Lactobacillus bulgaricus capable of producing exopolysaccharides and application of lactobacillus bulgaricus
By screening and isolating Lactobacillus bulgaricus LBA3, which produces high levels of extracellular polysaccharides, the problem of low extracellular polysaccharide production in lactic acid bacteria strains has been solved. This has resulted in high-yield and structurally diverse extracellular polysaccharides, promoting the innovative development of yogurt products.
Patent Information
- Application Number
- CN202511204748.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-27
- Publication Date
- 2025-12-05
AI Technical Summary
Existing lactic acid bacteria strains have low extracellular polysaccharide yields, which limits their large-scale promotion in industrial production and application. Furthermore, the polysaccharide structure is simple and cannot meet the needs of diversified products.
A strain of Lactobacillus bulgaricus LBA3, which produces a high amount of extracellular polysaccharides, was screened and isolated from the fermentation acid water of Rushan, a traditional dairy product in Yunnan. By optimizing culture conditions and metabolic regulation, the yield and structural diversity of extracellular polysaccharides were improved.
It significantly improves the yield and structural diversity of extracellular polysaccharides, reduces production costs, endows products with unique textural and functional properties, and supports the development of diversified yogurt products.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of microorganisms, and particularly relates to a strain of Lactobacillus bulgaricus producing exopolysaccharide and application thereof. BACKGROUND
[0002] Lactic acid bacteria (LAB) are essential in food fermentation processes, contributing to the production of various fermented products. Exopolysaccharides (EPS) secreted by LAB during growth and metabolism have numerous health benefits, including prebiotic effects, immune regulation, antibacterial properties, blood glucose and lipid regulation, and promotion of mineral absorption. They can stimulate the growth of beneficial intestinal bacteria, improve intestinal health, and enhance the texture and viscosity of dairy products.
[0003] However, current exopolysaccharide-producing LAB face many difficulties, with most LAB strains producing low amounts of exopolysaccharide. In industrial production, low production not only increases production costs but also severely limits its large-scale application. Compared to ordinary LAB strains, exopolysaccharide-producing LAB strains have significant advantages in innovative applications, particularly in terms of polysaccharide yield, composition, and viscosity production rate. After optimizing the culture conditions and selecting specific strains, their polysaccharide yield is significantly higher than that of ordinary strains.
[0004] Exopolysaccharide-producing strains and ordinary LAB strains differ significantly in polysaccharide metabolic pathways and molecular composition:
[0005] In terms of polysaccharide metabolic pathways, the main differences between the two lie in the gene clusters for polysaccharide synthesis, the regulation mechanisms of metabolic pathways, the structural and functional properties of products, and the sensitivity to environmental factors. For example, the gene clusters of exopolysaccharide-producing strains may contain more glycosyltransferase-encoding genes, enabling them to respond to environmental signals through more complex regulatory networks to drive exopolysaccharide synthesis.
[0006] In terms of polysaccharide molecular composition, the main differences lie in the structure and composition of polysaccharides. Exopolysaccharide-producing strains typically produce heteropolysaccharides, which are composed of various monosaccharides such as glucose, galactose, and mannose, and have complex branching structures and different glycosidic bond connection methods. In contrast, ordinary LAB strains may mainly produce homopolysaccharides, which are relatively simple in composition and typically consist of a single monosaccharide. Additionally, exopolysaccharide-producing strains exhibit significant differences in the molecular weight and viscosity properties of their polysaccharides. Some strains can produce high-molecular-weight polysaccharides up to several million Daltons, while ordinary strains have relatively low polysaccharide molecular weights. It is worth noting that the molecular structure of polysaccharides (such as glycosidic bond type, branching degree, monosaccharide composition, etc.) directly affects their viscosity.
[0007] Therefore, how to start from a large number of strain resources, systematically explore characteristic EPS-producing yogurt starters, and clarify their metabolic regulation mechanisms, is of great significance for the development of diversified yogurt products. This exploration not only can reduce the cost of industrial production by increasing the yield of exopolysaccharide, but also can endow the product with unique texture and functional properties by virtue of the diversity of polysaccharide structure, providing strong support for the innovative development of related industries. SUMMARY
[0008] In order to solve the above problems, the present application screens a strain of Lactobacillus bulgaricus LBA3 with high yield of extracellular polysaccharide (EPS) from the fermented acid water of traditional dairy products in Yunnan, China.
[0009] Therefore, the present application provides a Lactobacillus bulgaricus LBA3 with high yield of extracellular polysaccharide. The Lactobacillus bulgaricus provided by the present application is Lactobacillus delbrueckii subsp. bulgaricus, i.e. Lactobacillus bulgaricus LBA3, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC, address: No. 1, Beichen West Road, Chaoyang District, Beijing, China) on February 28, 2024, with the preservation number CGMCC NO: 29906.
[0010] The present application also provides a microbial agent containing the Lactobacillus bulgaricus and / or metabolites of the Lactobacillus bulgaricus.
[0011] The culture of the Lactobacillus bulgaricus is a substance obtained by culturing the Lactobacillus bulgaricus in a bacterial culture medium.
[0012] The active ingredient of the microbial agent can be the Lactobacillus bulgaricus and / or the metabolites of the Lactobacillus bulgaricus, and the active ingredient of the microbial agent can also contain other biological components or non-biological components.
[0013] According to the needs, surfactants (such as Tween 20, Tween 80, etc.), adhesives, stabilizers (such as antioxidants), pH regulators, etc. can be added to the microbial agent.
[0014] The metabolite can be obtained from the fermentation liquor of the Lactobacillus bulgaricus. The metabolite can be a sterile metabolite of the Lactobacillus bulgaricus or a metabolite containing bacteria of the Lactobacillus bulgaricus. The sterile metabolite of the Lactobacillus bulgaricus (sterile fermentation filtrate) can be prepared by culturing the Lactobacillus bulgaricus in a liquid culture medium, and filtering the Lactobacillus bulgaricus in the liquid culture (fermentation liquor) to obtain the sterile metabolite of the Lactobacillus bulgaricus. The metabolite containing bacteria of the Lactobacillus bulgaricus can be prepared by culturing the Lactobacillus bulgaricus in a liquid fermentation medium, and collecting the fermentation liquor, which is the metabolite containing bacteria of the Lactobacillus bulgaricus.
[0015] The application also provides the use of the Lactobacillus bulgaricus or the bacterial agent or the culture in the preparation of extracellular polysaccharide.
[0016] The application also provides the use of the Lactobacillus bulgaricus or the bacterial agent or the culture in the preparation of extracellular polysaccharide product.
[0017] The application also provides a preparation method of the bacterial agent.
[0018] The preparation method of the bacterial agent provided by the application comprises the following steps: taking the Lactobacillus bulgaricus as an active ingredient to obtain the bacterial agent.
[0019] The application also provides a method for preparing extracellular polysaccharide, which comprises the following steps: culturing the Lactobacillus bulgaricus in a culture medium, and obtaining extracellular polysaccharide from the fermentation product.
[0020] The application screens a Lactobacillus bulgaricus LBA3 with high yield of extracellular polysaccharide (EPS) from traditional dairy product Ruofan fermentation acid water in Yunnan, China. The crude extracellular polysaccharide extracted from the strain has a content of about 400 mg / L, which has great application value.
[0021] Biological material preservation information
[0022] Strain name: Lactobacillus bulgaricus LBA3
[0023] Classification name: Lactobacillus bulgaricus
[0024] Preservation agency: China General Microbiological Culture Collection Center
[0025] Abbreviation of preservation agency: CGMCC
[0026] Address of deposit institution: No. 3, Beichen West Road, Chaoyang District, Beijing
[0027] Date of deposit: February 28, 2024
[0028] Deposit number: CGMCC No. 29906. DETAILED DESCRIPTION
[0029] The present application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.
[0030] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.
[0031] In the quantitative experiments in the following examples, unless otherwise specified, three repeated experiments were set.
[0032] In the examples of the present application, the experimental data was subjected to one-way ANOVA by statistical software SPSS 26.0. The experimental results are expressed as mean ± SD.
[0033] Example 1, isolation and identification of strains and growth characteristics
[0034] 1. Isolation of strains
[0035] 5 g of milk fan acid was taken into 100 mL of acid MRS broth (pH = 5.4) and incubated at 37°C for 30 h, then spread on acid MRS medium (pH = 5.4) (potassium phosphate dibasic 0.2 g, sodium acetate anhydrous 0.5 g, yeast powder 0.5 g, magnesium sulfate 0.05 g, beef extract 1 g, ammonium citrate 0.2 g, tryptone 1 g, glucose 2 g, manganese sulfate 0.025 g, Tween 80 0.1 mL, agar 1.5 g, distilled water 100 mL; sterilized at 121°C for 15 min), incubated at 37°C under anaerobic conditions for 48 h, then single colonies with lactobacillus colony characteristics were picked from the culture medium, purified by multiple plate streaking to obtain strain LBA3, inoculated on slant and enriched in acid MRS broth. The enriched LBA3 was stored in 40% glycerol at -80°C.
[0036] 2. Strain identification
[0037] The strain LBA3 obtained in step 1 was inoculated in MRS medium and cultured for 24 h, and the bacterial DNA was extracted according to the instructions of Beijing Tiangeng Bacterial Genomic DNA Extraction Kit (item number: DP302). The universal primer was used to amplify the 16S rDNA genomic sequence. The PCR product was sent to Beijing Boshang Biotechnology Co., Ltd. for sequence determination, and the obtained 16S rDNA gene sequence was subjected to BLAST (https: / / blast.ncbi.nlm.nih.gov / ) comparison in NCBI. The results showed that the sequence had more than 99% homology with the 16S rDNA sequence of Lactobacillus bulgaricus, and the strain LBA3 was identified as Lactobacillus bulgaricus, which was referred to as Lactobacillus bulgaricus LBA3.
[0038] Example 2, Study on preparation of exopolysaccharide by using strain LBA3
[0039] 1. Preparation of Lactobacillus bulgaricus LBA3 inoculant
[0040] The Lactobacillus bulgaricus LBA3 stored in a glycerol tube at -80°C was thawed at room temperature, inoculated into sterilized acid MRS medium (pH = 5.4) at a 2% (V / V) inoculation amount, and cultured at 37°C for 12 h as an activated bacterial solution. The inoculation amount was 2% (V / V) for subculture, and the culture was carried out at 37°C for 12 h to obtain the LBA3 inoculant. The bacterial concentration of the bacterial suspension was 10 10 cfu / ml.
[0041] 2. Study on the ability of Lactobacillus bulgaricus LBA3 to produce exopolysaccharide
[0042] 2.1 Isolation of polysaccharide
[0043] After activation, the crude exopolysaccharide was produced in batches, and two activations were required for further culture. After fermentation for 18 h, 100 mL of fermentation broth was accurately taken, 4% (m / v) of trichloroacetic acid (TCA) was added, and the mixture was stirred at 1000 rpm for 2 h using a mechanical stirrer. Then, the mixture was centrifuged at 9000 rpm and 4°C for 30 min, and the supernatant was collected. The supernatant was mixed with anhydrous ethanol at a volume ratio of 1:3, stirred uniformly, and then placed in a refrigerator for 12 h. Then, the mixture was centrifuged at 9000 rpm and 4°C for 30 min, and the supernatant was discarded and the precipitate was collected.
[0044] The precipitate was dissolved with distilled water (DW) and poured into a dialysis bag with a molecular weight cut-off of 8000-14000 Da. DW was replaced every 12 hours, and the dialysis bag was stored at 4°C during the replacement. The crude exopolysaccharide solid after dialysis was collected by freeze-drying. The content of exopolysaccharide was determined by the phenol-sulfuric acid method. Specifically, 1 mg / mL LBA3 exopolysaccharide solution and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mg / mL glucose solution (as a standard) were prepared. 20 μL of LBA3 exopolysaccharide solution or different concentrations of glucose solution were added to a 96-well plate, with three replicates for each group. 20 μL of 5% phenol aqueous solution was added to each well, mixed thoroughly, and then 100 μL of concentrated sulfuric acid was added to each well, mixed thoroughly, and incubated at room temperature for 30 min. The absorbance at 480 nm of each well was detected using an enzyme-labeled instrument. The absorbance of the 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mg / mL glucose solution was used as the vertical coordinate (y), and the glucose concentration was used as the horizontal coordinate (x) to create a standard curve. According to the linear relationship between glucose concentration and absorbance, the exopolysaccharide obtained was named Y528E2F, and the content was 400 mg / L.
[0045] 2.2 Polysaccharide purification
[0046] Ion exchange column chromatography
[0047] (1) Pretreatment of ion exchange chromatography medium: DEAE sepharose FF gel suspension was poured into a Buchner funnel, the liquid was removed, and about 3 volumes of ultrapure water were used to wash, and the above operation was repeated several times until the filler had no alcohol smell. The gel was transferred to a beaker, and one-half to one volume of distilled water was added, and stirred to prepare for column packing.
[0048] (2) Column packing and equilibration: After pretreatment, the DEAE sepharose FF gel suspension was stirred and slowly added to the XK chromatography column (φ3.0×50 cm). After complete settlement, the column head was connected to a peristaltic pump, and the pump was started to flush the column with ultrapure water at a flow rate of 250 cm / h until the gel surface was stable. The column packing was completed.
[0049] (3) Sample loading and elution: The polysaccharide sample was dissolved in an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min to remove the precipitate, filtered with a 0.45 μm microporous filter, and the filtrate was loaded onto the equilibrated ion exchange chromatography column. The loading amount was 30% of the column volume. After the sample liquid entered the chromatography column, it was eluted and separated according to different salt concentrations (dH2O, 0.2 M NaCl, 0.5 M NaCl, 1.0 M NaCl) in sequence. Each concentration was eluted for 2 column volumes at a flow rate of 15 ml / min. The automatic fraction collector collected 100 tubes for each eluent gradient, with each tube collecting 10 ml. The anthrone-sulfuric acid method was used to track and detect the polysaccharide content in the eluent at 630 nm. The number of tubes was taken as the abscissa, and the absorbance was taken as the ordinate to obtain the polysaccharide elution curve. The eluent of the same elution peak area with higher peak height and better symmetry was collected, and the polysaccharide was enriched and purified by repeating the gel filtration column chromatography multiple times. The combined eluent components were concentrated under reduced pressure, dialyzed in a 3.5 KDa cutoff dialysis bag, vacuum freeze-dried, and the gel filtration column chromatography separated and purified polysaccharide was obtained.
[0050] Gel filtration column chromatography
[0051] (1) Gel filtration chromatography medium pretreatment: The gel filtration chromatography medium Chromdex 75 PG gel suspension was poured into a Buchner funnel, the liquid was removed, and about 3 volumes of ultrapure water were used to wash, and the above operation was repeated several times until the filler had no alcohol smell. It was transferred to a beaker, and one-half to one times the volume of the settled gel was added to the distilled water and stirred evenly for column loading.
[0052] (2) Column loading and equilibration: After the pretreated gel filtration chromatography medium gel suspension was stirred, it was slowly added to the XK chromatography column (φ 2.6 x 100 cm). After the gel completely settled, the column head was connected to the chromatography system, and the column was flushed with ultrapure water at a flow rate of 30 cm / h to stabilize the gel surface, and the column loading was completed.
[0053] (3) Sample loading and elution: The polysaccharide sample prepared by ion exchange column chromatography was dissolved in an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min to remove the precipitate, filtered with a 0.45 μm microporous filter, and the filtrate was loaded onto the equilibrated gel filtration column. The loading amount was 1% of the column volume. After the sample liquid entered the chromatography column, it was eluted and separated with 20 mM ammonium bicarbonate solution at a flow rate of 1.8 ml / min. The automatic fraction collector collected 4.5 ml per tube, and 1 column volume was eluted. The collected eluent was detected online by a differential detector. The eluent of the same elution peak area with higher peak height and better symmetry was collected, and the polysaccharide was enriched and purified by repeating the gel filtration column chromatography multiple times. The combined eluent components were concentrated under reduced pressure, vacuum freeze-dried, and the gel filtration column chromatography separated and purified polysaccharide was obtained.
[0054] 2.3 Polysaccharide purity identification and determination of molecular weight distribution
[0055] (1) Molecular weight calibration curve: The dextran standard of different molecular weight (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analysis standard) was weighed, and 0.05M NaCl solution was added to prepare a standard solution of 5mg / ml dextran, which was filtered with a 0.22μm microporous filter for use. HPGPC method was used, and high efficient gel permeation chromatography column was used for detection. The logarithmic value of the relative molecular mass of the standard was used as the vertical coordinate, and the retention time of the corresponding chromatographic peak was used as the horizontal coordinate for linear regression to obtain the calibration curve.
[0056] (2) Preparation of test sample solution: The purified polysaccharide sample was weighed, and 0.05M NaCl solution was added to prepare a test sample solution of 5mg / ml. The supernatant was filtered with a 0.22μm microporous filter, and then the sample was transferred to a 2ml sample bottle for use.
[0057] (3) Chromatographic method: HPGPC method was used, and high performance liquid chromatograph was used with differential detector, polymer matrix water-soluble SEC (GFC) chromatographic column Ohpak SB-803 HQ, Ohpak SB-804 HQ, Ohpak SB-805 HQ (8x300mm) column detection, mobile phase 0.05M NaCl solution, flow rate 0.65ml / min, column temperature 40°C, sample size 30μl.
[0058] 2.4 Experimental results
[0059] 2.4.1 Ion exchange column chromatography results
[0060] The crude polysaccharide sample was subjected to ion exchange column chromatography, and gradient elution was carried out according to different salt concentrations dH2O, 0.2M NaCl, 0.5M NaCl and 1.0M NaCl solution in turn. Anthrone-sulfuric acid method was used to determine the polysaccharide content of each tube. The polysaccharide content obtained by 0.2M NaCl elution component was significantly more than that of dH2O elution component, 0.5M NaCl and 1.0M NaCl solution elution component.
[0061] 2.4.2 Gel filtration column chromatography results
[0062] According to the elution curve and molecular weight test results obtained by ion column chromatography, component sample Y528-E2 was selected as the sample for gel filtration column chromatography. After gel filtration column chromatography, the elution curve was obtained, the elution components in the 120-135 min region were collected, concentrated under reduced pressure, vacuum freeze-dried, and repeatedly subjected to gel filtration column chromatography to obtain the separated and purified polysaccharide named Y528E2-F.
[0063] Example 3, Structure characterization of exopolysaccharide
[0064] 1. Extraction of exopolysaccharide sample
[0065] 5mg of Y528E2-F was weighed and 1mL of 2M trifluoroacetic acid was added. It was heated at 121°C for 2 hours. The mixture was subjected to nitrogen blowing, and the water was blown off and then washed with 3mL of methanol. It was blown dry again and this process was repeated twice. The mixture was poured into a chromatography bottle, and 5mL of DW was used for dissolution.
[0066] 2. 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization
[0067] In a centrifuge tube, 0.2mL of EPS solution was taken, 0.2mL of 0.5mol / L sodium hydroxide solution and 0.5mL of 0.5mol / L PMP methanol solution were added. Mix well by vortex, then react in 70°C water bath for 1h, then add 0.2mL of 0.5mol / L hydrochloric acid to neutralize the added sodium hydroxide, then add 1mL of chloroformic acid. Vortex and extract 3 times to remove excess PMP, then discard the chloroform. After discarding the chloroform, 0.3mL of solution was added to DW, and the volume was adjusted to 1mL.
[0068] Table 1, Proportion of monosaccharide composition
[0069]
[0070] Table 2, Comparison of the proportion of each monosaccharide in LBA3-produced exopolysaccharide Y528E2F and crude sample
[0071]
[0072] Table 3, Monosaccharide type produced by Lactobacillus bulgaricus LBA3 and its functional significance
[0073]
[0074] Table 4, Composition of monosaccharide residues
[0075]
[0076] Table 5, EPS production of several other strains
[0077]
[0078] The Lactobacillus bulgaricus LBA3 of the present application is derived from Yunnan sour milk fan, and has good adaptability to traditional fermentation process due to its long-term adaptation to fermentation environment. The strain is gram-positive and has typical metabolic characteristics of lactic acid bacteria, and can produce acid and synthesize exopolysaccharide. Table 1 shows the proportion of monosaccharide composition of the exopolysaccharide produced by LBA3, and mannose (Man) and glucose (Glc) are the core components (accounting for more than 85%), which is consistent with the typical glucomannan characteristics of lactic acid bacteria EPS. Table 2 is the comparison result of the proportion of each monosaccharide in the crude sample and the exopolysaccharide Y528E2F produced by LBA3, and the proportion of mannose and glucose is higher than that of the crude sample. The monosaccharide composition of exopolysaccharide affects the viscosity of yogurt, especially when the proportion of glucose in the monosaccharide composition increases, the thickening ability is significantly enhanced, which is beneficial to improve the viscosity of yogurt.
[0079] The exopolysaccharide produced by the strain has low uronic acid content (glucuronic acid + galacturonic acid = 0.176%), indicating that it is weakly acidic and more resistant to gastric acid degradation. The type and functional significance of monosaccharide are shown in Table 3, and the monosaccharide composition is significantly regulated by the acid water environment, and can interact with the flora to jointly shape the unique structure and functional characteristics of the milk fan. Therefore, the exopolysaccharide of the strain has definite application potential in the development of functional dairy products.
[0080] Table 4 shows that the exopolysaccharide produced by LBA3 contains various monosaccharide residues and connection modes, such as terminal mannose residues (t-Manp, relative molar ratio 33.639%), 1,2,6-linked mannose residues (22.448%), terminal glucose residues (t-Glcp, 11.534%), etc., which belongs to heteropolysaccharide. The rich monosaccharide composition and complex structure may endow it with more excellent biological activity and functional characteristics, such as immune regulation, antibacterial activity, etc. Table 5 collects the contents of exopolysaccharides produced by four common strains, indicating that LBA3 has high exopolysaccharide production capacity and research significance.
[0081] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.
Claims
1. A Lactobacillus bulgaricus producing exopolysaccharide, characterized in that, The preservation number thereof is CGMCC NO: 29906.
2. An inoculant characterized in that, The bacterial agent contains the Lactobacillus bulgaricus and / or the culture of the Lactobacillus bulgaricus according to claim 1.
3. The bacterial agent of claim 2, wherein The culture of the Lactobacillus bulgaricus is a substance obtained by culturing the Lactobacillus bulgaricus in a bacterial culture medium.
4. The bacterial agent of claim 2, wherein The active ingredient of the bacterial agent is the Lactobacillus bulgaricus and / or the culture of the Lactobacillus bulgaricus, and a surfactant, an adhesive, a stabilizer, a pH regulator are additionally added.
5. The bacterial agent of claim 4, wherein The surfactant is Tween 20 or Tween 80, and the stabilizer is an antioxidant.
6. The bacterial agent of claim 2, wherein The culture is obtained from the fermentation broth of the Lactobacillus bulgaricus; specifically, it is a sterile metabolite of the Lactobacillus bulgaricus or a bacterial-containing metabolite of the Lactobacillus bulgaricus.
7. The bacterial agent of claim 6, wherein The sterile metabolite of the Lactobacillus bulgaricus is prepared by culturing the Lactobacillus bulgaricus in a liquid culture medium, and filtering the Lactobacillus bulgaricus in the liquid fermentation broth to obtain the sterile culture of the Lactobacillus bulgaricus; the bacterial-containing metabolite of the Lactobacillus bulgaricus is prepared by culturing the Lactobacillus bulgaricus in a liquid fermentation culture medium, and collecting the fermentation broth, which is the bacterial-containing culture of the Lactobacillus bulgaricus.
8. The method for preparing the microbial agent according to any one of claims 1 to 7, characterized in that, It comprises the following step: the bacterial agent is prepared by taking the Lactobacillus bulgaricus as the active ingredient.
9. The use of the Lactobacillus bulgaricus according to claim 1 or the bacterial agent according to any one of claims 2 to 7 in the preparation of exopolysaccharide or in the preparation of an exopolysaccharide product.
10. A method of preparing an exopolysaccharide, characterized by, It comprises the following step: the Lactobacillus bulgaricus according to claim 1 is cultured in a culture medium, and exopolysaccharide is obtained from the fermentation broth.