CN112175053A - Bacteriocin produced by lactobacillus acidophilus NM, preparation method and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses bacteriocin produced by lactobacillus acidophilus NM, a preparation method and application thereof. The bacteriocin is synthesized by lactobacillus acidophilus NM with high yield in the fermentation growth period, can inhibit gram-positive bacteria and gram-negative bacteria, can be applied to the preparation of spectral antibacterial medicinal preparations or compositions, and is particularly applied to the preparation of medicines for inhibiting escherichia coli. The bacteriocin has strong heat resistance and acid resistance, can be degraded by protease, does not remain in human bodies, has high safety, and has the effect of broad-spectrum antibacterial activity.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to bacteriocin generated by lactobacillus acidophilus NM, a preparation method and application thereof.
Background
In recent years, the use of broad spectrum antibiotics in excess has led to the development of resistance to antibiotics by many pathogenic bacteria, and there has been great concern over how these pathogenic bacteria can be subsequently controlled. The huge potential of bacteriocin as a natural biological bacteriostatic agent in the therapeutic field has attracted people's attention. The bacteriocin is protein or polypeptide which is synthesized by ribosome in the metabolic process of bacteria and has antibacterial effect, and has better selective bactericidal effect. Decomposed by various enzymes in the digestive tract, does not accumulate in the animal body to cause adverse reaction, has no side effect, residue or drug resistance, does not pollute the environment, and has wide prospect in the biological pharmacy.
However, most bacteriocins have narrow antibacterial spectrum and can only act on gram-positive bacteria which are relatively close to the bacteriocins, and with the continuous and deep research, many researches find that lactobacillus acidophilus has broad-spectrum antibacterial activity, can inhibit not only gram-positive bacteria, but also gram-negative bacteria and fungi, and has wide application prospect. In addition, most lactobacillus bacteriocins have low yield and cannot meet the application requirements.
Therefore, a high-yield broad-spectrum bacteriocin needs to be developed, and a foundation is laid for the development and application of the bacteriocin.
Disclosure of Invention
According to an aspect of the present invention, there is provided bacteriocin produced by lactobacillus acidophilus NM, synthesized at high yield by lactobacillus acidophilus NM during the fermentation growth phase, capable of inhibiting both gram-positive and gram-negative bacteria.
The bacteriocin that lactobacillus acidophilus NM produced of this application, its beneficial effect is, the output of bacteriocin promotes, can satisfy the application demand, and it all has bacteriostatic action to gram-positive bacterium and gram-negative bacterium, is a kind of bacteriocin that has broad-spectrum antibacterial activity, has extensive application prospect.
The lactobacillus acidophilus NM is preserved in China general microbiological culture collection center with the preservation number: CGMCC No.4472, preservation date: 2010.12.14, deposit address: china general microbiological culture Collection center.
The preparation method of the bacteriocin generated by the lactobacillus acidophilus NM comprises the step of inoculating the activated lactobacillus acidophilus NM into an MRS liquid culture medium and culturing for 16-26 h at 37 ℃. Culturing lactobacillus acidophilus NM for 6h, and entering logarithmic phase; entering a stable period within 16-24 h; after 24h, the thalli grow to enter a decline period. When the thalli enters the logarithmic growth phase, bacteriocin is gradually produced, and the yield of the bacteriocin is continuously improved along with the increase of time. After 26h, the production of bacteriocin showed a downward trend.
In some embodiments, the bacteriocin produced by Lactobacillus acidophilus NM is prepared by inoculating activated Lactobacillus acidophilus in MRS broth for 20h at 37 ℃. The method has the beneficial effects that when the diameter of the bacteriocin inhibition zone is the largest when the bacteriocin inhibition zone is cultured for 20 hours and is kept stable, so that the bacteriocin can reach the highest yield after the bacteriocin is cultured for 20 hours, and the most effective effect can be achieved when the bacteriocin is applied.
In some embodiments, the method for the production of bacteriocins produced by lactobacillus acidophilus NM, characterized in that the MRS broth is adjusted to a PH < 7. With the increase of the pH value, the bacteriocin bacteriostatic activity of the lactobacillus acidophilus is gradually reduced, which shows that the bacterium has stronger bacteriostatic action under the acidic condition and has reduced bacteriostatic activity under the neutral or alkaline condition.
Application of bacteriocin generated by Lactobacillus acidophilus NM in preparing broad-spectrum antibacterial drugs. The bacteriocin produced by fermentation of Lactobacillus acidophilus NM can inhibit gram-positive and gram-negative bacteria, and therefore, the bacteriocin produced by fermentation of Lactobacillus acidophilus NM can be used for preparing bacteriostatic compositions and preparations.
In some embodiments, the bacteriocin produced by lactobacillus acidophilus NM can be used for preparing a medicament for inhibiting bacillus subtilis, staphylococcus aureus, micrococcus luteus, escherichia coli, salmonella, and listeria monocytogenes. And (3) uniformly coating the indicator bacterium liquid on corresponding solid culture media respectively, punching by using an oxford cup, adding bacteriocin, diffusing for 2h at 4 ℃, putting the mixture into an incubator at 37 ℃ for overnight culture, and measuring the diameter of a bacteriostatic circle, wherein the bacteriostatic circle has an inhibiting effect on the above all.
Further, the application of bacteriocin produced by Lactobacillus acidophilus NM in preparing medicines for inhibiting Escherichia coli.
Bacteriocins produced by Lactobacillus acidophilus NM are degraded by pepsin and trypsin. The bacteriocin has the beneficial effects that the bacteriocin is sensitive to protease, and does not cause side effects because of residue in vivo, so that the bacteriocin is safer and has no side effects when being used for preparing antibacterial drugs.
The bacteriocin produced by lactobacillus acidophilus NM has stronger heat resistance and acid resistance, can be degraded by protease, can not be remained in human body, and has high safety. The bacteriocin can inhibit gram-positive bacteria and gram-negative bacteria, and is a bacteriocin with broad-spectrum antibacterial activity.
Drawings
FIG. 1 is a schematic representation of the growth curve of Lactobacillus acidophilus and the yield curve of bacteriocin;
FIG. 2 is a graph showing the effect of different pH values on the bacteriostatic activity of bacteriocin;
FIG. 3 is a graph showing the effect of bacteriocin produced by Lactobacillus acidophilus NM on E.coli growth curves;
FIG. 4 is a graph showing the effect of bacteriocin produced by Lactobacillus acidophilus NM on E.coli nucleic acid efflux;
FIG. 5 is a graph showing the effect of bacteriocin produced by Lactobacillus acidophilus NM on E.coli protein efflux;
FIG. 6 is a graph showing the effect of bacteriocin produced by Lactobacillus acidophilus NM on the change in extracellular conductivity of E.coli.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
1. Culture of Lactobacillus acidophilus NM and indicator bacteria
Culturing lactobacillus acidophilus: inoculating activated Lactobacillus acidophilus into MRS liquid culture medium, and culturing at 37 deg.C for 24 h.
Culturing indicator bacteria: NA culture medium is used for micrococcus luteus, staphylococcus aureus, bacillus subtilis and salmonella, LB culture medium is used for escherichia coli, BHI culture medium is used for listeria monocytogenes, and the culture is carried out for 24h at 37 ℃.
Growth curve of lactobacillus acidophilus NM and determination of bacteriocin production:
lactobacillus acidophilus NM was inoculated into 100mL MRS liquid medium and cultured at 37 ℃ in a static manner. Sampling every 2h, measuring OD600, and determining the bacteriostatic activity of the fermentation supernatant by taking escherichia coli as an indicator bacterium. The results are shown in FIG. 1.
As can be seen from FIG. 1, Lactobacillus acidophilus NM was cultured for 6h and then entered logarithmic growth phase; entering a stable period within 16-24 h; after 24h, the thalli grow to enter a decline period. When the thalli enters the logarithmic growth phase, bacteriocin is gradually produced, and the yield of the bacteriocin is continuously improved along with the increase of time. When the culture is carried out for 20h, the diameter of the bacteriocin inhibition zone is maximum and is kept stable. After 26h, the production of bacteriocin showed a downward trend.
2. Analysis of the bacteriostatic Activity of bacteriocins produced by Lactobacillus acidophilus NM
The method comprises the steps of uniformly coating 100 mu L of indicator bacterium liquid on corresponding solid culture media respectively, punching a hole (with the hole diameter of 6mm) by using an Oxford cup, adding 50 mu L of bacteriocin per hole, diffusing at 4 ℃ for 2h, placing the indicator bacterium liquid in an incubator at 37 ℃ for overnight culture, and then measuring the diameter of a bacteriostatic circle, wherein the results are shown in Table 1.
TABLE 1 antibacterial spectra measurement results
As can be seen from Table 4, Lactobacillus acidophilus NM has obvious bacteriostatic effects on Bacillus subtilis, Staphylococcus aureus, Staphylococcus luteus, Escherichia coli, Salmonella and Listeria, which indicates that the bacteriocin generated by Lactobacillus acidophilus NM can inhibit both gram-positive bacteria and gram-negative bacteria, and is a bacteriocin with broad-spectrum bacteriostatic activity. And the diameter of the inhibition zone for measuring the escherichia coli can reach 20.11 +/-0.21, which shows that the lactobacillus acidophilus NM has stronger inhibition effect on the escherichia coli.
Organic acid influence is excluded: and adjusting the pH value of the MRS culture medium to be the same as that of the fermentation supernatant by using lactic acid and acetic acid, and then carrying out bacteriostatic activity determination.
Exclusion of hydrogen peroxide effects: adding catalase with the concentration of 5.0mg/mL dissolved in phosphate buffer solution with the pH value of 7.0 into the fermentation supernatant, taking out after water bath at 37 ℃ for 2h, and detecting the bacteriostatic activity.
Adjusting the pH value of the MRS culture medium to be the same as that of the fermentation supernatant by using lactic acid and acetic acid, and measuring the antibacterial activity of the MRS culture medium and the fermentation supernatant after the pH value is adjusted. The results are shown in Table 2.
TABLE 2 Elimination of organic acid and Hydrogen peroxide interference test results
As can be seen from Table 2, the Lactobacillus acidophilus NM fermentation supernatant still maintained inhibitory effect on Escherichia coli after removal of acid and hydrogen peroxide, so the bacteriostatic substance in the fermentation supernatant was preliminarily considered to be bacteriocin.
3. Analysis of the biological Properties of bacteriocins produced by Lactobacillus acidophilus NM
The equal volume of the fermentation supernatants were treated at different temperatures (40 deg.C, 60 deg.C, 80 deg.C, 100 deg.C and 121 deg.C) for 30min, after cooling, the bacteriostatic activity of bacteriocin was determined, and the untreated fermentation supernatants were used as controls, with the results shown in Table 3.
TABLE 3 Effect of temperature on bacteriocins
As can be seen from Table 3, the bacteriostatic activity did not change much after the treatment at 40-121 deg.C, indicating that the bacteriocin has strong heat resistance.
The pH of the fermentation supernatant was adjusted to 2.00, 3.00, 4.00, 500, 6.00, 7.00, 8.00 and 9.00 with 1mol/L HCl and NaOH solutions, respectively, and the bacteriocin inhibitory activity was determined. The results are shown in FIG. 2. As can be seen from FIG. 2, the bacteriocin inhibitory activity of Lactobacillus acidophilus gradually decreases with increasing pH, indicating that the bacterium has a strong inhibitory effect under acidic conditions and a decreased inhibitory activity under neutral or alkaline conditions.
Adjusting the fermentation supernatant to the optimum pH values of pepsin, papain, bromelain and trypsin, respectively adding the fermentation supernatant at the concentration of 1.00mg/mL, carrying out water bath at 37 ℃ for 2h, adjusting the pH value of the enzymolysis solution back to the pH value of the original fermentation solution, and measuring the bacteriostatic activity of bacteriocin, wherein the results are shown in Table 4.
TABLE 4 Effect of enzymes on bacteriocins
As can be seen from Table 4, the bacteriostatic activity of bacteriocin was decreased to different degrees after protease treatment, while the bacteriostatic activity of bacteriocin disappeared after pepsin and trypsin treatment, indicating that Lactobacillus acidophilus bacteriocin is the main bacteriostatic substance in the fermentation broth.
From the above analysis, it can be seen that: the bacteriocin has strong heat resistance and acid resistance, can be degraded by protease, does not remain in human body, and has high safety. The bacteriocin can inhibit gram-positive bacteria and gram-negative bacteria, and is a bacteriocin with broad-spectrum antibacterial activity.
4. Study on inhibitory effect of bacteriocin on Escherichia coli
The logarithmic phase of E.coli was diluted in LB liquid medium to a suspension with a viable count of 105CFU/mL, bacteriocin was added at the minimum inhibitory concentration, the suspension was cultured at 37 ℃ and sampled every 2 hours to determine OD600 values up to 24 hours, and growth curves were prepared using the control without bacteriocin, the results of which are shown in FIG. 3. As can be seen from FIG. 3, 2h starts from the start of the logarithmic phase of growth, 12h to 22h are stationary phases, and 24h enters the decay phase. With the addition of lactobacillus acidophilus bacteriocin, the growth of the escherichia coli is obviously inhibited.
Taking 4.0mL of escherichia coli liquid at logarithmic phase, centrifuging at 4500r/min for 10min, washing the precipitate with 5mmol/L HEPES-NaCl buffer solution with pH 7.00, and suspending in the buffer solution to prepare bacterial suspension with viable count of 107 CFU/mL. 1.0mL of the suspension was added with 1.0mL of 10mmol/L glucose and 1.0mL of bacteriocin with the minimum inhibitory concentration, incubated at 37 ℃ and absorbance values at 260nm and 280nm were measured at 3h intervals until 12h of incubation, and the results are shown in FIGS. 4 and 5.
Diluting the Escherichia coli in logarithmic phase with deionized water until viable count is 105CFU/mL, adding bacteriocin with minimum inhibitory concentration, sampling every 2h, centrifuging at 4000r/min for 10min, diluting the supernatant 20 times, and measuring conductivity value, the result is shown in FIG. 6.
As shown in FIGS. 3-6, the bacteriocin has a strong inhibitory effect on Escherichia coli, mainly causing damage to cell membranes, and leaking substances such as ions, nucleic acids, proteins and the like in cells, thereby causing the death of Escherichia coli.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (8)
1. Bacteriocins produced by lactobacillus acidophilus NM characterized by being synthesized by lactobacillus acidophilus NM in high yield during the fermentation growth phase and capable of inhibiting gram-positive and gram-negative bacteria.
2. The preparation method of the bacteriocin produced by the lactobacillus acidophilus NM is characterized in that the activated lactobacillus acidophilus NM is inoculated into an MRS liquid culture medium and cultured for 16-26 h at 37 ℃.
3. The method of preparing bacteriocin produced by lactobacillus acidophilus NM according to claim 2, characterized by inoculating activated lactobacillus acidophilus NM in MRS broth for 20h at 37 ℃.
4. The method of claim 2, wherein the MRS liquid medium is adjusted to pH < 7.
5. Application of bacteriocin generated by Lactobacillus acidophilus NM in preparing broad-spectrum antibacterial drugs.
6. The use according to claim 5, in the manufacture of a medicament for inhibiting Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Salmonella, and Listeria monocytogenes.
7. Use according to claim 6, for the preparation of a medicament for inhibiting Escherichia coli.
8. Bacteriocin produced by lactobacillus acidophilus NM according to claim 1, characterized by the capacity to be degraded by pepsin and trypsin.
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| CN113717884A (en) * | 2021-08-20 | 2021-11-30 | 内蒙古普泽生物制品有限责任公司 | Development and application of lactobacillus acidophilus NM for inhibiting alpha-glucosidase |
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| CN107868764A (en) * | 2017-11-27 | 2018-04-03 | 内蒙古普泽生物制品有限责任公司 | A kind of compound probiotic powder preparation for strengthening immunity of pets and preparation method thereof |
| CN109999059A (en) * | 2019-04-18 | 2019-07-12 | 内蒙古普泽生物制品有限责任公司 | A kind of gynaecology's Tiny ecosystem inhibiting-bacteria preparation and its application |
| CN110885359A (en) * | 2019-12-27 | 2020-03-17 | 上海源耀农业股份有限公司 | Bacteriocin produced by lactobacillus acidophilus and separation and purification method thereof |
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| CN107868764A (en) * | 2017-11-27 | 2018-04-03 | 内蒙古普泽生物制品有限责任公司 | A kind of compound probiotic powder preparation for strengthening immunity of pets and preparation method thereof |
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| CN110885359A (en) * | 2019-12-27 | 2020-03-17 | 上海源耀农业股份有限公司 | Bacteriocin produced by lactobacillus acidophilus and separation and purification method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113717884A (en) * | 2021-08-20 | 2021-11-30 | 内蒙古普泽生物制品有限责任公司 | Development and application of lactobacillus acidophilus NM for inhibiting alpha-glucosidase |
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