CN114752518B - Bacillus licheniformis XCL-09 and application thereof - Google Patents
Bacillus licheniformis XCL-09 and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a bacillus licheniformis XCL-09 and application thereof, the preservation number of the bacillus licheniformis XCL-09 is CCTCC NO: M20211497, the bacillus licheniformis XCL-09 can not only efficiently inhibit alpha-glucosidase, but also efficiently antagonize staphylococcus aureus, fusarium graminearum, aspergillus niger and trichomonas, the growth rate of the bacillus licheniformis XCL-09 is high, and the sporulation 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 inhibiting 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 increasingly valued by people. The phenomenon of obesity in pets is becoming more and more serious with the increase of nutrition, lack of exercise, ingestion of high fat, high carbohydrate, etc. (Zhu Yali, zhang Anzhi, liang Lin, wang Mingyan, zhang Jianwei, cui Shangjin. Potential use of probiotics in obesity in pets [ J ]. Modern livestock veterinarians, 2020 (09): 52-57.). The obesity of the pet not only can influence the behavior activity of the pet, often causes diseases such as hyperglycemia, hyperlipidemia and the like, but also is easy to cause other organ 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 intestinal tracts in the form of monosaccharides, polysaccharide substances such as starch are degraded under the action of saliva and pancreatic alpha-amylase to generate oligosaccharides and disaccharides, and then are degraded by alpha-glucosidase to glucose to be absorbed. The alpha-glucosidase inhibitor can inhibit the generation and absorption of glucose, thereby inhibiting the digestion and absorption of starch, and maintaining the blood sugar at a certain level smoothly and slowly. Zhu Yun the bacillus subtilis can be fermented to produce the alpha-glucosidase inhibitor, and has important potential in controlling the weight and reducing blood glucose of pets (Zhu Yunping, cheng Yongjiang, liu Haijie, li Lite, eight rolls of unfortunately two. Research on producing alpha-glucosidase inhibitor by fermenting bean dregs of different strains [ J ]. Chinese grain and oil journal, 2008 (04): 70-74).
Meanwhile, the abuse of antibiotics seriously threatens the health and environment of animals, and the feed industry in China comprehensively carries out forbidden orders. Under such circumstances, probiotic products having antibacterial function are receiving increasing attention from the pet food industry as antibiotic substitutes, and the maintenance of animal intestinal health and flora balance by inhibition of pathogenic bacteria by probiotics is one of the most potential antibiotic substitute products at present, and plays an important role in the health of pets (Hu Huiling, li Huixia. Development of application of probiotics in pets [ J ]. Animal husbandry industry, 2021 (08): 61-66.). Therefore, screening for probiotics with highly potent antimicrobial activity would be of great significance. Bacillus licheniformis is a potential probiotic, and no report of inhibiting alpha-glucosidase by the Bacillus licheniformis is currently available. It would be of great significance if bacillus licheniformis could be obtained that both inhibited alpha-glucosidase and had high-efficiency bacteriostatic activity. Thus, the present invention aims to screen and obtain a novel bacillus licheniformis strain which can inhibit both alpha-glucosidase and harmful microorganisms.
Disclosure of Invention
The invention aims to provide bacillus licheniformis which can inhibit alpha-glucosidase efficiently and inhibit harmful microorganisms.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The invention obtains a strain which can not only inhibit alpha-glucosidase but also antagonize staphylococcus aureus, fusarium graminearum, aspergillus niger and rope blue fungus through separation and screening, and the strain is identified as bacillus licheniformis (Bacillus licheniformis) according to morphological characteristics and 16S rRNA gene sequence analysis, is named as bacillus licheniformis XCL-09 and is preserved in China Center for Type Culture Collection (CCTCC) for 11 months and 29 days in 2021, and the preservation number is CCTCC NO: m20211497.
Use of bacillus licheniformis XCL-09 for inhibiting alpha-glucosidase or for preparing an alpha-glucosidase inhibitor: the inhibition rate of the fermentation supernatant of the bacillus licheniformis XCL-09 diluted by 100 times to the alpha-glucosidase is 51%, which shows that the bacillus licheniformis XCL-09 has high-efficiency alpha-glucosidase inhibition activity.
Application of bacillus licheniformis XCL-09 in inhibiting staphylococcus aureus, aspergillus niger, blue-light fungus and fusarium graminearum: in the specific embodiment of the invention, compared with other strains of bacillus licheniformis, the bacteriostasis ring of bacillus licheniformis XCL-09 on staphylococcus aureus, aspergillus niger, blue-light fungus and fusarium graminearum shows that the bacteriostasis activity of the bacillus licheniformis XCL-09 has obvious advantages.
The invention also provides an optimized culture medium of bacillus licheniformis XCL-09, which can remarkably improve the sporulation rate, wherein the culture medium comprises 30-45 g/L of corn starch, 40-60 g/L of bean pulp, 0-1.5 g/L of K 2HPO4·3H2 O, 0.75-1.125 g/L of MgSO 4·7H2 O and 0.001-0.0015 g/L of MnSO 4·H2 O.
The bacillus licheniformis XCL-09 provided by the invention has the following beneficial effects:
(1) The bacillus licheniformis capable of inhibiting the alpha-glucosidase efficiently is obtained for the first time.
(2) The strain can inhibit alpha-glucosidase and can also efficiently antagonize staphylococcus aureus, fusarium graminearum, aspergillus niger and blue-like bacteria.
Drawings
Fig. 1: microscopic morphology of bacillus licheniformis XCL-09.
Fig. 2: bacillus licheniformis XCL-09 colony morphology.
Fig. 3: phylogenetic tree constructed based on the 16S rRNA gene sequence.
Fig. 4: the bacillus licheniformis XCL-09 has antibacterial effect on staphylococcus aureus, aspergillus niger, blue-light-rope bacteria and fusarium graminearum.
Detailed Description
Example 1: separation and screening of bacillus licheniformis XCL-09
Adding 1g of fermented soybean paste from different regions into a sterilized test tube filled with 9mL of sterile water, sealing with a cotton plug, oscillating for 2min, heating at 80 ℃ in a water bath for 15min, standing and cooling, sucking 0.1mL of supernatant to dilute 10 -2、10-3, respectively taking 0.1mL of diluted and coated LB plates, culturing at 37 ℃ for 24h, selecting single bacterial colony, inoculating into LB seed liquid culture medium at 37 ℃ and culturing at 180r/min for 12h. Transferring the seed solution into sterilized 50g of sterilized soybean according to an inoculation amount of 3%, standing at 37 ℃ for culturing for 36h, adding water for extraction, centrifuging to obtain 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 LNaAc-HAc buffer solution and 15 mu L of alpha-glucosidase, carrying out water bath at 37 ℃ for 30min, carrying out boiling water bath for 10min, detecting the content of glucose by using a glucose measuring kit, and calculating the alpha-glucosidase inhibition rate to obtain 6 strains with high-efficiency alpha-glucosidase inhibition activity; further carrying out antibacterial activity rescreening on the 6 strains, manufacturing a Sahnikovia culture medium plate containing fusarium graminearum, aspergillus niger and rope-shaped blue fungus and a LB plate containing staphylococcus aureus, punching by using a puncher, sucking 20 mu L of seed liquid into an agar hole by using a pipetting gun, culturing the mould plate in a 30 ℃ biochemical incubator for 36 hours to observe the size of a bacteriostasis zone, and culturing the bacterial plate in a 37 ℃ biochemical incubator for 12 hours to observe the size of the bacteriostasis zone. The re-screening can obtain the strain XCL-09 which can inhibit the alpha-glucosidase and antagonize bacteria (staphylococcus aureus) and fungi (fusarium graminearum, aspergillus niger and blue-light fungus) with high efficiency.
Example 2: identification of Strain XCL-09
As shown in FIG. 1, the bacterial strain XCL-09 has a straight-bar shape, gram-positive staining, and the spores are approximately circular and are grown on the tip. As shown in FIG. 2, the colonies were flat, irregular in edge, matt, opaque and spread on the lawn surface on LB solid medium. The optimum growth temperature is 30-37 ℃, and the pH is suitable for 6.8-7.2.
The 16S rRNA gene segment of the strain XCL-09 is amplified, purified and recovered by PCR, and DNA sequencing work is completed by the Optic and New technology Co., ltd. The sequence similarity analysis was performed on the measured sequence by using the Blastn program of NCBI, the 16S rRNA gene sequence of a representative strain of similar species was selected from the Genbank database and the ribosome database, the phylogenetic analysis was performed by MEGA 6.0 software, and a phylogenetic tree based on the 16S rRNA gene sequence was constructed by the adjacency method (Neighbor training) for checking the Bootstrap support rate (Bootstrap) for 1000 times. Sequencing of the purified product is carried out by the Beijing qing department biotechnology company, the 16S rRNA gene sequence of the strain XCL-09 obtained by sequencing has 1475 bases, the homology analysis result shows that the 16S rRNA gene sequence of the strain XCL-09 is highly similar to that of bacillus licheniformis (Bacilluslicheniformis), and a phylogenetic tree is constructed through MEGA 6.0 software, and the phylogenetic tree is shown as a figure 3, and shows that the strain XCL-09 belongs to bacillus licheniformis. By combining morphological characteristics and 16S rRNA gene sequence analysis, the strain is identified as bacillus licheniformis (Bacillus licheniformis), named bacillus licheniformis XCL-09 and is preserved in China Center for Type Culture Collection (CCTCC) at the year 2021, 11 and 29, and the preservation number is CCTCC NO: m20211497.
Example 3: bacillus licheniformis XCL-09 inhibition alpha-glucosidase capability assay
Transferring Bacillus licheniformis XCL-09 into 50g sterilized soybean according to 3% inoculum size, standing at 37deg.C for 36h, extracting fermentation product with water, centrifuging to obtain 100 times diluted supernatant, collecting 10 μl of diluted solution, adding 50 μl of 2g/L maltose, 45 μl NaAc-HAc buffer, 15 μl α -glucosidase, water-bathing at 37deg.C for 30min, boiling water-bathing for 10min, and detecting glucose content with glucose determination kit. The glucose concentration and the alpha-glucosidase inhibition rate were calculated according to the following formula: glucose (mmol/L) =calibrator concentration x sample tube absorbance (a)/calibrator tube absorbance (a), α -glucosidase inhibition = 100% × (blank glucose concentration-sample glucose concentration)/blank glucose concentration. The result shows that the bacillus licheniformis XCL-09 fermented soybeans are diluted by 100 times, the inhibition rate of the bacillus licheniformis XCL-09 fermented soybeans reaches 51%, and the bacillus licheniformis XCL-09 has high-efficiency alpha-glucosidase inhibition activity. The bacillus licheniformis capable of inhibiting the alpha-glucosidase is obtained for the first time, and the inhibition activity is stronger after dilution by 100 times.
Example 4: antibacterial Activity assay of Bacillus licheniformis XCL-09
The bacillus licheniformis XCL-09 single colony is inoculated into LB seed liquid culture medium for culturing for 12h at 37 ℃ and 180 r/min. Punching holes on a Sahnikovia plate containing fusarium graminearum, aspergillus niger and blue-like bacteria and a staphylococcus aureus LB plate respectively by using a sterilized puncher, sucking 20 mu L of bacillus licheniformis XCL-09 bacterial liquid into an agar hole by using a pipette, wherein fungi are cultured in a biochemical incubator at 30 ℃ for 36 hours to observe the size of a bacteriostasis zone, and the bacteria plates are cultured in the biochemical incubator at 37 ℃ for 12 hours to observe the size of the bacteriostasis zone. The bacillus licheniformis with antibacterial activity stored in the earlier stage of the laboratory is used as a control bacterium, and the size of the inhibition zone is shown in table 1 and fig. 4. Compared with the control bacteria, the bacillus licheniformis XCL-09 has obvious advantages on the inhibition activity of staphylococcus aureus, fusarium graminearum, aspergillus niger and blue-tooth fungus.
TABLE 1 size of inhibition zone of Bacillus licheniformis XCL-09 against different microorganisms
Example 5: fermentation culture of bacillus licheniformis XCL-09
A. Strain activation
Inoculating frozen and preserved bacillus licheniformis XCL-09 to LB solid culture medium (containing 10 g/L peptone, 5g/L yeast extract, 10 g/L NaCl, 15 g/L agar and pH 7.0), scraping the lawn, inoculating to LB liquid culture medium, and culturing at 37deg.C, 180r/min for 12 hr; sucking a certain volume of bacterial liquid, and storing the bacterial liquid in a glycerol storage tube, wherein the volume of glycerol in the glycerol storage tube is 20%; freezing and preserving at-80 ℃;
b. Seed liquid culture
Transferring the frozen and preserved strain to LB solid medium plate, culturing at 37 deg.C for 24-36 hr, and activating; transferring the activated seeds into a triangular flask seed culture medium (containing 10 g/L peptone, 5g/L yeast extract, 10 g/L NaCl, 15 g/L agar and pH 7.0), and culturing at 37 ℃ for 14-16 hours at 180r/min to mid-log growth;
c. Comparing the viable count, the spore count and the spore formation rate of the bacillus licheniformis under different culture mediums
Culture medium 1: the fermentation medium was prepared according to the addition of 1 liter of medium: 30 g of corn starch; 40 g of bean pulp; k 2HPO4·3H2 O1 g; mgSO 4·7H2 O0.75 g; mnSO 4·H2 O0.001 g, distilled water to 1000mL, pH7.0, was supplemented, 150mL fermentation broth was prepared and packed in 250mL triangular flasks, and the liquid loading amount per flask was 50mL. Inoculating seed liquid according to 3% inoculum size, rotating 180r/min, fermenting at 37deg.C for 48 hr.
Culture medium 2: the fermentation medium was prepared according to the addition of 1 liter of medium: 37.5 g of corn starch; 50 g of soybean meal; k 2HPO4·3H2 O1.25 g; mgSO 4·7H2 O0.9375 g; mnSO 4·H2 O0.00125 g, distilled water to 1000mL, pH7.0, was supplemented, 150mL fermentation broth was prepared and packed in 250mL triangular flasks, and the liquid loading amount per flask was 50mL. Inoculating seed liquid according to 3% inoculum size, rotating 180r/min, fermenting at 37deg.C for 48 hr.
Culture medium 3: the fermentation medium was prepared in an amount of 1 liter of medium: 45 g of corn starch; 60 g of bean pulp; k 2HPO4·3H2 O1.5 g; mgSO 4·7H2 O1.125 g; mnSO 4·H2 O0.0015 g, distilled water to 1000mL, pH7.0, was supplemented, 150mL fermentation broth was prepared and packed in 250mL triangular flasks, each flask containing 50mL of liquid. Inoculating seed liquid according to 3% inoculum size, rotating 180r/min, fermenting at 37deg.C for 48 hr.
Culture medium 4: the fermentation medium was prepared in an amount of 1 liter of medium: 52.5 g of corn starch; 70 g of soybean meal; k 2HPO4·3H2 O1.75 g; mgSO 4·7H2 O1.3125 g; mnSO 4·H2 O0.00175 g, distilled water to 1000mL, pH7.0, was supplemented, 150mL fermentation broth was prepared and packed in 250mL triangular flasks, and the liquid loading amount per flask was 50mL. Inoculating seed liquid according to 3% inoculum size, rotating 180r/min, fermenting at 37deg.C for 48 hr.
Culture medium 5: the fermentation medium was prepared in an amount of 1 liter of medium: 60 g of corn starch; 80 g of bean pulp; k 2HPO4·3H2 O2 g; mgSO 4·7H2 O1.5 g; mnSO 4·H2 O0.002 g was supplemented with distilled water to 1000mL, pH7.0, and 150mL of fermentation broth was prepared and packed in 250mL triangular flasks with 50mL of liquid per flask. Inoculating seed liquid according to 3% inoculum size, rotating 180r/min, fermenting at 37deg.C for 48 hr.
D. Viable count and spore count
And (3) 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 bacterial content of the sample dilution is 10-300CFU/mL, taking 0.1mL of diluted and coated LB plate, culturing at 37 ℃ for 24 hours, and counting to obtain the viable count of bacillus licheniformis. Adding 1mL of the fermentation liquor into a sterilized test tube filled with 9mL of sterile water, sealing with a cotton plug, oscillating for 2min, heating at 80 ℃ in a water bath kettle for 15min, cooling, carrying out continuous gradient dilution until the estimated bacterial content of the sample dilution is 10-300CFU/mL, ending, taking 0.1mL of diluted and coated LB plate, culturing at 37 ℃ for 24h, and counting to obtain the spore number of bacillus licheniformis. The counting results are shown in Table 2. As can be seen from Table 2, the strain can keep a higher viable count in different culture mediums, wherein the sporulation rate of the culture mediums 1, 2 and 3 reaches more than 45%, and the sporulation rate of the culture medium 2 reaches 91% at the highest.
TABLE 2 viable count and spore count of different media
Claims (4)
1. Bacillus licheniformis (Bacillus licheniformis) XCL-09 is characterized in that the preservation number is CCTCC NO: M20211497.
2. Use of bacillus licheniformis XCL-09 according to claim 1 for the preparation of an alpha-glucosidase inhibitor.
3. Use of bacillus licheniformis XCL-09 according to claim 1 for the preparation of a formulation for inhibiting staphylococcus aureus, aspergillus niger, blue-light-forming bacteria, fusarium graminearum.
4. Use of a fermentation medium for culturing bacillus licheniformis XCL-09 according to claim 1, wherein said fermentation medium comprises corn starch 30-45 g/L, bean pulp 40-60 g/L, K 2HPO4·3H2 O1-1.5 g/L, mgSO 4·7H2 O0.75-1.125 g/L, mnSO 4·H2 O0.001-0.0015 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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