains no animal or plant-derived extracts or undefined ingredients, and is suitable for both large-volume and small-volume (e.g., 200 μL) high-throughput anaerobic liquid culture of Bifidobacterium strains, with good culture results.

[0026] Example 4: Evaluation of the culture and growth performance of Bifidobacterium strains Eight strains of Bifidobacteria isolated from the intestines of infants from different regions were selected as test subjects, including: Source of Chinese babies: B. short GZX43; Source of American babies: B. dentium LFYP24 B. pseudocatenulatum LFYP29 B. of a young man LFYP80 B. short LFYP81, B. longum subsp. long LFYP82 and B. longum subsp. child ATCC 15697; Source of Japanese babies: B. bifidum JCM 1254.

[0027] Each strain was anaerobically activated overnight at 37°C in enrichment medium (LYHBHI), and the OD was adjusted. 600 After reaching 1.0, the sample was inoculated into a CDM at a volume ratio of 1:100. The CDM was configured with the following single carbon source conditions: 1% glucose, 1% fructose, 1% lactose, 1% maltose, or 1% galactooligosaccharides (GOS) (w / v), and the remaining raw material components were the same as in Example 1. LYHBHI was used as a positive control.

[0028] The composition and content of the raw materials used in the preparation of LYHBHI, calculated per 1L, are shown in Table 2 below: Table 2 LYHBHI Raw Material Components and Contents

[0029] The above-mentioned Brain-heart infusion broth, product number: CM1135, Oxoid; The above-mentioned yeast extract, product number: A515245-0500, is manufactured by Sangon Biotech (Shanghai) Co., Ltd. The above-mentioned cellobiose, C7252, is from Sigma-Aldrich. The above-mentioned maltose, product number: M5885, Sigma-Aldrich; The above-mentioned cysteine ​​(Cysteine), catalog number: C1276, Sigma-Aldrich; The above-mentioned ferric heme (Hemin), catalog number: 51280, Sigma-Aldrich; Prepare LYHBHI enrichment medium according to the above content and autoclave it.

[0030] All bacterial strains were cultured in 96-well shallow-well microtiter plates, 200 μL per well, anaerobic incubation at 37°C for 72 hours, and the optical density (OD) at 600 nm was automatically recorded every 15 minutes using a microplate reader. 600 The value (mixed by medium-speed shaking before measurement) is used to monitor the growth of the strain.

[0031] The data are presented as a scatter plot, showing the mean ± standard error (SEM) of three biological replicates. The results are as follows: Figure 1 As shown in A-1H. The results indicate that Bifidobacteria from different sources can grow normally in CDMs with different single carbon sources; and, under more suitable carbon source conditions, the growth indicators of the strains are comparable to those in LYHBHI.

[0032] Comparative Example 1 A minimal culture medium for Bifidobacterium (BMM) is prepared by dissolving the raw materials in sterile water, with specific raw material components and contents as described in reference 4 (Sakaguchi et al., 2013).

[0033] strain B. short GZX43 B. dentium LFYP24 B. pseudocatenulatum LFYP29 B. of a young man LFYP80 B. short LFYP81 and B. longum subsp. long LFYP82 was anaerobically activated overnight in LYHBHI at 37°C to adjust OD. 600 After reaching 1.0, the culture was inoculated into BMM at a volume ratio of 1:100. LYHBHI was used as a positive control. All strains were cultured in 96-well shallow-well microtiter plates, 200 μL per well, anaerobic at 37°C for 72 hours. The optical density (OD) at 600 nm was automatically recorded every 15 minutes using a microplate reader. 600 The value (mixed by medium-speed shaking before measurement) is used to monitor the growth of the strain.

[0034] The data are presented as a scatter plot, showing the mean ± standard error (SEM) of three biological replicates. The results are as follows: Figure 2 As shown in A-2F, the results showed that the strain hardly grew in liquid BMM, indicating that BMM cannot support the liquid culture of Bifidobacterium.

[0035] In summary, the CDM of the present invention, as a liquid culture medium with 21 clearly defined components, is easy to prepare, highly versatile, and can effectively support the liquid culture of Bifidobacteria.

[0036] The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit it. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

Claims

1. A chemically defined culture medium for supporting the growth of Bifidobacteria, characterized in that, It includes a carbon source, salts, amino acids, vitamins, and water; the carbon source is selected from at least one of glucose, fructose, lactose, maltose, and galactooligosaccharides; the salts include sodium acetate, potassium hydrogen phosphate, ammonium acetate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium chloride, and manganese sulfate monohydrate; the amino acids include alanine, cystine, cysteine, tryptophan, and asparagine; and the vitamins include pyridoxine hydrochloride, niacin, calcium pantothenate, thiamine hydrochloride, riboflavin, para-aminobenzoic acid, folic acid, and biotin.

2. The chemically defined culture medium for supporting the growth of Bifidobacteria as described in claim 1, characterized in that, Based on per 1L, the composition and content of each raw material used in the preparation of the chemically defined culture medium are as follows: carbon source 5-20g, sodium acetate 15-35g, dipotassium hydrogen phosphate 1.5-4.0g, ammonium acetate 1.0-3.5g, magnesium sulfate heptahydrate 100-400mg, ferrous sulfate heptahydrate 5-20mg, sodium chloride 5-20mg, manganese sulfate monohydrate 3-12mg, alanine 100-400mg, cystine 100- 400mg, cysteine ​​100-400mg, tryptophan 100-400mg, asparagine 50-200mg, pyridoxine hydrochloride 600-2000μg, nicotinic acid 300-1000μg, calcium pantothenate 200-700μg, thiamine hydrochloride 100-400μg, riboflavin 100-400μg, para-aminobenzoic acid 5-20μg, folic acid 5-20μg, biotin 2-8μg, balance water.

3. The chemically defined culture medium for supporting the growth of Bifidobacteria as described in claim 2, characterized in that, The composition and content of each raw material used in the preparation of the chemically defined culture medium per 1L are as follows: carbon source 10g, sodium acetate 25g, dipotassium hydrogen phosphate 2.5g, ammonium acetate 2.0g, magnesium sulfate heptahydrate 200mg, ferrous sulfate heptahydrate 10mg, sodium chloride 10mg, manganese sulfate monohydrate 6.75mg, alanine 200mg, cystine 200mg, cysteine ​​200mg, tryptophan 200mg, asparagine 100mg, pyridoxine hydrochloride 1200μg, nicotinic acid 600μg, calcium pantothenate 400μg, thiamine hydrochloride 200μg, riboflavin 200μg, para-aminobenzoic acid 10μg, folic acid 10μg, biotin 4μg, and the remainder is water.

4. The chemically defined culture medium for supporting the growth of Bifidobacteria as described in claim 2, characterized in that, The chemical composition specifies that the pH of the culture medium is 6.7-7.

2.

5. The use of the chemically defined culture medium for supporting the growth of Bifidobacteria as described in any one of claims 1-4 in the culture of Bifidobacteria.

6. The application as described in claim 5, characterized in that, The Bifidobacterium is selected from at least one of the following: Bifidobacterium breve ( Bifidobacterium breve Bifidobacterium longum ( Bifidobacterium longum Bifidobacterium densiflorum ( Bifidobacterium dentium Bifidobacterium pseudosporidis ( ), Bifidobacterium pseudosporidis ( ) Bifidobacterium pseudocatenulatum Bifidobacterium adolescentis ( ) Bifidobacterium adolescentis Bifidobacterium bifidum ( Bifidobacterium bifidum ).

7. The application as described in claim 6, characterized in that, The Bifidobacteria were selected from: B. breve GZX43 B. dentium LFYP24 B. pseudocatenulatum LFYP29 B. adolescentis LFYP80 B. breve LFYP81, B. longum subsp. longum LFYP82, B. longum subsp. infantis ATCC 15697 or B. bifidum JCM 1254.

8. The application as described in claim 5, characterized in that, The specific steps include: activating Bifidobacteria and adjusting OD. 600 Up to 1.0, inoculate at a volume ratio of 1:100 into a chemically defined medium for supporting the growth of Bifidobacteria and culture anaerobically.

9. The application as described in claim 8, characterized in that, The culture temperature is controlled at 35-42℃.

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