Patent Application
20230348531
The present application claims the benefit of Chinese Patent Application No. 202210476564.7 filed on Apr. 30, 2022, the contents of which are incorporated by reference herein in its entirety.
The present disclosure belongs to the technical field of food biotechnology, and specifically relates to isolation and purification of a bacteriocin produced by Lactobacillus sakei and an antibacterial use thereof and lactic acid bacteria used.
Food spoilage can be seen everywhere in our daily life, especially for foods with relatively high water content, such as fish, meat, vegetables, etc., which usually deteriorate in a very short time and cannot be eaten, thus causing great waste. Generally, the causes for the food spoilage are as follows: first, the microbial action, food contamination caused by food-borne such as Stappathogens Hylococcus aureus and Bacillus subtilis; second, the enzymes in food; third, the humidity and temperature of the air. While microbial contamination is the main cause of the food spoilage. Although emergence of a food preservative can effectively alleviate the food spoilage problem caused by microorganisms, with the improvement of human living standards and medical standards, it is found that many food additives used today, especially chemical additives, have a risk of carcinogenesis, teratogenesis and even mutation if they are consumed for a long time. Therefore, development of a novel non-toxic and harmless food preservative has become a focus of research in the food field.
Lactic acid bacteria are generally recognized as non-toxic microorganisms and can be used in food. The lactic acid bacteria have been used as a starter to produce fermented products and some strains with a probiotic function have been used for producing probiotic products. The vast majority of lactic acid bacteria can produce a variety of inhibitory substances, among which bacteriocin is a protein or polypeptide with an inhibitory effect that is produced by ribosome, which has attracted extensive attention due to its advantages such as no toxicity, no residue and no drug resistance. At present, only Nisin is a bacteriocin allowed to be used as a food additive worldwide. Since the Nisin can only inhibit gram-positive pathogens and has no inhibitory effect on gram-negative bacteria such as Escherichia coli, its application is limited, so it is urgent to find new bacteriocin with broad-spectrum inhibitory effect.
The problem to be solved by the present disclosure is to provide a Lactobacillus sakei bacteriocin obtained by isolation and purification, an antibacterial use thereof and lactic acid bacteria used.
To solve the above technical problems, the present disclosure provides an isolation and purification method of Lactobacillus sakei ZFM225 bacteriocin, which includes the steps of:
As an improvement of the isolation and purification method of the Lactobacillus sakei ZFM225 bacteriocin of the present disclosure, the step 1) is as follows:
As a further improvement of the isolation and purification method of the Lactobacillus sakei ZFM225 bacteriocin of the present disclosure, the step 2) is as follows: Ammonium sulfate powder is added to the fermentation supernatant until saturation is 70±2%, stirring is performed at 4±1° C. for 10 to 14 h, and collected precipitate is centrifuged to obtain the crude protein;
As a further improvement of the isolation and purification method of the Lactobacillus sakei ZFM225 bacteriocin of the present disclosure, the step 3) is as follows:
As a further improvement of the isolation and purification method of the Lactobacillus sakei ZFM225 bacteriocin of the present disclosure, the step 3) is as follows:
A corresponding collected eluate of 34.5% to 37.5% mobile phase B elution is collected to obtain a Lactobacillus sakei ZFM225 bacteriocin solution.
The present disclosure further provides a use of the Lactobacillus sakei ZFM225 bacteriocin solution: the bacteriocin solution has inhibitory activity on both gram-positive bacteria and gram-negative bacteria.
The gram-positive bacteria include Micrococcus luteus 10209, Staphylococcus aureus D48, Staphylococcus muscae, Staphylococcus carnosus pCA 44, and Staphylococcus carnosus pet 20.
The gram-negative bacteria include Escherichia coli DH5 α, Salmonella paratyphi A CMCC 50093, Salmonella choleraesuis ATCC 13312, and Pseudomonas aeruginosa ATCC 47085.
The present disclosure is specific as follows:
Preservation Name: The Lactobacillus sakei ZFM225, Preservation Authority: China Center for Type Culture Collection, Preservation Address: Wuhan University, Wuhan, China, Preservation No.: CCTCC NO:M 2016669, and Preservation Date: Nov. 23, 2016.
Compared with the prior art, the present disclosure has the following advantages.
To sum up, the present disclosure obtains the novel Lactobacillus sakei ZFM225 bacteriocin by establishing an isolation and purification technology, and determines broad-spectrum antibacterial characteristics, thermal stability, and enzyme sensitivity of the bacteriocin, which has a potential to be applied to food preservation.
Specific implementations of the embodiments of the present disclosure are described in further detail in the following with reference to the accompanying drawings.
The present disclosure is further described below with reference to specific embodiments, and the advantages and characteristics of the present disclosure become more apparent with the description. However, these examples are only exemplary and do not constitute any limit to the scope of the present disclosure.
(1) Screening of Lactic Acid Bacteria from Raw Milk Samples
Isolated samples came from raw milk of the Hangzhou dairy farm. After collection, the isolated samples were gradient-diluted tenfold with normal saline. For each gradient, 100 μL of the isolated sample was taken and spread onto plates of a lactic acid bacteria screening medium (MRS medium containing 2% calcium carbonate). The isolated sample was spread onto three plates for each gradient, and is cultured upside down at 37° C. for 48 h. Single colonies with an obvious calcium dissolving zone were selected and inoculated into a 10 mL MRS liquid medium for activation. Then a bacterial solution was spread onto the MRS screening medium for streaking, which was repeated for multiple times, to obtain single colonies with the obvious calcium dissolving zone. These single colonies were picked out for Gram staining and catalase test. The single colonies preliminarily identified as lactic acid bacteria were inoculated into the MRS medium, cultured at 37° C. for 24 h, activated and numbered, and preserved at −80° C. Lactic acid bacteria that may produce the calcium dissolving zone were screened out. As shown in
(2) Preliminary Screening of Lactic Acid Bacteria with Inhibitory Effect
Preparation of corresponding fermentation supernatant from strains 01 to 12: A loopful of strains was taken and added to a 10 mL MRS medium, and was cultured at 30° C. for 24 h. After the culture, the cultured strains were centrifuged at 8000 r/min at 4° C. for 30 min. Thalli were discarded. Supernatant substances were filtered with a 0.22 μM filter membrane to remove impurities, to obtain corresponding fermentation supernatant.
The inhibitory activity of the fermentation supernatant was tested by using an Oxford cup agar diffusion method, and a strain with a best inhibitory effect was selected for subsequent experiments. In a biosafety cabinet, the indicator bacteria were added with an inoculation amount of 1% (v/v) into an LB semi-solid medium that had been fully heated and melted and kept at a constant temperature of 55° C. After being shaken and mixed well, the indicator bacteria were poured into a disposable culture dish that had been evenly placed with sterile Oxford cups. The Oxford cups were carefully pulled out after the mixture was fully cooled and solidified, and 100 μL of each fermentation supernatant to be tested was added into all wells. A dish was allowed to stand for half an hour at 4° C. to make the fermentation supernatant fully diffuse, and then was put in an incubator for culture for 12 h. An inhibitory zone diameter was measured with a vernier caliper and its transparency was observed. Three parallels were made on the fermentation supernatant of each strain.
The inhibitory activity of the 12 lactic acid bacteria numbered 01 to 12 obtained in step (1) on the Micrococcus luteus 10209 and the Escherichia coli DH5α is shown in Table 1, and it was analyzed from results that the strain 03 had a best inhibitory effect on the two indicator bacteria, and therefore, the strain 0.3 was selected for the subsequent experiments.
Micrococcus
luteus
Escherichia
coli
(3) Re-Screening of Bacteriocin Producing Lactic Acid Bacteria
In addition to bacteriocin-like inhibitory substances, lactic acid bacteria may also produce substances with the inhibitory activity, such as an organic acid and hydrogen peroxide in their fermentation process. Therefore, to determine that it was the bacteriocin to have the broad-spectrum inhibitory effect in the lactic acid bacteria, it was necessary to eliminate an inhibitory interference effect of the organic acid and the hydrogen peroxide on the indicator bacteria. In addition, lactic acid bacteria bacteriocin was a protein substance produced by the lactic acid bacteria in their fermentation process. Therefore, the fermentation supernatant may be treated with protease to see a change of the inhibitory activity, so as to determine whether there was a protein inhibitory substance or not.
1) Elimination of Interference of Organic Acid
The pH of the fermentation supernatant was adjusted to 5 with 1 M NaOH. The pH of the MRS liquid medium was adjusted to the same pH with a lactic acid and an acetic acid. Taking the original fermentation supernatant (pH 4.23) as control, and taking the Micrococcus luteus 10209 as the indicator bacteria, its inhibitory activity was determined by using the above Oxford cup agar diffusion method. The bacteriostasis experiment results were shown in
2) Elimination of Interference of Hydrogen Peroxide
10 mL of fermentation supernatant (process parameters of concentration: 37° C. and vacuum) was concentrated with a rotary evaporator to obtain 5 mL of concentrated fermentation supernatant.
A catalase working solution with a concentration of 2.5 to 5.0 mg/mL was prepared. The concentrated fermentation supernatant was made to be mixed with the catalase working solution in a ratio of 1:1. Taking the fermentation supernatant with no catalase added as blank control, and the mixture was treated in a water bath at 37° C. for 2 h. Its inhibitory activity was determined with the Oxford cup agar diffusion method and their differences were compared. The results were as shown in
3) Determination of Bacteriocin Producing Substances
Enzyme solutions of proteinase K, trypsin and pepsin were prepared, 5 mg/mL for each enzyme solution. The pH of the fermentation supernatant was adjusted to optimum pH of each protease. Each corresponding enzyme solution was added to make its final concentration 1 mg/mL, and was treated in a water bath at 37° C. for 2 h. The pH was adjusted to the original pH. Taking the fermentation supernatant without enzyme treatment as the blank control, bacteriostasis experiments were conducted, and their differences were compared. The results were shown in
(4) Identification of Bacteriocin Producing Lactic Acid Bacteria
As shown in
Preservation Name: The Lactobacillus sakei ZFM225, Preservation Authority: China Center for Type Culture Collection, Preservation Address: Wuhan University, Wuhan, China, Preservation No.: CCTCC NO:M 2016669, and Preservation Date: Nov. 23, 2016.
The following indicator bacteria used for determination of the inhibitory activity were all the Micrococcus luteus 10209.
(1) Influence of Culture Temperature
The Lactobacillus sakei ZFM225 was inoculated with an inoculation amount of 1% (v/v) into the MRS liquid medium, and underwent stationary culture at 25° C., 30° C., 37° C. and 45° C. for 24 h. Its OD600 value and inhibitory activity (expressed by the inhibitory zone diameter) were determined. The results showed that optimum fermentation temperature was 37° C. Under this condition, the inhibitory zone diameter was maximum, which was 16.70 mm.
(2) Influence of Culture Time
The Lactobacillus sakei ZFM225 was inoculated with an inoculation amount of 1% (v/v) into the MRS liquid medium, with culture temperature of 30° C. Samples were taken at time points of 0, 2, 4, 6, 8, 10, 14, 18, 24, 30, 36, 42, and 48 h of the culture time. Their OD values and inhibitory activity (expressed by the inhibitory zone diameter) were determined. It was determined that optimum fermentation time was 24 h. At this time, the inhibitory zone diameter was maximum, which was 16.04 mm.
(3) Influence of Inoculation Amount
The Lactobacillus sakei ZFM225 was inoculated with an inoculation amount of 1%, 1.5%, 2%, 2.5% and 3% (v/v) into the MRS liquid medium, and underwent stationary culture at 37° C. for 24 h. Its OD value and inhibitory activity (expressed by the inhibitory zone diameter) were determined. It was determined that optimum inoculation amount was 2%. At this time, the inhibitory zone diameter was maximum, which was 16.47 mm.
(4) Influence of Initial pH
The initial pH of the MRS liquid medium was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5. The Lactobacillus sakei ZFM225 was inoculated with an inoculation amount of 2% (v/v) into the MRS liquid medium adjusted to different pH values, and underwent stationary culture at 37° C. for 24 h. Its OD value and inhibitory activity (expressed by the inhibitory zone diameter) were determined. It was determined that optimum initial pH was 6.5. At this time, the inhibitory zone diameter was maximum, which was 16.77 mm.
It was finally determined that optimal culture conditions of the bacteriocin produced by the Lactobacillus sakei ZFM225 were: the bacteriocin was inoculated with the inoculation amount of 2% (v/v) into the MRS liquid medium with pH 6.5, and underwent stationary culture at 37° C. for 24 h. Under this condition, the potency of the fermentation supernatant was 266 IU/mL, which was increased by 1.8 times in comparison with 146 IU/mL before optimization.
Note: Parameters before optimization: The inoculation amount was 1% (v/v), the pH of the MRS liquid medium was 7, and stationary culture was performed at 30° C. for 24 h.
(1) Preparation of Fermentation Supernatant of Lactobacillus sakei ZFM225
The Lactobacillus sakei ZFM225, preserved at −80° C., streaked onto an MRS solid medium plate, and cultured at 37° C. After the single colonies grew out, single colonies were picked out therefrom and put in the 10 mL MRS liquid medium. The picked out single colonies were cultured to their growth logarithmic phases. The cultured single colonies were inoculated with an inoculation amount of 2% (v/v) into the 20 mL MRS liquid medium with pH 6.5, and were subcultured to its logarithmic phase to obtain an inoculum. Before use, the thalli concentration of the inoculum was adjusted to OD600=0.6 with the normal saline. The inoculum was inoculated by the optimal culture condition, the inoculation amount of 2% (v/v), into the 1 L MRS liquid medium with pH 6.5, and was cultured at 37° C. for 24 h. The obtained fermentation broth was centrifuged at 8000 r/min and 4° C. for 30 min to obtain the fermentation supernatant (lactic acid bacteria fermentation supernatant), which was placed at 4° C. for later use.
(2) Ammonium Sulfate Precipitation
1 L of the lactic acid bacteria fermentation supernatant was taken, and ammonium sulfate powder was slowly added until saturation was 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% (at 25° C., ammonium sulfate powder saturation was 707 g/L). The mixture was placed in a chromatography cabinet at 4° C. and stirred overnight, and was centrifuged at 8000 r/min and 4° C. for 30 min and precipitate was collected. The precipitate was dissolved in deionized water (the concentration was 5.56 mg/mL) to obtain a protein salt solution. The activity was detected by the Oxford cup agar diffusion method. The indicator bacterium was the Micrococcus luteus 10209. The results showed that crude protein precipitated at 70% ammonium sulfate concentration had the best inhibitory effect, and the inhibitory zone diameter was 15.45 mm. Therefore, crude protein extract was obtained for subsequent experiments after the crude protein precipitated at the 70% ammonium sulfate concentration was desalted by using the sephadex column G-10 (Φ1.6×50).
The 70% ammonium sulfate precipitation: about 500 g ammonium sulfate powder was added to the 1 L of lactic acid bacteria fermentation supernatant.
The desalination was carried out by using the sephadex column G-10 specifically as follows: The crude protein precipitated at 70% ammonium sulfate concentration was filtered with 0.22 μM aqueous system filter membrane. 2 mL of the filtered crude protein was taken each time and added into the sephadex column G-10. Elution was performed with ultra-pure water at a flow rate of 1 mL/min for 60 min. The corresponding eluate of the 60 min elution was collected to obtain the crude protein extract. Then elution was further performed with the ultra-pure water at a flow rate of 1 mL/min for 120 min, ammonium sulfate in the sephadex column was all removed, and this part of the eluate was discarded.
(3) Cation Exchange Chromatography
5 mL of the crude protein extract obtained in the step (2) was taken, and added to a chromatographic column system (AKTA purifier 100, GE Healthcare, Sweden), and the cation exchange chromatographic column (HiPrepTMSP XL 16/10, GE Healthcare) was used. A buffer solution was obtained from 30 mM sodium acetate by adjusting pH to 4, and performing 0.22 μM suction filtration, and ultrasonic treatment for 20 min. An eluent was obtained from 30 mM sodium acetate and 1 M sodium chloride by adjusting pH to 4, and performing 0.22 suction filtration, and ultrasonic treatment for 20 min.
Specifically, that was, after 5 mL of the crude protein extract was loaded, rinsing was performed with the buffer solution at a flow rate of 1 mL/min. The use amount of the buffer solution was 40 mL. Then gradient elution was performed with a mixture of the buffer solution and the eluent. The use amount of the gradient eluent was 30 mL. The flow rate was constant at 1 mL/min.
As shown in
Note: The desalination with the sephadex column G-10 can refer to the desalination of the above step (2).
(4) Reversed-Phase High-Performance Liquid Chromatography
The elution peaks of the step (3) were further purified by using preparative C18 reversed-phase high-performance liquid chromatography (high-performance liquid chromatograph waters 2998, USA). The chromatographic column was Sunfire™ Prep C18 (5 μm, 10×100 mm). The protein preliminary purified solution (a sample with inhibitory activity) obtained in the step (3) was diluted with the ultra-pure water to an appropriate concentration (that was, diluted to 0.5 to 1 mg/mL). The diluted protein preliminary purified solution was placed at 4° C. for later use after being filtered with the 0.22 μM filter membrane. An elution program was set, as shown in Table 2. An injection volume was 5 mL, the mobile phase A was the ultra-pure water (containing 0.05% TFA, v/v), and the mobile phase B was the acetonitrile (containing 0.05% TFA, v/v). The purification results are shown in
Note: The corresponding collected eluate of the 34.5% to 37.5% mobile phase B elution was about 1 mL.
(5) Identification of Purity of Bacteriocin by Analytical HPLC
About 5 mg of freeze-dried bacteriocin powder was obtained after about 1 mL of corresponding collected eluate of 34.5% to 37.5% mobile phase B elution obtained in the above step 4) was freeze-dried at −80° C., and was redissolved with 5 mL of the ultra-pure water. Purity detection was performed with analytical HPLC (high-performance liquid chromatograph waters 2998, USA). The chromatographic column was Sunfire™ Prep C18 (5 μm, 4.6×250 mm). The elution program was as shown in Table 3. The mobile phase was the same as that in the above step (4), and the injection volume was 30 μL. It may be seen from
(6) Determination of Molecular Weight of Bacteriocin by SDS-PAGE
The above peak 2 with the relatively high antibacterial activity, purified by the preparative HPLC, underwent rotary evaporation (37° C.) for being concentrated to a concentration of 2 to 5 mg/mL (the concentration was detected by the BCA protein quantitative kit), and the molecular weight of the bacteriocin was estimated by using SDS-PAGE electrophoresis. The results were shown in
A total of 17 bacteria such as the gram-positive bacteria, the gram-negative bacteria and molds were selected as the indicator bacteria. Among them, the Micrococcus luteus 10209 was purchased from the China Center of Industrial Culture Collection (CICC); Staphylococcus aureus D48, Staphylococcus warneri, Staphylococcus muscae, Staphylococcus carnosus pCA 44, and Staphylococcus carnosus pet 20 were all presented by Professor Eefjan Breukink of Utrecht University in the Netherlands; Escherichia coli DH5α was purchased from the Sangon Biotechnology (Shanghai) Co., Ltd; Bacillus subtilis BAS2, Salmonella paratyphi A CMCC 50093, Salmonella paratyphi B CMCC50094, Salmonella enterica subsp. arizonae CMCC (B) 47001, Salmonella typhimurium CMCC 50015, and Candida albicans were all purchased from the National Center for Medical Culture Collections (CMCC); Pseudomonas aeruginosa ATCC 47085, Salmonella choleraesuis ATCC 13312 and Aspergillus niger ATCC 13073 were all purchased from the American Type Culture Collection (ATCC).
The inhibitory activity of the bacteriocin produced by ZFM225 was tested by an Oxford Cup inhibitory zone experiment. The specific experimental method was the same as that in the step (2) of Embodiment 1. The results are shown in Table 4, which showed that the bacteriocin produced by the Lactobacillus sakei ZFM225 was a bacteriocin with broad-spectrum inhibitory activity, which had relatively high inhibitory activity on most gram-positive bacteria, and also had certain inhibitory activity on these several gram-negative bacteria, including Escherichia coli DH5 α, Salmonella paratyphi A CMCC 50093, Salmonella choleraesuis ATCC 13312 and Pseudomonas aeruginosa ATCC 47085, but had no inhibitory effect on the molds. The bacteriocin had a relatively good inhibitory effect on some common food-borne pathogens, such as Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. It was reported that the bacteriocin produced from the Lactobacillus sakei mainly inhibited growth of gram-positive pathogen Listeria. While the Lactobacillus sakei ZFM225 bacteriocin of the present disclosure had the inhibitory activity on these several gram-negative bacteria, including Escherichia coli DH5 α, Salmonella paratyphi A CMCC 50093, Salmonella choleraesuis ATCC 13312 and Pseudomonas aeruginosa ATCC 47085. Therefore, the Lactobacillus sakei ZFM225 bacteriocin of the present disclosure made up for the defect of the adverse inhibitory effect of most existing bacteriocins produced from the Lactobacillus sakei on gram-negative pathogens.
Therefore, as a biological preservative, the Lactobacillus sakei ZFM225 bacteriocin had potential application value in food safety.
Micrococcus
luteus CICC10209
Staphylococcus
aureus D48
Staphylococcus
warneri
Staphylococcus
muscae
Staphylococcus
carnosus pCA 44
Staphylococcus
carnosus pet 20
Escherichia
coli DH5α
Bacillus
subtilis BAS2
Salmonella
paratyphi A CMCC 50093
Salmonella
paratyphi B CMCC50094
Salmonella
enterica subsp. arizonae
Salmonella
choleraesuis ATCC 13312
Salmonella
typhimurium CMCC 50015
Pseudomonas
aeruginosa ATCC 47085
Candida
albicans
Aspergillus
niger ATCC 13073
Taking the Micrococcus luteus 10209 and the Staphylococcus aureus D48 as the indicator bacteria, the minimum inhibitory concentration of the bacteriocin against these two indicator bacteria was determined by a 96-well titration plate method. After the indicator bacteria were inoculated into an LB liquid medium after being activated for overnight culture, the LB liquid medium was diluted until its OD600 was 0.05 for later use.
The freeze-dried bacteriocin powder was redissolved with 0.05% acetic acid and was gradient-diluted to 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, 0.031 mg/mL, 0.015 mg/mL, 0.007 mg/mL, to obtain bacteriocin ZFM225 solutions of different concentrations. 100 μL of indicator bacteria suspension and bacteriocin diluent were added into a 96-well ELISA Plate in sequence, and underwent stationary culture at 37° C. for 24 h. Absorbance values of thalli at different bacteriocin concentrations were determined with a microplate reader at 600 nm, and a bacterial solution without the bacteriocin was taken as the control. If the OD600 value did not increase after 24 h of culture, then the corresponding bacteriocin concentration under this condition was the minimum inhibitory concentration.
The bacteriocin was mixed with the indicator bacteria after being diluted to different concentration gradients, and the mixtures were placed at the optimum growth temperature of the indicator bacteria for 24 h. Its absorbance value at 600 nm was determined. The results were as shown in
(1) Temperature Stability of Lactobacillus sakei ZFM225 Bacteriocin
The bacteriocin ZFM225 solution (2 mg/mL) obtained in Embodiment 3 above was treated at 4° C., 37° C., 50° C., 60° C., 80° C. and 100° C. for 30 min. Taking Micrococcus luteus 10209 as the indicator bacteria, the inhibitory activity of treated bacteriocins in each group was determined with the Oxford cup method. It may be seen from
(2) PH Stability of Lactobacillus sakei ZFM225 Bacteriocin
The pH of THE bacteriocin ZFM225 solution (2 mg/mL) was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 with 1 M HCl and NaOH. Taking the Micrococcus luteus 10209 as the indicator bacteria, the inhibitory activity of the bacteriocin adjusted to different pH values was detected with by the Oxford cup method. As shown in
(3) Enzyme Sensitivity of Lactobacillus sakei ZFM225 Bacteriocin
Enzymes used in experiments to test the sensitivity of bacteriocin to the enzymes were pepsin (pH 2.0), papain (pH 7.0), trypsin (pH 5.4) and protease K (pH 7.6). Each enzyme in a corresponding optimum pH buffer solution added with the bacteriocin to make its final concentration reach 1 mg/ml. An equal amount of freeze-dried bacteriocin powder (2 mg) was added into each portion. The mixture was placed at 37° C. for 4 h to make them fully react. Then, the fully reacted mixture was treated at 100° C. for 5 min to make the enzyme inactivated. The pH of the mixture was adjusted back to the initial pH with 1 M HCl and NaOH solution. Taking an untreated bacteriocin solution as the blank control, and taking the Micrococcus luteus 10209 as the indicator bacteria, the inhibitory activity of the bacteriocin was determined by an Oxford cup diffusion method.
As shown in
Finally, it should further be noted that the above enumeration is only a few specific embodiments of the present disclosure. Obviously, the present disclosure is not limited to the foregoing embodiments, and there may further be many modifications. All deformations that a person of ordinary skill in the art can directly derive or associate from the content disclosed in the present disclosure shall be considered as the protection scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210476564.7 | Apr 2022 | CN | national |
