Bacillus coagulans strain from the south china sea deep sea and application thereof

Abstract

This invention provides a strain of *Bacillus coagulans* derived from the deep sea of ​​the South China Sea and its applications. The *Bacillus coagulans* strain was deposited on February 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located in Guangzhou, with accession number GDMCC No: 64379. The *Bacillus coagulans* strain of this invention exhibits low-temperature delayed growth characteristics, is easy to culture, and can produce spores even at low temperatures in seawater. It also demonstrates strong thermostability. Furthermore, it produces highly active lactase and protease, and the prepared probiotic powder exhibits strong storage stability.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of Bacillus coagulans originating from the deep sea of ​​the South China Sea and its application. Background Technology

[0002] According to the World Health Organization, probiotics are defined as "live microorganisms that, when given in adequate amounts, are beneficial to the host." Common probiotics include lactic acid bacteria, bifidobacteria, Bacillus, and yeast. However, most probiotics derived from the human gut and fermented products are heat-sensitive, limiting their application in high-temperature processing technologies, and they also have poor shelf-life stability.

[0003] Bacillus coagulans is a Gram-positive, rod-shaped, sporulatory, aerobic or facultative anaerobic bacterium belonging to the genus Bacillus. It possesses probiotic characteristics, is heat-resistant, and easy to store. The lactic acid it produces can lower the pH value in the intestine, inhibiting the growth of harmful bacteria. Furthermore, it secretes various nutrients such as amino acids and vitamins, as well as enzymes such as proteases and amylases. It can also work synergistically with other probiotics such as Lactobacillus to promote intestinal peristalsis and accelerate the digestion and absorption of food.

[0004] Lactose intolerance refers to a series of symptoms such as diarrhea and bloating caused by the inability of the gut to digest and absorb lactose after consuming cow's milk or breast milk, due to a lack of enzymes in the gut to break down lactose. Probiotics can alter the intestinal pH, promoting the digestion of lactose into β-galactose and positively impacting intestinal activity and the overall colonic microbiota. This effect not only helps improve lactose intolerance symptoms but also enhances overall gut health.

[0005] This invention screened a new species of Bacillus coagulans from the ocean and conducted a preliminary evaluation of its biological activity, aiming to provide candidate strains for scientific research and product development of marine probiotics. The prepared freeze-dried probiotic powder can be applied to the production of functional fermented foods, and has broad market prospects in the future. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a strain of Heyndrickxia coagulans from the deep sea of ​​the South China Sea. The strain of Heyndrickxia coagulans was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on February 28, 2024, with the deposit address in Guangzhou and the deposit number GDMCC No: 64379. The Heyndrickxia coagulans was isolated from the deep sea of ​​the South China Sea.

[0007] Preferably, the Bacillus coagulans DNA sequence is SEQ ID NO.1.

[0008] Preferably, the Bacillus coagulans powder is used to alleviate lactose intolerance and promote protein digestion.

[0009] Preferably, the probiotic powder prepared by Bacillus coagulans is used.

[0010] Preferably, the probiotic powder has strong storage stability.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. This invention isolates and identifies a strain of Bacillus coagulans (Heyndrickxia coagulans). Through genome sequence analysis and phylogenetic tree construction, the strain is identified as Bacillus coagulans.

[0013] 2. Compared with the prior art, the Bacillus coagulans in this invention has the characteristics of low-temperature delayed growth, is easy to cultivate, can produce spores even at low temperatures in seawater, and has strong heat resistance expressed by Bacillus coagulans.

[0014] 3. The Bacillus coagulans in this invention produces lactase and protease with high activity, and can alleviate lactose intolerance and promote protein digestion.

[0015] 4. The probiotic powder prepared by Bacillus coagulans in this invention has extremely strong storage stability. Attached Figure Description

[0016] Figure 1 This is a colony diagram of strain BC04 in Example 1 of the present invention;

[0017] Figure 2 This is a microscopic image of strain BC04 from Example 1 of the present invention;

[0018] Figure 3 This is an agarose gel electrophoresis image of the PCR amplification product of strain BC04 in Example 2 of the present invention;

[0019] Figure 4 This is the phylogenetic tree of strain BC04 in Example 2 of the present invention;

[0020] Figure 5 This is an example of the growth of strain BC04 in Example 3 of the present invention at different temperatures;

[0021] Figure 6 Example 5 of this invention illustrates the effect of different freeze-drying protectants on the survival rate of Bacillus coagulans BCO4.

[0022] Figure 7a Example 6 of the present invention illustrates the effect of Bacillus coagulans BCO4 bacterial powder on lactose(a) content in milk at different time points.

[0023] Figure 7b This is Example 6 of the present invention, showing the effect of Bacillus coagulans BCO4 bacterial powder on protein (b) content at different times;

[0024] Figure 7c This shows the change in the number of viable bacteria in milk at different times for strain BC04 in Example 6 of the present invention (c). Detailed Implementation

[0025] Example 1: Obtaining the Heyndrickxia coagulans strain.

[0026] 1. Screening of strains

[0027] Seawater samples were collected from the deep sea of ​​the South China Sea. Each sample was cultured, serially diluted, and plated onto MRS agar plates. The plates were numbered and anaerobically incubated at 45°C for 24 hours. Single colonies with good growth were selected and inoculated into test tubes for further incubation. The colonies were clearly defined, raised, whitish in color, moist, and round in shape, appearing as short, thin rods under a microscopic view (e.g.,...). Figure 1 ,2).

[0028] Example 2: Gene sequence analysis of *Heyndrickxia coagulans* strain

[0029] 1. Bacterial DNA genome extraction:

[0030] The culture medium of the strain obtained in Example 1 was centrifuged and the precipitate was collected for DNA extraction.

[0031] 2. Perform PCR amplification of 16S rDNA on this strain:

[0032] PCR amplification: PCR amplification was performed using primers 27F and 1492R. The 27F sequence was 5′-AGAGTTTGATCCTGGCTCAG-3′, and the 1492R sequence was 5′-GGTTACCTTGTTACGACTT-3′. The PCR reaction mixture consisted of 25 μL TAQ enzyme, 2 μL 27F primer, 2 μL 1492R primer, 5 μL bacterial template, and 16 μL sterile water. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 60 s, 56℃ annealing for 60 s, 72℃ extension for 90 s, 30 cycles; 72℃ extension for 7 min, and termination at 4℃. Gel electrophoresis and PCR product recovery: The PCR products were identified by 1.5% agarose gel electrophoresis. The target PCR products were recovered and purified using a rapid agarose gel DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd.) and then sequenced as 16S rDNA. PCR products were identified by 1% agarose gel electrophoresis (e.g.) Figure 3 ). Observe whether the target band is amplified.

[0033] 3. 16S rDNA sequencing and homology analysis

[0034] The target PCR product was recovered and purified using a rapid agarose gel DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd.). The purified PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the gene sequence is shown in SEQ ID NO: 1. The obtained sequence was submitted to NCBI's GENBANK database to obtain similar sequences. Comparative analysis was performed using BLAST tools and DNAMAN software, and a phylogenetic tree was constructed using the Neighbor-Joining method, as shown below. Figure 4 As shown.

[0035] Phylogenetic analysis revealed that this strain is highly related to Heyndrickxia coagulans and others, thus identifying it as a marine-derived Bacillus coagulans, which we named Heyndrickxia coagulans BC04.

[0036] The coagulant bacillus obtained from the above screening was named Heyndrickxiacoagulans, strain number BC04. It was deposited on February 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located in Guangzhou, with accession number 64379.

[0037] Example 3 Temperature tolerance test of Bacillus coagulans BC04

[0038] The fermentation broth was centrifuged at 4000 r / min and 4℃ for 6 min, the supernatant was discarded, the cells were washed twice with PBS, and 1% was inoculated into MRS culture medium. The samples were placed in shakers at 20, 30, 37, 40, 45, 50 and 55℃ and cultured at 200 rpm. Samples were taken at 0, 10, 22, 34 and 46 h, and the optical density (OD) value was measured at a wavelength of 600 nm.

[0039] from Figure 5 As can be seen, Bacillus coagulans BC04 can grow at temperatures ranging from 20 to 50°C, with a maximum tolerance temperature of 50°C. It cannot grow at 55°C, and the bacterial concentration (OD) of strain BC04 decreases with increasing temperature. 600nm The growth rate of Bacillus coagulans BC04 decreased. At 20–30°C, it began to grow after 22 hours. At 37–50°C, it began to grow after 10 hours, and its growth tended to stabilize at 32 hours. In summary, Bacillus coagulans BC04 exhibits the characteristic of delayed growth at low temperatures, and its later growth is better at lower temperatures.

[0040] Enzyme production characteristics of strain 4 in Example 4

[0041] 1. Methods for determining lactase activity

[0042] The strain BC04 was incubated in the fermentation broth for 20 h, and then disrupted using a cell disruptor (on for 1 second, off for 1 second, power 150W) for 30 min. The supernatant was collected by centrifugation to obtain crude enzyme solution. The enzyme activity of lactase produced by the strain was determined according to the method in "Technical Guidelines for Determination of Industrial Enzyme Preparations" (GB / T 35538).

[0043] 2. Methods for determining the enzyme activity of proteases

[0044] The strain BC04 was incubated in the fermentation broth for 20 hours, and then disrupted using a cell disruptor (1 second on, 1 second off, 150W power) for 30 minutes. The supernatant was collected by centrifugation to obtain crude enzyme solution. The enzyme activity of the protease produced by the strain was determined according to the method of "Determination of the Activity of Acidic and Neutral Proteases in Feed Additives - Spectrophotometric Method" (GB / T28715).

[0045] As shown in Table 1, Bacillus coagulans BC04 can produce lactase, acidic protease, and neutral protease, with enzyme activities of 11.2±0.06, 16.24±0.19, and 21.59±0.83 U / mL, respectively.

[0046] Table 1 Enzyme production analysis of strain BC04

[0047]

[0048] Example 5: Preparation of probiotic powder of Bacillus coagulans BC04

[0049] (1) Scale-up culture of Bacillus coagulans BCO4

[0050] Bacillus coagulans BC04 was inoculated into sterile liquid culture medium at a 1% (v / v) inoculum and cultured at 200-400 rpm and 37°C for 16-46 hours to obtain activated seed culture liquid. The seed culture liquid was then inoculated into fermentation medium at a 0.5-4% (v / v) inoculum and cultured at a constant temperature of 200-400 rpm. The pH was maintained at 6.0 ± 0.2 during fermentation. Samples were taken every 4 hours to measure OD. Fermentation was considered complete when OD remained constant or decreased, yielding a high-density culture of Bacillus coagulans BC04 with colony counts of 1-5 × 10⁻⁶. 10 The CFU / mL concentration was above 1. The fermentation medium consisted of: lactose 5–20 g / L, glucose 0.5–2 g / L, yeast extract 5–20 g / L, peptone 3–15 g / L, beef extract 1–5 g / L, potassium dihydrogen phosphate 1–4 g / L, manganese sulfate 0.05–0.2 g / L, and ferrous chloride 0.1–0.4 g / L. Compared to the initially cultured Bacillus coagulans, the colony count increased by 30.45 times.

[0051] Table 2. Fermentation medium formulation for strain BC04

[0052]

[0053]

[0054] (2) Preparation of probiotic powder of Bacillus coagulans BCO4

[0055] The fermentation broth of Bacillus coagulans BC04, after cultivation, was centrifuged at 4000 rpm for 30 min. The supernatant was discarded, and the bacterial sludge was collected. The bacterial sludge was washed 2-3 times with sterile physiological saline. The washed bacterial sludge was then mixed separately with skim milk powder (1%, 5%, 10%, 15%, 20%, m / v), fructooligosaccharides (1%, 5%, 10%, 15%, 20%, m / v), and monosodium glutamate (1%, 5%, 10%, 15%, 20%, m / v) as a preservative, with the dry matter ratio of bacterial sludge to preservative being... For example, the ratio of bacteria to 1:1.2 (m:m) was mixed evenly and then pre-frozen at -80℃ for 12 hours. The pre-frozen bacterial cells were then placed in a freeze dryer and freeze-dried for 36 hours. After freeze-drying, the sample was pulverized using a grinder to obtain the freeze-dried bacterial powder. The survival rate was calculated based on the number of viable bacteria before and after freeze-drying to determine the optimal ratio of the freeze-drying agent. Through comparison, the optimal ratio was determined to be 10% skim milk powder, 10% fructooligosaccharides, and 5% monosodium glutamate, with a survival rate of 87.64%, which is more than 10 times higher than the survival rate of bacterial sludge without the use of freeze-drying protectant.

[0056] Survival rate % = (Total viable bacteria after freeze-drying / Total viable bacteria before freeze-drying) × 100%

[0057] (3) Storage stability of Bacillus coagulans BCO4 powder

[0058] The bacterial powders without and with a compound lyophilization protectant were stored at -20°C, 4°C, and 25°C, respectively. The viable cell counts were measured after 0 and 30 days, and the survival rate was calculated. Table 3 shows that the survival rate of the bacterial powder with the compound protectant remained almost unchanged after one month of storage at -20°C and 4°C, and reached 90.35% at 25°C. In contrast, the survival rate of the lyophilized bacterial powder without the protectant was only half at 4°C and 30.31% at 25°C, indicating a greater loss of activity. Therefore, the lyophilized bacterial powder with the compound protectant exhibited stronger storage stability.

[0059] Table 3. Survival rates of freeze-dried bacterial powders with and without added compound preservatives after 30 days of storage at different temperatures.

[0060]

[0061] Example 6: The Digestive Effect of Bacillus coagulans BCO4 Powder on Milk

[0062] An experiment was conducted to investigate the effects of Bacillus coagulans BC04 on alleviating lactose intolerance and promoting protein digestion, using whole milk as an example. 250 mL of whole milk was taken and divided into portions containing 0.5%, 1%, and 1.5% of the bacterial powder with added compound preservative. The mixture was incubated in a 37°C water bath for 6 hours. Samples were taken every 2 hours to determine lactose and protein content. Lactose was determined according to GB 5009.8-2023, and protein was determined according to SN / T3926-2014.

[0063] As shown in Figures 7(a) and (b) regarding the changes in lactose and protein content over time, the lactose and protein content in milk gradually decreased with increasing time, and the greater the amount of BC04 added, the more significant the decrease in lactose and protein content. Figure 7(c) showing the change in the viable count of strain BC04 over time indicates that with an added bacterial powder concentration of 0.5%–1.5%, the viable count increased with increasing time, and the more bacteria added, the greater the growth. The gradual decrease in lactose and protein content in milk may be due to the growth of strain BC04 in milk, where its whole cells and crude enzyme solution decompose lactose and protein. A higher bacterial quantity and growth density result in more whole cells and crude enzyme solution, leading to a more significant decomposition of lactose and protein. At 6 hours, adding 1.5% bacterial powder to milk resulted in a lactose decomposition rate of 77.51% and a protein decomposition rate of 70.87%.

[0064] In summary, Bacillus coagulans BC04 can reduce the lactose and protein content in milk, thus alleviating lactose intolerance and promoting protein digestion.

[0065] sequence

[0066] SEQ ID NO:1

[0067]

Claims

1. A strain of *Heyndrickxia coagulans* BC04 derived from the deep sea of ​​the South China Sea, characterized by: The Bacillus coagulans strain was deposited on February 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located in Guangzhou, with accession number GDMCC No: 64379; the Bacillus coagulans strain was derived from deep water in the South China Sea.

2. The Bacillus coagulans strain from the deep sea of ​​the South China Sea according to claim 1, characterized in that: The DNA sequence of the Bacillus coagulans is SEQ ID NO.

1.

3. The application of the Bacillus coagulans strain from the deep sea of ​​the South China Sea according to claim 1, characterized in that: The application of the Bacillus coagulans powder in the preparation of products that alleviate lactose intolerance and promote protein digestion.

4. A probiotic powder, characterized in that: The invention comprises a strain of Bacillus coagulans from the deep sea of ​​the South China Sea as described in claim 1 or 2, and a composite freeze-drying protectant; the composite freeze-drying protectant comprises skim milk powder, fructooligosaccharides and monosodium glutamate.

5. The probiotic powder according to claim 4, characterized in that: In the composite freeze-drying protectant, the amount of skim milk powder added is 10% (m / v), the amount of fructooligosaccharide added is 10% (m / v), and the amount of monosodium glutamate added is 5% (m / v); and the mass ratio of the Bacillus coagulans sludge to the dry matter of the composite freeze-drying protectant is 1:1.2.

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