CN114752518A - Bacillus licheniformis XCL-09 and application thereof - Google Patents
Bacillus licheniformis XCL-09 and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus licheniformis XCL-09 and application thereof, wherein the preservation number of the strain is CCTCC NO: M20211497, the bacillus licheniformis XCL-09 can efficiently inhibit alpha-glucosidase and efficiently antagonize staphylococcus aureus, fusarium graminearum, aspergillus niger and ceruleus funiculorum, the strain has a high growth rate, and the spore formation rate is as high as 91%.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to bacillus licheniformis and application thereof in inhibition of alpha-glucosidase and pathogenic microorganisms.
Background
With the development of social economy and the improvement of the quality of life of people, the number of pets in China is continuously increased, and the nutrition and health of the pets are more and more valued by people. With the increase of nutrition, lack of exercise and high intake of fat and carbohydrate of pets, the phenomenon of obesity of pets is more and more serious (Zhuyali, Zhang Guangzhi, Lijing, Wang Ming Yan, Zhang Jianwei, Chi Shang jin. the potential application value of probiotics in obesity of pets [ J ]. modern animal husbandry and veterinary medicine 2020(09): 52-57.). The obesity of the pet not only affects the behavior and activity of the pet, often causes diseases such as hyperglycemia and hyperlipidemia, but also easily causes other visceral diseases along with the extension of the attack time, so that the physical and mental health of the pet is seriously threatened. Carbohydrates in food are mainly absorbed in the intestinal tract in the form of monosaccharides, and polysaccharide substances such as starch are degraded under the action of saliva and pancreatic alpha-amylase to generate oligosaccharides and disaccharides, and then degraded into glucose by alpha-glucosidase to be absorbed. The alpha-glucosidase inhibitor can inhibit the production and absorption of glucose, thereby inhibiting the digestion and absorption of starch, and leading the blood sugar to be stably and slowly maintained at a certain level. Zhu Yu Ping et al found that Bacillus subtilis could be fermented to produce alpha-glucosidase inhibitor, and has important potential in controlling pet weight and reducing blood sugar (Zhu Ping, Yong Qiang, Liu Hai Jie, Li, eight Hao II. research on producing alpha-glucosidase inhibitor from bean dregs fermented by different strains [ J ]. Chinese food and oil Commission, 2008(04): 70-74.).
Meanwhile, the abuse of antibiotics seriously threatens the health and the environment of animals, and the feed industry in China has comprehensively implemented banning resistance orders. Under such circumstances, probiotic products with antibacterial function are receiving increasing attention from the pet food industry as antibiotic substitutes, maintaining the intestinal health and flora balance of animals by means of probiotics inhibiting pathogenic bacteria, and are one of the most potential antibiotic substitute products at present, and play an important role in the health of pets (huiling, lihuixima. application and research of probiotics in pets progress [ J ] animal husbandry industry, 2021(08): 61-66.). Therefore, it would be of great significance to screen for probiotics with highly effective antibacterial activity. Bacillus licheniformis is a potential probiotic bacterium, and the alpha-glucosidase inhibition by Bacillus licheniformis is not reported at present. If the bacillus licheniformis which can inhibit the alpha-glucosidase and has high-efficiency bacteriostatic activity can be obtained, the bacillus licheniformis has great significance. Therefore, the invention aims to screen and obtain a novel bacillus licheniformis strain capable of inhibiting alpha-glucosidase and harmful microorganisms.
Disclosure of Invention
The invention aims to provide a bacillus licheniformis capable of effectively inhibiting alpha-glucosidase and inhibiting harmful microorganisms.
In order to achieve the purpose, the invention adopts the following technical scheme:
the strain can effectively inhibit alpha-glucosidase and efficiently antagonize staphylococcus aureus, fusarium graminearum, aspergillus niger and cercospora funiculosum, is identified as Bacillus licheniformis (Bacillus licheniformis) according to morphological characteristics and 16S rRNA gene sequence analysis, is named as Bacillus licheniformis XCL-09, is preserved in China Center for Type Culture Collection (CCTCC) in 11.29.2021, and has a preservation number of CCTCC NO: m20211497.
The application of bacillus licheniformis XCL-09 in inhibiting alpha-glucosidase or preparing alpha-glucosidase inhibitor comprises the following steps: the alpha-glucosidase inhibition rate of the fermentation supernatant of 100 times diluted bacillus licheniformis XCL-09 is 51 percent, which indicates that the bacillus licheniformis XCL-09 has high-efficiency alpha-glucosidase inhibition activity.
The application of bacillus licheniformis XCL-09 in inhibiting staphylococcus aureus, aspergillus niger, ceruloplasmin and fusarium graminearum is as follows: in the specific embodiment of the invention, the results of comparing the inhibition zones of the bacillus licheniformis XCL-09 and other strains of bacillus licheniformis on staphylococcus aureus, aspergillus niger, ceruloides funiculosum and fusarium graminearum show that the antibacterial activity of the bacillus licheniformis XCL-09 has obvious advantages.
The invention also provides an optimized culture medium of the bacillus licheniformis XCL-09, which can obviously improve the sporulation rate and comprises 30-45 g/L of corn starch, 40-60 g/L of soybean meal and K2HPO4·3H2O1-1.5 g/L, MgSO4·7H2O0.75-1.125 g/L, MnSO4·H2O0.001-0.0015 g/L.
The bacillus licheniformis XCL-09 provided by the invention has the following beneficial effects:
(1) the bacillus licheniformis capable of efficiently inhibiting the alpha-glucosidase is obtained for the first time.
(2) The strain can inhibit alpha-glucosidase, and can efficiently antagonize staphylococcus aureus, fusarium graminearum, aspergillus niger and cercospora funiculosum.
Drawings
FIG. 1: microscopic morphology of Bacillus licheniformis XCL-09.
FIG. 2: bacillus licheniformis XCL-09 colony morphology chart.
FIG. 3: and (3) constructing a phylogenetic tree based on the 16S rRNA gene sequence.
FIG. 4: bacillus licheniformis XCL-09 has antibacterial effect on Staphylococcus aureus, Aspergillus niger, ceruleus funiculosum and Fusarium graminearum.
Detailed Description
Example 1: separation and screening of Bacillus licheniformis XCL-09
Adding 1g fermented soybean paste from different regions into sterilized test tube containing 9mL sterile water, sealing with cotton plug, oscillating for 2min, heating in water bath at 80 deg.C for 15min, standing, cooling, absorbing 0.1mL supernatant, diluting 10 -2、10-30.1mL of diluted and coated LB plate is respectively taken, cultured for 24h at 37 ℃, a single colony is selected and inoculated into an LB seed liquid culture medium, and cultured for 12h at 37 ℃ and 180 r/min. Transferring the seed solution into 50g of sterilized soybeans according to the inoculation amount of 3%, standing and culturing at 37 ℃ for 36h, adding water, extracting, centrifuging to obtain a supernatant, and measuring the alpha-glucosidase inhibition rate of the supernatant: taking 10 mu L of supernatant diluted by 100 times, adding 50 mu L of 2g/L maltose, 45 mu L of an Ac-HAc buffer solution and 15 mu L of alpha-glucosidase, carrying out water bath at 37 ℃ for 30min, boiling the water bath for 10min, detecting the glucose content by using a glucose determination kit, and calculating the inhibition rate of the alpha-glucosidase to obtain 6 strains with high-efficiency alpha-glucosidase inhibition activity; further carrying out antibacterial activity re-screening on the 6 strains to prepare the strain containing fusarium graminearum,Aspergillus niger, a Sabouraud's culture medium plate of the blue fungus rope and an LB plate containing staphylococcus aureus, a hole puncher is used for punching, a liquid transfer gun is used for sucking 20 mu L of seed liquid into an agar hole, the mould plate is cultured in a 30 ℃ biochemical incubator for 36h to observe the size of a bacteriostatic zone, and the bacterial plate is cultured in a 37 ℃ biochemical incubator for 12h to observe the size of the bacteriostatic zone. And re-screening to obtain the strain XCL-09 which can effectively inhibit alpha-glucosidase and antagonize bacteria (staphylococcus aureus) and fungi (fusarium graminearum, aspergillus niger and cercospora funiculosum).
Example 2: identification of Strain XCL-09
As shown in FIG. 1, the strain XCL-09 has a straight rod-shaped thallus, a gram-positive gram stain and approximately circular spores which are terminal. As shown in FIG. 2, the colonies on LB solid medium were flat, irregular in edges, and dull, opaque, and spread on the lawn surface. The optimal growth temperature is 30-37 ℃, and the pH is 6.8-7.2.
16S rRNA gene segments of the strain XCL-09 are amplified, purified and recovered by PCR, and DNA sequencing work is completed by the inventor' S biotechnology limited company. The determined sequence was subjected to sequence similarity analysis using the Blastn program of NCBI, 16S rRNA gene sequences of representative strains of the close species were selected from the Genbank database and the ribosome database, phylogenetic analysis was performed by MEGA6.0 software, and a phylogenetic tree based on the 16S rRNA gene sequences was constructed using the Neighbor Joining method (Bootstrap Joining), and the number of repeated sampling for checking Bootstrap support rate (boottrap) was 1000. Sequencing the purified product by Beijing Optimalaceae biotechnology company, wherein the 16S rRNA gene sequence of the strain XCL-09 obtained by sequencing has 1475 basic groups, the sequence is shown as SEQ ID NO.1, the homology analysis result shows that the 16S rRNA gene sequence of the strain XCL-09 is highly similar to that of Bacillus licheniformis (Bacillus licheniformis), a phylogenetic tree is constructed by MEGA6.0 software, and the phylogenetic tree is shown as figure 3, which shows that the strain XCL-09 belongs to the Bacillus licheniformis. By combining morphological characteristics and 16S rRNA gene sequence analysis, the strain is identified as Bacillus licheniformis (Bacillus licheniformis), named as Bacillus licheniformis XCL-09, which is preserved in China Center for Type Culture Collection (CCTCC) at 11 months and 29 months in 2021 with the preservation number of CCTCC NO: m20211497.
Example 3: analysis of alpha-glucosidase inhibition capability of Bacillus licheniformis XCL-09
Transferring bacillus licheniformis XCL-09 into 50g of sterilized soybeans according to the inoculation amount of 3%, standing and culturing for 36h at 37 ℃, adding water to the fermentation product for extraction and centrifugation to obtain supernatant diluted by 100 times, taking 10 mu L of diluent, adding 50 mu L of 2g/L maltose, 45 mu L of NaAc-HAc buffer solution and 15 mu L of alpha-glucosidase, carrying out water bath at 37 ℃ for 30min, boiling the water bath for 10min, and detecting the glucose content by using a glucose determination kit. The glucose concentration and the alpha-glucosidase inhibition rate are calculated according to the following formulas: glucose (mmol/L) is calibration solution concentration × sample tube absorbance (a)/calibration tube absorbance (a), and α -glucosidase inhibition is 100% × (blank glucose concentration-sample glucose concentration)/blank glucose concentration. The result shows that the alpha-glucosidase inhibition rate of the 100-fold dilution of the bacillus licheniformis XCL-09 fermented soybeans reaches 51 percent, which indicates that the bacillus licheniformis XCL-09 has high-efficiency alpha-glucosidase inhibition activity. The bacillus licheniformis capable of inhibiting alpha-glucosidase is obtained for the first time, and the activity of inhibiting bacillus licheniformis is stronger after 100-time dilution.
Example 4: bacillus licheniformis XCL-09 antibacterial activity analysis
Inoculating single colony of Bacillus licheniformis XCL-09 into LB seed liquid culture medium, and culturing at 37 deg.C and 180r/min for 12 h. Respectively punching holes on a Sha's plate containing fusarium graminearum, aspergillus niger and ceruloid hyphomycetes and a staphylococcus aureus LB plate by using sterilized puncher, sucking 20 mu L of bacillus licheniformis XCL-09 bacterial liquid into the agar holes by using a liquid transfer gun, culturing the fungi in a 30 ℃ biochemical incubator for 36h to observe the size of a bacteriostatic ring, and culturing the bacterial plate in a 37 ℃ biochemical incubator for 12h to observe the size of the bacteriostatic ring. Bacillus licheniformis with antibacterial activity preserved in the early stage of the laboratory is taken as a control bacterium, and the size of the inhibition zone is shown in table 1 and figure 4. Compared with the contrast bacteria, the bacillus licheniformis XCL-09 has obvious advantages on the inhibitory activity of staphylococcus aureus, fusarium graminearum, aspergillus niger and ceruloides.
TABLE 1 inhibition zone size of Bacillus licheniformis XCL-09 to different microorganisms
Example 5: fermentation culture of bacillus licheniformis XCL-09
a. Bacterial activation
Inoculating the frozen Bacillus licheniformis XCL-09 to LB solid culture medium (containing peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, pH7.0), scraping the lawn, and culturing at 37 deg.C for 12 hr at 180 r/min; sucking a certain volume of bacterium liquid and storing the bacterium liquid into a glycerol storage tube, wherein the volume of glycerol in the tube is 20% finally; freezing and preserving at-80 ℃;
b. Seed liquid culture
Transferring the frozen strain to an LB solid culture medium plate, and culturing at 37 ℃ for 24-36 hours to activate the strain; transferring the activated seeds into a triangular flask seed culture medium (containing 10 g/L of peptone, 5 g/L of yeast extract, 10 g/L of NaCl, 15 g/L of agar and pH7.0), and culturing at 37 ℃ and 180r/min for 14-16 hours to the middle logarithmic growth phase;
c. comparing the viable count, spore count and spore formation rate of Bacillus licheniformis in different culture media
Culture medium 1: preparing a fermentation medium according to the addition of 1 liter of the culture medium: 30 g of corn starch; 40 g of soybean meal; k is2HPO4·3H2O1 g; MgSO (MgSO)4·7H20.75 g of O; MnSO4·H2O0.001 g, adding distilled water to 1000mL, pH7.0, preparing 150mL fermentation liquor, and subpackaging 250mL triangular bottles with liquid filling amount of 50mL per bottle. Inoculating the seed liquid according to the inoculation amount of 3%, rotating the table at 180r/min, fermenting at 37 deg.C for 48 h.
Culture medium 2: preparing a fermentation medium according to the addition of 1 liter of the culture medium: 37.5 g of corn starch;50 g of soybean meal; k2HPO4·3H21.25 g of O; MgSO (MgSO)4·7H20.9375 g of O; MnSO4·H20.00125 g of O, supplementing distilled water to 1000mL, pH7.0, preparing 150mL of fermentation liquor, and subpackaging 250mL of triangular bottles with the liquid filling amount of 50mL per bottle. Inoculating the seed liquid according to the inoculation amount of 3%, rotating the table at 180r/min, fermenting at 37 deg.C for 48 h.
And (3) a culture medium: preparing a fermentation medium according to the addition amount of each 1 liter of the culture medium: 45 g of corn starch; 60 g of bean pulp; k is2HPO4·3H21.5 g of O; MgSO (MgSO) in vitro4·7H2O1.125 g; MnSO4·H20.0015 g of O, supplementing distilled water to 1000mL, pH7.0, preparing 150mL of fermentation liquor, and subpackaging 250mL of triangular bottles with the liquid filling amount of 50mL per bottle. Inoculating the seed liquid according to the inoculation amount of 3%, rotating the table at 180r/min, fermenting at 37 deg.C for 48 h.
And (4) a culture medium: preparing a fermentation medium according to the addition amount of each 1 liter of the culture medium: 52.5 g of corn starch; 70 g of soybean meal; k2HPO4·3H21.75 g of O; MgSO (MgSO)4·7H2O1.3125 g; MnSO4·H20.00175 g of O, supplementing distilled water to 1000mL, pH7.0, preparing 150mL of fermentation liquor, and subpackaging 250mL of triangular bottles with the liquid filling amount of 50mL per bottle. Inoculating the seed liquid according to the inoculation amount of 3%, rotating the table at 180r/min, fermenting at 37 deg.C for 48 h.
And (5) culture medium: preparing a fermentation medium according to the addition amount of each 1 liter of the culture medium: 60 g of corn starch; 80 g of soybean meal; k2HPO4·3H2O2 g; MgSO (MgSO)4·7H21.5 g of O; MnSO4·H2O0.002 g, adding distilled water to 1000mL, pH7.0, preparing 150mL fermentation liquor, and subpackaging 250mL triangular bottles with liquid filling amount of 50mL per bottle. Inoculating the seed liquid according to the inoculation amount of 3%, rotating the table at 180r/min, fermenting at 37 deg.C for 48 h.
d. Viable and spore counts
And adding 1mL of the fermentation liquor into a sterilized test tube filled with 9mL of sterile water, carrying out continuous gradient dilution until the estimated bacteria content of the sample diluent is 10-300CFU/mL, taking 0.1mL of the diluted sample, coating an LB flat plate, culturing at 37 ℃ for 24h, and counting to obtain the viable count of the bacillus licheniformis. Adding 1mL of the fermentation liquid into a sterilized test tube filled with 9mL of sterile water, sealing the test tube by using a cotton plug, oscillating for 2min, heating the test tube in a water bath kettle at 80 ℃ for 15min, cooling, carrying out continuous gradient dilution until the estimated bacteria content of a sample diluent is 10-300CFU/mL, taking 0.1mL of the diluted sample diluent, coating an LB (Luria-Bertani) flat plate, culturing at 37 ℃ for 24h, and counting to obtain the number of spores of the bacillus licheniformis. The counting results are shown in table 2. As can be seen from Table 2, the strains can keep higher viable count in different culture media, wherein the sporulation rate of the culture media 1, 2 and 3 is more than 45%, and the sporulation rate of the culture medium 2 is the highest and reaches 91%.
TABLE 2 viable count and spore count of different media
Claims (5)
1. Bacillus licheniformis (B.licheniformis:Bacillus licheniformis) XCL-09, wherein the preservation number is CCTCC NO: M20211497.
2. Use of bacillus licheniformis XCL-09 according to claim 1 for inhibiting α -glucosidase.
3. Use of bacillus licheniformis XCL-09 as described in claim 1 for the preparation of α -glucosidase inhibitors.
4. Use of bacillus licheniformis XCL-09 according to claim 1 for inhibiting staphylococcus aureus, aspergillus niger, cyanobacteria funiculosum, fusarium graminearum.
5. The fermentation culture medium of the bacillus licheniformis XCL-09 is characterized by comprising 30-45 g/L of corn starch, 40-60 g/L of soybean meal and K2HPO4·3H2O1-1.5 g/L, MgSO4·7H2O 0.75-1.125 g/L, MnSO4·H2O0.001-0.0015 g/L.
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| CN102433270A (en) * | 2011-11-03 | 2012-05-02 | 江南大学 | Organic solvent resistant bacillus licheniformis for producing glucosidase and application of bacillus licheniformis |
| WO2017222142A1 (en) * | 2016-06-20 | 2017-12-28 | 강원대학교 산학협력단 | Bacillus licheniformis strain ny1505 for mass producing α-glucosidase inhibitor |
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| CN102433270A (en) * | 2011-11-03 | 2012-05-02 | 江南大学 | Organic solvent resistant bacillus licheniformis for producing glucosidase and application of bacillus licheniformis |
| WO2017222142A1 (en) * | 2016-06-20 | 2017-12-28 | 강원대학교 산학협력단 | Bacillus licheniformis strain ny1505 for mass producing α-glucosidase inhibitor |
| JP2019517810A (en) * | 2016-06-20 | 2019-06-27 | カンウォン ナショナル ユニバーシティ−インダストリー コーポレーション ファウンデーション | Bacillus licheniformis NY1505 strain producing a large amount of α-glucosidase inhibitor |
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