Bacillus coagulans from deep sea of South China Sea and application thereof

Abstract

The invention provides bacillus coagulans from deep sea of South China Sea and application of the bacillus coagulans, the lactobacillus plantarum strain is preserved in Guangdong Microbiological Culture Collection Center (GDMCC) on February 28, 2024, the preservation address is Guangzhou, and the preservation number is GDMCC No: 64379; the bacillus coagulans disclosed by the invention has the growth characteristic of low-temperature delay, is easy to culture and can generate spores in seawater at low temperature, and the high temperature resistance expressed by the bacillus coagulans is high. And lactase and protease with high activity are produced, and the prepared probiotic powder has relatively strong storage stability.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Bacillus coagulans strain originating from the deep sea of ​​the South China Sea and applications thereof. Background Art

[0002] According to the World Health Organization, probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a beneficial effect on the host", and common probiotics include lactic acid bacteria, bifidobacteria, bacillus and yeast. However, most probiotics derived from the human intestine and fermented products are heat-sensitive, which limits their application in high-temperature product processing and has poor shelf life stability.

[0003] Bacillus coagulans is a Gram-positive, rod-shaped, spore-forming, aerobic or facultative anaerobic bacterium in the genus Bacillus. Bacillus coagulans has the characteristics of probiotics, high temperature resistance, and easy storage. The lactic acid it produces can lower the pH value in the intestine and inhibit the reproduction of harmful bacteria. In addition, it can secrete a variety of nutrients such as amino acids, vitamins, and enzymes such as proteases and amylases. It can also play a synergistic role with probiotics such as lactobacilli to promote small intestinal peristalsis and accelerate intestinal digestion and absorption of food.

[0004] "Lactose intolerance" refers to a series of symptoms such as diarrhea and bloating caused by the inability to digest and absorb lactose in the intestines due to the lack of enzymes in the intestines to break down lactose after consuming milk or breast milk. Probiotics can change the pH value of the intestines, promote the digestion of lactose into β-galactose, and have a positive impact on intestinal activity and the overall colon microbiome. This effect not only helps to improve the symptoms of lactose intolerance, but also enhances the overall health of the intestines.

[0005] The present invention screened a new species of Bacillus coagulans from the ocean and conducted a preliminary evaluation of its biological activity in order to provide candidate strains for scientific research and product development of marine probiotics. The prepared probiotic freeze-dried powder can be used in 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 originating from the deep sea of ​​the South China Sea. The Bacillus coagulans strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on February 28, 2024, with a storage address in Guangzhou and a storage number of GDMCC No: 64379; the Bacillus coagulans was isolated from the deep sea water 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 for alleviating lactose intolerance and promoting protein digestion.

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

[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. The present invention isolated and identified a strain of Heyndrickxia coagulans, and determined that the strain was Bacillus coagulans through genome sequence analysis and construction of a phylogenetic tree.

[0013] 2. Compared with the prior art, the Bacillus coagulans of the present invention has the characteristic of delayed growth at low temperature, is easy to culture, can produce spores even in the low temperature of seawater, and has strong high temperature resistance expressed by Bacillus coagulans.

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

[0015] 4. The probiotic powder prepared by Bacillus coagulans in the present invention has extremely strong storage stability. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0020] Figure 5 The growth of strain BC04 at different temperatures in Example 3 of the present invention;

[0021] Figure 6 The effects of different lyophilization protective agents on the survival rate of Bacillus coagulans BC04 in Example 5 of the present invention;

[0022] Figure 7a This is the effect of Bacillus coagulans BC04 powder on the lactose (a) content in milk at different times according to Example 6 of the present invention;

[0023] Figure 7b This is the effect of Bacillus coagulans BC04 powder on the content of protein (b) at different times in Example 6 of the present invention;

[0024] Figure 7c The figure shows the change of the number of viable bacteria of strain BC04 in milk at different times according to Example 6 of the present invention (c). DETAILED DESCRIPTION

[0025] Example 1 Obtaining Heyndrickxia coagulans strain.

[0026] 1. Screening of strains

[0027] Some seawater samples were collected from the deep seawater of the South China Sea, and the collected samples were cultured one by one, and then spread on MRS plate culture medium after gradient dilution. The corresponding plates were numbered and placed in a 45℃ constant temperature incubator for anaerobic culture for 24 hours. The single colonies with good growth were selected and placed in test tubes for re-culture to preserve the bacteria. The colonies have obvious outlines, are convex, white in color, moist, and round in shape. They appear as thin short rods (such as Figure 1 , 2).

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

[0029] 1. Bacterial DNA genome extraction:

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

[0031] 2. PCR amplification of 16S rDNA of the strain:

[0032] PCR amplification: Primers 27F and 1492R were used for PCR amplification. The sequence of 27F was 5′-AGAGTTTGATCCTGGCTCAG-3′, and the sequence of 1492R was 5′-GGTTACCTTGTTACGACTT-3′. The PCR reaction system was: 25 μL of TAQ enzyme, 2 μL of 27F primer, 2 μL of 1492R primer, 5 μL of bacterial liquid template, and 16 μL of sterile water. The PCR amplification program was: 94°C pre-denaturation for 3 min; 94°C denaturation for 60 s, 56°C annealing for 60 s, 72°C extension for 90 s, 30 cycles; 72°C extension for 7 min, and 4°C termination of the reaction. Gel electrophoresis and PCR product gel recovery: PCR products were identified by 1.5% agarose gel electrophoresis. The target PCR product was recovered and purified using the Quick Agarose Gel DNA Recovery Kit (Beijing Biotech Biotechnology Co., Ltd.) and 16S rDNA sequencing was performed. PCR products were identified by 1% agarose gel electrophoresis ( 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 quick agarose gel DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd.), and the purified PCR product was sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The gene sequence is shown in SEQ ID NO: 1. The measured sequence was submitted to GENBANK of NCBI in the United States to obtain similar sequences, and the BLAST tool and DNAMAN software were used for comparative analysis and the Neighbor-Joining method was used to construct a phylogenetic tree, as shown in Figure 4 shown.

[0035] The phylogenetic tree revealed that the strain was highly related to Heyndrickxia coagulans and others, so it was determined to be a marine-derived Bacillus coagulans, which we named Heyndrickxia coagulans BC04.

[0036] The Bacillus coagulans obtained by the above screening was named Bacillus coagulans (Heyndrickxiacoagulans), strain number BC04, and its preservation date was February 28, 2024, and it was preserved in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), the preservation address is Guangzhou, and the preservation number is 64379.

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

[0038] The fermentation broth was centrifuged at 4000 r / min and 4°C for 6 min, the supernatant was discarded, the bacteria were washed twice with PBS, inoculated into MRS culture medium at 1%, the samples were cultured in a shaker at 200 rpm at 20, 30, 37, 40, 45, 50, and 55°C, and 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 It can be seen that Bacillus coagulans BC04 can grow at 20-50℃, with the highest tolerance temperature being 50℃, and cannot grow at 55℃. As the temperature increases, the bacterial concentration OD of strain BC04 600nm At 20-30°C, Bacillus coagulans BC04 began to grow after 22 hours. At 37-50°C, Bacillus coagulans BC04 began to grow after 10 hours, and the growth tended to be stable at 32 hours. In summary, Bacillus coagulans BC04 has the characteristics of delayed growth at low temperature, and its later growth is better at low temperature.

[0040] Example 4 Enzyme production characteristics of strains

[0041] 1. Lactase enzyme activity determination method

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

[0043] 2. Method for determining the enzyme activity of protease

[0044] The strain BC04 was incubated in the fermentation broth for 20 h, disrupted with a cell disruptor (on 1 s, off 1 s, power 150 W) for 30 min, and the supernatant was centrifuged to obtain the crude enzyme solution. The enzyme activity of the protease produced by the strain was determined by referring to the method of "Spectrophotometric method for determination of acidic and neutral protease activity of feed additives" (GB / T28715).

[0045] As shown in Table 1, Bacillus coagulans BC04 can produce lactase, acid and neutral proteases, and their enzyme activities are 11.2±0.06, 16.24±0.19, and 21.59±0.83 U / mL, respectively.

[0046] Table 1 Analysis of enzyme production of strain BC04

[0047]

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

[0049] (1) Scale-up culture of Bacillus coagulans BC04

[0050] Inoculate Bacillus coagulans BC04 in a sterile liquid culture medium at an inoculum rate of 1% (v / v), and culture at 200-400rpm and 37°C for 16-46 hours to obtain an activated seed culture liquid. Inoculate the seed culture liquid in a fermentation medium at an inoculum rate of 0.5-4% (v / v), and culture at a constant temperature of 200-400rpm. During the fermentation process, the pH is maintained at 6.0±0.2. Take a sample every 4 hours and measure the OD. When the OD remains unchanged or decreases, the fermentation endpoint is reached, and a high-density culture liquid of Bacillus coagulans BC04 is obtained, and the number of colonies can reach 1 to 5×10 10 CFU / mL or more. The fermentation medium composition is: lactose 5-20g / L, glucose 0.5-2g / L, yeast extract 5-20g / L, peptone 3-15g / L, beef extract 1-5g / L, potassium dihydrogen phosphate 1-4g / L, manganese sulfate 0.05-0.2g / L, ferrous chloride 0.1-0.4g / L, the colony count is increased by 30.45 times compared with the initial culture of Bacillus coagulans.

[0051] Table 2 Strain BC04 fermentation medium formula

[0052]

[0053]

[0054] (2) Preparation of probiotic powder of Bacillus coagulans BC04

[0055] The fermented bacterial liquid of Bacillus coagulans BC04 after cultivation was centrifuged at 4000rpm for 30min, the supernatant was discarded, the bacterial sludge was collected, and the bacterial sludge was washed with sterile physiological saline for 2-3 times, and the washed bacterial sludge was mixed with skim milk powder (1%, 5%, 10%, 15%, 20%, m / v), oligofructose (1%, 5%, 10%, 15%, 20%, m / v), sodium glutamate (1%, 5%, 10%, 15%, 20%, m / v) and protective agents respectively to make the dry matter ratio of bacterial sludge to protective agent be 1.5~2.0%. The ratio of the two ingredients is 1:1.2 (m:m). After mixing evenly, the mixture is pre-frozen in a -80°C refrigerator for 12 hours. The pre-frozen bacteria are placed in a freeze dryer and freeze-dried for 36 hours. After freeze drying, the sample is crushed with a grinder to obtain the freeze-dried bacterial powder. The survival rate is calculated based on the number of live bacteria before and after freeze drying to determine the optimal ratio of the freeze-dried agent. The optimal ratio is determined by comparison to be 10% skim milk powder, 10% oligofructose and 5% sodium glutamate, with a survival rate of 87.64%, which is more than 10 times higher than the survival rate of the bacterial mud without using a freeze-drying protective agent.

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

[0057] (3) Storage stability of Bacillus coagulans BC04 powder

[0058] The above-mentioned bacterial powder without adding freeze-dried protective agent and adding composite freeze-dried protective agent were stored at -20, 4, and 25°C, and the number of live bacteria at 0 and 30 days was measured to calculate the survival rate. Table 3 shows that the survival rate of the bacterial powder with added composite protective agent is almost unchanged when stored at -20°C and 4°C for one month, and the survival rate is 90.35% at 25°C, while the survival rate of the freeze-dried bacterial powder without adding protective agent is only half at 4°C, and the survival rate is 30.31% at 25°C, with a large loss of activity. Therefore, the freeze-dried bacterial powder with added composite protective agent has strong storage stability.

[0059] Table 3 Survival rate of freeze-dried bacterial powder without and with composite protective agent stored at different temperatures for 30 days

[0060]

[0061] Example 6 Digestion of milk by Bacillus coagulans BC04 powder

[0062] The experiment of Bacillus coagulans BC04 relieving lactose intolerance and promoting protein digestion takes whole milk as an example. Take 250mL of whole milk, add 0.5%, 1% and 1.5% of bacterial powder with compound protective agent, and place it in a water bath at 37℃ for 6h. Samples are taken every 2h to test the lactose content and protein content. Lactose is determined according to GB 5009.8-2023, and protein is determined according to SN / T3926-2014.

[0063] As shown in Figure 7 (a) and (b) of the changes in lactose and protein content over time, the lactose and protein content in milk gradually decreases with time, and the more BC04 is added, the more obvious the decrease in lactose and protein content. As shown in Figure 7 (c) of the changes in the number of live bacteria of strain BC04 over time, when the added bacterial powder is 0.5-1.5%, the number of live bacteria increases with time, and the more bacteria are added, the more they grow. The gradual decrease in the lactose and protein content in milk may be because the strain BC04 grows in milk, and its whole cells and crude enzyme solution decompose lactose and protein. The more bacteria are added, the higher the growth density, the more whole cells and crude enzyme solution are produced, and the more obvious the decomposition of lactose and protein is. At 6h, the decomposition rate of lactose by adding 1.5% bacterial powder to milk is 77.51%, and the decomposition rate of protein is 70.87%.

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

[0065] sequence

[0066] SEQ ID NO:1

[0067]

Claims

1. A strain of Heyndrickxia coagulans from the deep sea of ​​South China Sea, characterized in that: The Bacillus coagulans strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on February 28, 2024, with the storage address in Guangzhou and the storage number GDMCC No: 64379; the Bacillus coagulans is derived from the deep sea water of the South China Sea.

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

1.

3. Application of a strain of Bacillus coagulans from the deep sea of ​​South China Sea, characterized in that: The application of the Bacillus coagulans powder in alleviating lactose intolerance and promoting protein digestion.

4. Application of a strain of Bacillus coagulans from the deep sea of ​​South China Sea, characterized in that: The probiotic powder prepared by the Bacillus coagulans.

5. The use of a strain of Bacillus coagulans from the deep sea of ​​South China Sea according to claim 4, characterized in that: The probiotic powder has strong storage stability.

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