Bacillus coagulans MC-A9 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus coagulans MC-A9 and application thereof. The newest classification name of the bacillus coagulans is bacillus coagulans MC-A9, the bacillus coagulans are preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241012. According to the bacillus coagulans MC-A9 and the application thereof in the fermented milk, the bacillus coagulans MC-A9 has excellent uric acid degradation capacity, antibacterial capacity and ACE activity inhibition capacity, the fermented milk prepared by using the bacillus coagulans MC-A9 as an auxiliary leavening agent has the advantages of excellent flavor, good viscosity and the like, the product quality and safety can be improved, and the fermented milk has the advantages of being good in fermentation effect and the like. Wide application prospects and economic values are realized in the fields of foods and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a Bacillus coagulans MC-A9 and its applications. Background Art

[0002] Bacillus coagulans is a Gram-positive, facultative anaerobic probiotic bacterium that can produce lactic acid and spores. Bacillus coagulans was discovered in spoiled milk in the early 19th century and has the dual characteristics of lactic acid bacteria and bacillus. Its former name was Bacillus coagulans, and the latest taxonomic proposal is the species name Heyndrickxia coagulans. Bacillus coagulans not only has health care characteristics but also has strong stress resistance such as high temperature resistance, acid resistance, and bile salt resistance. In addition, it has a strong inhibitory effect on pathogenic bacteria in the intestine. Bacillus coagulans is used in industries such as food, medicine, animal husbandry, aquaculture, and health care, and this strain is safe and beneficial for human consumption. For example, Patent 202410614448.6 discloses a Bacillus coagulans and its applications, and the Bacillus coagulans LBK has the effect of alleviating oxidative stress caused by acute cadmium exposure and can regulate the amino acid metabolism in the cecum to be normal. Patent 202410192397.2 discloses the application of a Bacillus coagulans in the preparation of a product for regulating immunity or improving renal function, and the strain has the probiotic effect of regulating the body's immunity and improving renal function in the body.

[0003] Fermented milk is considered the most suitable matrix for delivering probiotics in food. Compared with traditional probiotics, there is broad prospect in developing spore-forming probiotic fermented milk. Bacillus coagulans has attracted attention due to its high survival rate in various food matrices and beneficial effects on human health. Therefore, it is of great significance to develop fermented milk containing Bacillus coagulans. Summary of the Invention

[0004] The present invention provides a Bacillus coagulans MC-A9 and its applications to solve the problems raised in the above background art.

[0005] In the first aspect, a Bacillus coagulans MC-A9 is provided:

[0006] It was deposited at the China Center for Type Culture Collection on May 20, 2024, with the deposit number CCTCC NO: M20241012.

[0007] In the second aspect, an application of the above-mentioned Bacillus coagulans MC-A9 as an auxiliary starter in fermented milk is provided.

[0008] The beneficial effects brought by the technical solution provided by the present invention include:

[0009] The present invention provides a strain of Bacillus coagulans MC-A9 and its applications, which have excellent uric acid degradation ability, antibacterial ability, and ACE activity inhibition ability. The fermented milk prepared using the Bacillus coagulans MC-A9 described in the present invention as an auxiliary starter has excellent flavor and good viscosity, etc., and can improve the product quality and safety, and has broad application prospects and economic value in the fields of food, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0011] Figure 1 It is the phylogenetic tree of the 16S rDNA sequence of Bacillus coagulans MC-A9;

[0012] Figure 2 It is the (left) Gram staining map and (right) colony morphology map of Bacillus coagulans MC-A9;

[0013] Figure 3 It is the picture of the uric acid degradation zone of Bacillus coagulans MC-A9;

[0014] Figure 4 It is the picture of the antibacterial zone of Candida albicans by Bacillus coagulans MC-A9;

[0015] Figure 5 It is the picture of the antibacterial zone of Escherichia coli by Bacillus coagulans MC-A9. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] See Figures 1 - 5 As shown, the present invention provides a strain of Bacillus coagulans MC-A9 and its applications.

[0018] Specifically, the Bacillus coagulans MC-A9 is classified and named as Bacillus coagulans MC-A9, and was deposited at the China Center for Type Culture Collection on May 20, 2024, with the deposit number CCTCC NO: M 20241012.

[0019] Example 1: Molecular biological identification of Bacillus coagulans MC-A9

[0020] The Bacillus coagulans MC-A9 is derived from a fermented vegetable sample in Qixiaying Town, Zhuozi County, Wulanchabu City, Inner Mongolia Autonomous Region.

[0021] The gene sequence of Bacillus coagulans MC-A9 is shown in SEQ ID NO.1.

[0022] The pure bacteria of Bacillus coagulans MC-A9 were inoculated into MRS liquid medium and cultured with shaking at 37°C until the logarithmic phase. The bacterial sludge was collected, and after repeated freezing and thawing, amplification was carried out using the universal bacterial 16S rDNA primers 27F (sequence: 5'AGAGTTTGACCTGGCTAG-3' as SEQ ID NO.2) and 1495R (sequence: 5'-CTACGGCTCCTTGTTCGA-3' as SEQ ID NO.3). After the amplification products were detected by 0.1% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After comparing the obtained sequencing results with the sequences in the database, it had a high homology with the genus Heyndrickoda coagulans and belonged to Bacillus coagulans (Heyndrickoda coagulans).

[0023] The 16S rDNA gene sequence (SEQ ID NO.1) of the Bacillus coagulans MC-A9 is:

[0024]

[0025] Example 2: Uric acid degradation ability of Bacillus coagulans MC-A9

[0026] (1) Plate method: Using the Oxford cup method, pour the prepared uric acid solid medium into a plate. After solidification, pull out the Oxford cup, add the bacterial solution into the hole, and incubate at 37°C for 48 h. As shown in Figure 3 , the size of the uric acid dissolution zone was observed to be 12.60 ± 0.36 mm;

[0027] (2) High performance liquid chromatography (HPLC): Take an appropriate amount of the secondary bacterial solution, centrifuge to collect the bacterial cells, wash them 3 times with PBS buffer, and then resuspend them in UA phosphate buffer. After static incubation at 37°C for 4 d, centrifuge the obtained bacterial solution at 8000 r / min for 10 min at 4°C. Pipette 900 μL of the supernatant, filter and sterilize the supernatant with a 0.22 aqueous filter membrane (a filter membrane with a pore size of 0.22 μm), and then mix it evenly with 100 μL of the terminator perchloric acid for HPLC analysis. The results showed that the uric acid degradation rate was 23.73 ± 0.87%.

[0028] Example 3: ACE activity inhibition ability of Bacillus coagulans MC-A9

[0029] Use a bacterial angiotensin-converting enzyme (ACE) ELISA detection kit. Add specimens, standards, and HRP-labeled detection antibodies to the coated microwells pre-coated with angiotensin-converting enzyme (ACE) antibodies in sequence, incubate and wash thoroughly. Color with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The color depth is positively correlated with angiotensin-converting enzyme (ACE) in the sample. Measure the absorbance (OD value) with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The measured ACE activity inhibition ability was 75.1 U / L.

[0030] Example 4:

[0031] As shown in Figure 4 and Figure 5 , Bacillus coagulans MC-A9 showed antagonistic effects against the common intestinal pathogenic fungi Candida albicans Y54 ( Figure 4 ) and Escherichia coli T22 ( Figure 5 ).

[0032] Example 5: Fermentation performance of Bacillus coagulans MC-A9

[0033] Furthermore, the Bacillus coagulans MC-A9 and a commercially available Chr. Hansen starter culture were inoculated into MRS medium at a ratio of 1:1, with an inoculation amount of 5%, a white sugar addition amount of 4%, and an inulin addition amount of 6%, and fermented and cultured at a constant temperature of 43°C until the fermentation ended when the pH value reached 4.6 to obtain fermented milk. After the fermented milk was placed in a 4°C refrigerator for 12 hours of after-ripening, the sensory evaluation method was as shown in Table 1:

[0034] Table 1

[0035]

[0036]

[0037] The sensory scoring criteria refer to GB 19302-2010 "National Food Safety Standard Fermented Milk" with slight modifications. Ten professional sensory evaluation personnel (50% male and 50% female) conducted sensory evaluation on the mixed fermented milk of Bacillus coagulans MC-A9. The evaluation indicators included color (20 points), taste and odor (40 points), texture (20 points), and whey separation (20 points). The full score of the sensory score was 100 points. Each sample was tasted in parallel 3 times, and the calculated sensory score was 91.8 points;

[0038] 300 mL of the fermented milk after fermentation was taken and placed in a special cup for texture analyzer measurement, and the hardness of the fermented milk containing Bacillus coagulans MC-A9 was found to be 34.27 ± 3.66 g, the viscosity was 62.24 ± 5.03 g*s, and the adhesiveness was -16.75 ± 1.86 s.

[0039] The overall fermentation characteristics of the fermented milk added with Bacillus coagulans MC-A9 are shown in Table 2:

[0040] Table 2

[0041]

[0042] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A strain of Bacillus coagulans MC-A9, characterized in that: The deposit number of the Bacillus coagulans MC-A9 is CCTCC NO: M 20241012, the deposit date is May 20, 2024, and it is deposited in the China Center for Type Culture Collection.

2. Use of the Bacillus coagulans MC-A9 as claimed in claim 1 as an auxiliary fermentation agent in fermented milk.

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