CN106701834B - Preparation method of bacteriostatic paenibacillus fermentation liquor extract - Google Patents
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Abstract
The invention provides a preparation method of a bacteriostatic Paenibacillus sp fermentation broth extract. The preparation method comprises the following steps: culturing Paenibacillus polymyxa (Paenibacillus polymyxa) CGMCC No.10062 in cow milk, and collecting supernatant of fermentation liquid. The bacteriostatic paenibacillus fermentation liquor extract prepared by the preparation method provided by the invention has broad-spectrum and high-efficiency bacteriostatic effects, and can effectively inhibit or kill pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method of a bacteriostatic paenibacillus fermentation liquid extract.
Background
Food is extremely easy to deteriorate and decay due to the pollution of external microorganisms in the processes of processing, preservation, transportation and the like, and the traditional food preservation method generally comprises pickling, smoking, cellar storage, wine stain and the like. With the continuous development of scientific technology, chemically synthesized preservatives become one of the most important additives in the food industry. However, research shows that the chemical synthetic preservatives not only affect the original flavor of food, but also have potential safety hazards such as carcinogenicity, teratogenicity or food poisoning, and therefore, the search and development of natural and safe antibacterial agents which can effectively inhibit or kill pathogenic bacteria in food undoubtedly can promote the progress of the food and medicine industries.
Paenibacillus sp is widely present in soil, plant stems and leaves, fruits, root systems and natural fermented foods, and part of the Paenibacillus sp can generate efficient and rich antibacterial substances, has strong inhibition effect on various pathogenic bacteria, and has important prospects in biological control and food preservation. Meanwhile, the paenibacillus has the advantages of fast growth and reproduction, easy survival, strong stress resistance, simple nutritional requirement and the like, and is beneficial to preparing thalli or specific metabolites thereof on a large scale. Thus, the search and development of natural, safe antimicrobial agents that effectively inhibit or kill pathogenic bacteria in food products has prompted advances in the food and pharmaceutical industries.
Disclosure of Invention
The invention aims to solve the technical problems that a chemically synthesized preservative in the field of traditional Chinese medicines and foods in the prior art can influence the original flavor of the foods, potential safety hazards such as cancer induction, teratogenicity or food poisoning exist on human bodies, and a natural and safe bacteriostatic agent is absent, and provides a preparation method of a bacteriostatic paenibacillus fermentation liquor extract. The preparation method can efficiently prepare the bacteriostatic paenibacillus fermentation liquor extract; the bacteriostatic paenibacillus fermentation liquor extract has broad-spectrum and high-efficiency bacteriostatic effect, and can effectively inhibit or kill pathogenic bacteria in food.
The invention adopts one of the technical schemes: a preparation method of a bacteriostatic Paenibacillus fermentation liquor extract comprises the following steps: fermenting Paenibacillus polymyxa CGMCC No.10062 in cow milk, and collecting supernatant of fermentation liquid.
In the invention, the paenibacillus polymyxa is preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 26 days in 2014, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, accession number: CGMCC No.10062, the name of the culture is BD3736, and the classification name is Paenibacillus polymyxa.
In the present invention, the milk may be conventional milk in the art, preferably whole milk and/or skim milk, more preferably skim milk. . The milk may be raw milk or reconstituted milk. The milk solids content may be as conventional in the art, preferably 2-15%, more preferably 10-15%, most preferably 10%, said percentage being the mass volume percentage of the mass of said solids to the volume of said milk.
In the present invention, the temperature of the fermentation may be a temperature conventional in the art, preferably 25 to 35 ℃, more preferably 30 ℃. The fermentation time may be a time period conventional in the art, preferably 1 to 7 days, more preferably 4 to 6 days, most preferably 5 days; the fermentation is preferably a shaking culture, and the rotation speed of the shaking culture can be the rotation speed conventional in the field, and is preferably 180 r/min.
In the present invention, the collection of the supernatant of the fermentation broth may be a routine procedure in the art, and preferably comprises the following steps: inactivating the paenibacillus polymyxa CGMCC No.10062 in the fermentation liquor, centrifuging and collecting supernatant. The inactivation may be performed by conventional procedures in the art, such as high temperature water bath, filtration sterilization, ultra high pressure sterilization, etc., preferably by water bath at 50-80 deg.C for 5-15min, cooling, more preferably by water bath at 60 deg.C for 10min, and cooling to room temperature. The centrifugation may be performed as is conventional in the art, preferably at 9000r/min, and preferably for 25 min.
In the present invention, the preparation method preferably further comprises the following steps: adding salt into the fermentation liquor for precipitation and centrifugation, and collecting the precipitate. The salt may be a salt conventional in the art, preferably ammonium sulfate. The precipitation time may be conventional in the art, preferably from 4 to 24 hours. The final saturation of the salt may be as conventional in the art, preferably 40-90%, more preferably 80%, said percentage being the mass volume percentage of the mass of the salt to the total volume of the broth and salt. The rotation speed of the centrifugation can be the rotation speed conventional in the field, and is preferably 9000 r/min; the time for the centrifugation may be of a length conventional in the art, preferably 25 min.
In the present invention, the preparation method preferably further comprises the following steps: purifying the precipitate, the purifying the precipitate being performed after the collecting the precipitate. The purification preferably comprises the steps of: resuspending the pellet, dialyzing, and passing through gel filtration chromatography. The solution used for the resuspension may be a solution conventional in the art, preferably ultrapure water. The dialysis may be dialysis as is conventional in the art, and the dialysis belt preferably may have a molecular weight cut-off of 3500Da and 1000 Da; the temperature of the dialysis is preferably 4 ℃; the dialysis time is preferably 4-24 h. The chromatographic column for gel filtration chromatography can be a chromatographic column conventional in the art, preferably a chromatographic column for screening molecular weight in the range of 100Da-1500Da, more preferably Sephadex G-15.
In the present invention, the bacteriostatic Paenibacillus fermentation broth extract preferably comprises proteins with molecular weight between 25kDa and 37kDa and proteins with molecular weight less than 10kDa, more preferably proteins with molecular weight less than 10 kDa.
In the invention, the bacteriostatic paenibacillus fermentation liquor extract can preferably inhibit gram-negative bacteria and gram-positive bacteria. The gram-negative bacteria preferably include Escherichia coli (Escherichia coli), Shigella flexneri (Shigella flexneri), and Salmonella enteritidis (Salmonella enteritidis); the gram-positive bacteria preferably include Listeria monocytogenes (Listeria monocytogenes), Micrococcus luteus (Micrococcus luteus) and Bacillus subtilis (Bacillus subtilis).
In the invention, the bacteriostatic activity of the bacteriostatic paenibacillus fermentation liquor extract is not influenced by temperature. Specifically, the bacteriostatic activity of the bacteriostatic paenibacillus fermentation liquor extract is not affected after being respectively treated in water bath at 60 ℃, 80 ℃ and 100 ℃ for 30min or at 121 ℃ for 15 min.
In the invention, the bacteriostatic activity of the bacteriostatic paenibacillus fermentation liquor extract is not influenced by the pH value. Specifically, after the mixture is placed at room temperature for 2 hours at the pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 respectively, the bacteriostatic activity of the bacteriostatic paenibacillus fermentation liquor extract is not affected.
In the invention, the bacteriostatic activity of the bacteriostatic paenibacillus fermentation liquor extract is influenced by pepsin or proteinase K, and is not influenced by lipase or trypsin.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the bacteriostatic paenibacillus fermentation liquor extract prepared by the preparation method of the bacteriostatic paenibacillus polymyxa fermentation liquor extract generated by the paenibacillus polymyxa provided by the invention has broad-spectrum and high-efficiency bacteriostatic effects, and can effectively inhibit or kill pathogenic bacteria in food.
Biological material preservation information
In the invention, the paenibacillus polymyxa is preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 26 days in 2014, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, accession number: CGMCC No.10062, the name of the culture is BD3736, and the classification name is Paenibacillus polymyxa.
Drawings
FIG. 1 shows the bacteriostatic effect of the Paenibacillus fermentation broth extract on different kinds of bacteria.
FIG. 2 shows the ultraviolet absorption results of different collection tubes at 280nm after Sephadex G-15 column chromatography of the bacteriostatic Paenibacillus fermentation broth extract.
FIG. 3 shows the bacteriostatic activity of different collecting tube eluents on salmonella after Sephadex G-15 column chromatography of the bacteriostatic Paenibacillus fermentation broth extract.
FIG. 4 shows the protein content of the eluate from different collection tubes after Sephadex G-15 column chromatography of the Bacteroides fermentation broth extract for bacteriostasis.
FIG. 5 shows SDS-PAGE electrophoresis results of different collection tube eluents after Sephadex G-15 column chromatography of the bacteriostatic Paenibacillus fermentation broth extract of the invention and ammonium sulfate precipitation. Wherein, M: a protein Marker; 23-26 represent the collection tube number for the Sephedx G-15 column chromatography.
FIG. 6 shows the bacteriostatic effect of each band of the bacteriostatic Paenibacillus fermentation liquor extract on Salmonella enteritidis after SDS-PAGE electrophoresis.
FIG. 7 shows the stability of the bacteriostatic Paenibacillus fermentation broth extract of the present invention to different temperature treatments.
FIG. 8 shows the Paenibacillus fermentation broth extract for bacteriostasis of the present inventionStability of different pH treatments. The vertical coordinate in the figure is R2-r2(R is the radius of the inhibition zone generated by adding buffers with different pH values into the sample, and R is the radius of the inhibition zone generated by adding buffers with different pH values).
FIG. 9 shows the stability of the bacteriostatic Paenibacillus fermentation broth extract of the present invention to different enzyme treatments.
FIG. 10 shows the bacteriostatic effect of the bacteriostatic Paenibacillus fermentation broth extract prepared by the preparation method of the invention on Salmonella enteritidis after different saturation ammonium sulfate settlement.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The overnight period of the present invention is a term conventional in the art, i.e., 4 to 24 hours, and the room temperature is a term conventional in the art, i.e., 25 ℃.
The components of the culture medium used in the invention are as follows:
LB culture medium: tryptone (or casein hydrolysate) 1 g; 0.5g of yeast extract; NaCl 0.5g, water 100mL, 115 ℃, 15min of sterilization.
LB solid medium: 1.8g of agar was added to the above LB medium.
BHI medium: tryptone (or casein hydrolysate) 1 g; 0.5g of yeast extract; NaCl 0.5g, water 100mL, 115 ℃, 15min of sterilization.
BHI solid medium: 1.8g of agar was additionally added to the above BHI medium.
The apparatus used in the present invention is as follows:
sephadex G-15: purchased from GE Healthcare.
The strains used in the invention:
escherichia coli (Escherichia coli), Shigella flexneri (Shigella flexneri), Salmonella enteritidis (Salmonella enteritidis), Listeria monocytogenes (Listeria monocytogenes), Micrococcus luteus (Micrococcus luteus) and Bacillus subtilis (Bacillus subtilis) are all purchased from China general microbiological culture Collection center (CGMCC).
In the following examples, the solid content percentage of each cow's milk is the mass volume percentage of the solid mass in the cow's milk to the cow's milk volume.
Example 1 extraction of antibacterial substance derived from Paenibacillus polymyxa (Paenibacillus polymyxa) BD3736
1. Culture of BD3736 Strain
The BD3736 strain preserved in 20% glycerol was inoculated on LB solid medium and placed in an incubator at 30 ℃ for 48 hours under atmospheric conditions. Then, scraping a ring of single colony in 15mL sterilized and defatted milk with the solid content of 10 percent, and carrying out constant temperature shaking culture at 30 ℃ and 180r/min for 24h to obtain BD3736 strain seed fermentation liquor.
And (3) filling 130mL of sterilized skim milk with the solid content of 10% into a 500mL triangular flask, inoculating the BD3736 strain seed fermentation broth according to the inoculation amount of 4% (v/v), and oscillating at the constant temperature of 180r/min for 5 days at 30 ℃ to obtain the BD3736 strain fermentation broth.
2. Preparation of BD3736 strain fermentation broth extract
The fermentation liquor of the BD3736 strain is subjected to water bath at 60 ℃ for 10min, cooled to room temperature, centrifuged at 9000r/min for 25min, and the supernatant is collected. Under the action of magnetic stirring, slowly adding ammonium sulfate powder to ensure that the final saturation degree of salt ions reaches 80 percent, wherein the percentage is the mass volume percentage of the salt accounting for the total volume of the supernatant and the salt, and precipitating overnight. Then, the mixture is centrifuged at 9000r/min for 25min, and the precipitate is collected. And (3) re-suspending the precipitate into deionized water to obtain a BD3736 strain fermentation liquor extract crude product.
Example 2 bacteriostatic profile of BD3736 Strain fermentation broth extract
1. Preparation of indicator bacterium plate
The pathogenic bacteria preserved by 20 percent of glycerol are respectively transferred to BHI solid culture medium and cultured for 48h at 30 ℃. Then, a ring of single colony is scraped and transferred into liquid BHI, and cultured for 24h at 30 ℃ and 180 r/min. Centrifuging at 12000rpm for 5min, collecting thallus, diluting with sterile water, and making into bacterial suspension. Benefit toCounting with a blood counting chamber, and controlling the final bacteria concentration to 106cfu/mL。
And (3) cooling the BHI solid culture medium to 55 ℃, uniformly mixing the BHI solid culture medium with the prepared indicator bacterium suspension, pouring the mixture into a flat plate, and pouring about 20mL of culture medium into each culture dish. And solidifying and fully airing to obtain the indicator bacterium plate.
2. Bacterial inhibition spectrum of BD3736 strain fermentation liquor extract
Escherichia coli (Escherichia coli), Salmonella enteritidis (Salmonella enteritidis) and Shigella flexneri (Shigella flexneri) are used as gram-negative bacteria, and Bacillus subtilis, Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus) and Micrococcus luteus are used as gram-positive bacteria. An oxford cup was placed on the indicator plate, and then 100. mu.L of the crude BD3736 strain fermentation broth extract obtained in example 1 was added to detect whether or not it had inhibitory effect.
The experimental result is shown in figure 1, and the result shows that the BD3736 strain fermentation broth extract crude product has relatively obvious inhibition effect on escherichia coli, Shigella flexneri, salmonella enteritidis, Listeria monocytogenes, glomerulosclerosis gamboge and Bacillus subtilis, and has no inhibition effect on staphylococcus aureus. Therefore, the BD3736 strain fermentation broth extract crude product has broad-spectrum and high-efficiency antibacterial effect.
Example 3 purification of fermentation broth extract of BD3736 Strain
(1) The BD3736 strain fermentation broth extract crude product obtained in example 1 was dialyzed overnight at 4 ℃ in dialysis bags with the cut-off values of 3500Da and 1000Da respectively, and the samples in the bags were collected. And (5) freeze-drying the dialyzate to obtain freeze-dried powder.
(2) Dissolving the freeze-dried powder obtained in the step (1) by using ultrapure water to the mass concentration of 200mg/mL and the sample loading amount of 10mL, and performing chromatographic separation on a Sephadex G-15 (the column volume is about 800mL) gel column. The eluent was ultrapure water with pH 7.0 and the flow rate was 3 mL/min. One tube was collected every 4min, approximately 12mL per tube. The protein is detected by a protein ultraviolet detector at 280 nm. The results of the experiment are shown in FIG. 2.
The above collected sample was evaporated to dryness of the aqueous phase. The diameter of the inhibition zone for salmonella is measured, and the experimental result is shown in figure 3. The results show that the two collecting tubes (A, B respectively) of 14-16 and 23-30 show obvious bacteriostatic activity, and the elution time of the two collecting tubes is relatively close, A is 64min, B is 96min, which indicates that the molecular weights are relatively close.
(3) And (3) respectively detecting the protein content of the different collecting pipes obtained in the step (2) by using a protein quantitative kit. The results are shown in FIG. 4.
Example 4 analysis of the relative molecular masses of extracts from fermentation broths of strain BD3736
(1) SDS-PAGE electrophoresis.
Samples collected from collection tubes numbered 23-26 in example 3 were spotted on SDS-PAGE gels and the results are shown in FIG. 5. The result shows that two obvious color bands appear after the sample is subjected to electrophoresis, and the molecular weights of the two color bands are respectively 37 kD-25 kD and below 10 kDa.
(2) And (3) repeatedly soaking the two strips obtained in the step (1) with sterile distilled water, and testing the bacteriostatic action of different separated strips. The results are shown in FIG. 6 and show that the band appearing below the 10kD region has bacteriostatic activity.
Example 5 temperature stability of BD3736 Strain fermentation broth extract
The crude BD3736 strain fermentation broth extract prepared in example 1 was treated in water bath at 60 deg.C, 80 deg.C and 100 deg.C for 30min and 121 deg.C for 15min, and the non-heat treated CK sample was used as a control. Using the indicator bacterium (Salmonella enteritidis) plate prepared as described in example 2, 100. mu.L of each of the above samples was added to an Oxford cup to measure the zone diameter. The experimental result is shown in figure 7, and the result shows that the bacteriostatic activity of the crude product of the bacteriostatic paenibacillus fermentation liquor extract produced by the BD3736 strain is not obviously changed after the crude product is treated at the temperature of 60 ℃, 80 ℃ and 100 ℃ for 30 min; the activity was not lost even after treatment at 121 ℃ for 15 min. Therefore, the bacteriostatic paenibacillus fermentation liquor extract of the BD3736 strain has better heat stability.
Example 6 stability of fermentation broth extract of BD3736 Strain to pH value
The crude product of BD3736 strain fermentation broth extract prepared in example 1 was adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 with buffers of different pH ranges, and left at room temperature for 2h, and the zone diameter was measured using the indicator (Salmonella enteritidis) plate prepared in example 2 with the corresponding pH buffer as a control, and 100. mu.L of the sample was added to an Oxford cup.
The experimental result is shown in figure 8, and the result shows that the BD3736 strain fermentation liquid extract can tolerate a wider range of pH value, and the bacteriostatic activity is higher than that of acid under an alkaline condition.
Example 7 enzyme stability of fermentation broth extract of BD3736 Strain
5mg/mL pepsin, trypsin, proteinase K and lipase (pH 1.5, 7.5 and 6.5) were prepared, 100. mu.L each was put into a centrifuge tube, and 400. mu.L of the BD3736 strain fermentation broth extract crude product prepared in example 1 was added to make the final enzyme concentration 1 mg/mL. Performing enzymolysis in constant temperature water bath at optimum temperature of each protease (40 deg.C, 37 deg.C, 55 deg.C and 26 deg.C for pepsin, trypsin, proteinase K and lipase respectively) for 4 hr, and inactivating enzyme in 100 deg.C boiling water bath for 5 min. The pH was then adjusted back to 6.0, and the volume was made up to the same volume with 20mmol/mL PBS buffer, using 100. mu.L ultrapure water plus 400. mu.L bacteriocin as a control. Using the indicator bacterium (Salmonella enteritidis) plate prepared as described in example 2, 100. mu.L of each of the above samples was added to an Oxford cup to measure the zone diameter. The experimental results are shown in FIG. 9, and the results show that the fermentation liquor extract of the BD3736 strain is partially sensitive to pepsin and proteinase K and is not sensitive to lipase and trypsin. Therefore, the BD3736 strain fermentation liquid extract is presumed to be protein substances.
Example 8 extraction of antibacterial substance derived from Paenibacillus polymyxa (Paenibacillus polymyxa) BD3736
1. Culture of BD3736 Strain
The BD3736 strain preserved in 20% glycerol was inoculated on LB solid medium and placed in an incubator at 30 ℃ for 48 hours under atmospheric conditions. Then, scraping a ring of single colony in 15mL sterilized and defatted milk with the solid content of 10 percent, and carrying out constant temperature shaking culture at 30 ℃ and 180r/min for 24h to obtain BD3736 strain seed fermentation liquor.
And (3) filling 130mL of sterilized fresh milk into a 500mL triangular flask, inoculating the BD3736 strain seed fermentation broth according to the inoculum size of 4% (v/v) for 25 ℃, and oscillating at the constant temperature of 180r/min for 5 days to obtain the BD3736 strain fermentation broth. The solid content of the sterilized fresh milk is 11%.
2. Preparation of BD3736 strain fermentation broth extract
The fermentation liquor of the BD3736 strain is subjected to water bath at 50 ℃ for 15min, cooled to room temperature, centrifuged at 9000r/min for 25min, and the supernatant is collected. Under the action of magnetic stirring, slowly adding ammonium sulfate powder to enable the final saturation degree of salt ions to reach 80%, wherein the percentage is the mass volume percentage of the salt accounting for the total volume of the supernatant and the salt. The precipitate was allowed to settle overnight. Then, the mixture is centrifuged at 9000r/min for 25min, and the precipitate is collected. And (3) re-suspending the precipitate into deionized water to obtain a BD3736 strain fermentation liquor extract crude product.
The inhibition zone diameter of the paenibacillus fermentation broth extract inhibited in bacteria on the indicator plate (salmonella) was determined. The results show that the zone of inhibition is 14.5mm in diameter.
Example 9 extraction of antibacterial substance derived from Paenibacillus polymyxa (Paenibacillus polymyxa) BD3736
1. Culture of BD3736 Strain
The BD3736 strain preserved in 20% glycerol was inoculated on LB solid medium and placed in an incubator at 30 ℃ for 48 hours under atmospheric conditions. Then, a ring of single colony is scraped in 15mL of sterilized skim milk with the solid content of 10 percent, and the mixture is subjected to constant temperature shaking culture at 30 ℃ and 180r/min for 24 hours to prepare the BD3736 strain seed fermentation liquid.
And (3) filling 130mL of sterilized skim milk into a 500mL triangular flask, inoculating the BD3736 strain seed fermentation broth according to the inoculum size of 4% (v/v) for 25 ℃, and oscillating at the constant temperature of 180r/min for 5 days to obtain the BD3736 strain fermentation broth. The solid content of the sterilized skim milk is 2%.
2. Preparation of BD3736 strain fermentation broth extract
The fermentation liquor of the BD3736 strain is subjected to water bath at 50 ℃ for 15min, cooled to room temperature, centrifuged at 9000r/min for 25min, and the supernatant is collected. Under the action of magnetic stirring, slowly adding ammonium sulfate powder to enable the final saturation degree of salt ions to reach 80%, wherein the percentage is the mass volume percentage of the salt accounting for the total volume of the supernatant and the salt. The precipitate was allowed to settle overnight. Then, the mixture is centrifuged at 9000r/min for 25min, and the precipitate is collected. And (3) re-suspending the precipitate into deionized water to obtain a BD3736 strain fermentation liquor extract crude product.
The inhibition zone diameter of the paenibacillus fermentation broth extract inhibited in bacteria on the indicator plate (salmonella) was determined. The results show that the zone of inhibition is 13mm in diameter.
Example 10 extraction of antibacterial substance derived from Paenibacillus polymyxa (Paenibacillus polymyxa) BD3736
1. Culture of BD3736 Strain
The BD3736 strain preserved in 20% glycerol was inoculated on LB solid medium and placed in an incubator at 30 ℃ for 48 hours under atmospheric conditions. Then, scraping a ring of single colony in 15mL sterilized and defatted milk with the solid content of 10 percent, and carrying out constant temperature shaking culture at 30 ℃ and 180r/min for 24h to obtain BD3736 strain seed fermentation liquor.
And (3) filling 130mL of sterilized skim milk into a 500mL triangular flask, inoculating the BD3736 strain seed fermentation broth according to the inoculum size of 4% (v/v) for 35 ℃, and oscillating at constant temperature of 180r/min for 5 days to obtain the BD3736 strain fermentation broth. The solid content of the sterilized skim milk is 15%.
2. Preparation of BD3736 strain fermentation broth extract
The fermentation liquor of the BD3736 strain is subjected to water bath at 80 ℃ for 5min, cooled to room temperature, centrifuged at 9000r/min for 25min, and the supernatant is collected. Under the action of magnetic stirring, slowly adding ammonium sulfate powder to enable the final saturation degree of salt ions to reach 80%, wherein the percentage is the mass volume percentage of the salt accounting for the total volume of the supernatant and the salt. The precipitate was allowed to settle overnight. Then, the mixture is centrifuged at 9000r/min for 25min, and the precipitate is collected. And (3) re-suspending the precipitate into deionized water to obtain a BD3736 strain fermentation liquor extract crude product.
The inhibition zone diameter of the paenibacillus fermentation broth extract inhibited in bacteria on the indicator plate (salmonella) was determined. The results show that the zone of inhibition is 14mm in diameter.
Example 11 optimization of fermentation time of BD3736 Strain fermentation broth extract
130ml of a fermentation broth of sterilized skim milk having a solid content of 10% filled in a 500ml Erlenmeyer flask as described in example 1 was continuously fermented and sampled every 1 day.
Sterile oxford cups were placed on the indicator plate described in example 2 at equal intervals, 100 μ L of the fermentation broth was added to the oxford cups, 2 replicates for each sample, and the diameter (mm) of the zone of inhibition was measured after incubation of the well-applied petri dish at 30 ℃ for 36 h. The results are shown in Table 1.
TABLE 1 bacteriostatic effect of fermentation supernatant of BD3736 strain on Salmonella, Escherichia coli and Shigella flexneri at different times
Note: determination standard of bacteriostatic zone test result: the diameter (mm) of the bacteriostatic zone is more than 20: is extremely sensitive; the diameter (mm) of the bacteriostatic zone is 15-20: high sensitivity; the diameter (mm) of the inhibition zone is 10-14: medium sensitivity; the diameter (mm) of the bacteriostatic circle is less than 10: low sensitivity; the diameter (mm) of the inhibition zone is 0: it is not sensitive.
As can be seen from Table 1, the bacteriostatic activity of the fermentation supernatant of the BD3736 strain at day 5 is obviously strongest, the average diameter of the bacteriostatic zone is about 13.08mm, and the bacteriostatic Paenibacillus fermentation liquor extract has the strongest bacteriostatic action on Salmonella enteritidis.
Example 12 optimization of ammonium sulfate precipitation of BD3736 Strain fermentation broth extract
130ml of sterilized skim milk containing 10% solids in a 500ml Erlenmeyer flask as described in example 1 was cultured at 30 ℃ at 180r/min for 5 days.
And (3) carrying out water bath on the fermentation liquor at 60 ℃ for 10 min. After cooling to room temperature, 9000r/min, centrifuging for 25min, and collecting supernatant.
Slowly adding ammonium sulfate powder into the supernatant under the action of a magnetic stirrer to ensure that the final saturation degree of salt ions reaches 40%, standing overnight, centrifuging at 9000r/min for 25min, and collecting precipitate; slowly adding ammonium sulfate powder into the supernatant after precipitation under the action of a magnetic stirrer to ensure that the final saturation degree of salt ions reaches 60 percent, standing overnight, centrifuging at 9000r/min for 25min, and collecting the precipitate; slowly adding ammonium sulfate powder into the supernatant after precipitation under the action of a magnetic stirrer to ensure that the final saturation degree of salt ions reaches 80%, standing overnight, centrifuging at 9000r/min for 25min, and collecting the precipitate; slowly adding ammonium sulfate powder into the supernatant after precipitation under the action of a magnetic stirrer to ensure that the final saturation degree of salt ions reaches 90%, standing overnight, centrifuging at 9000r/min for 25min, and collecting the precipitate. The precipitates obtained in stages are resuspended by using deionized water, and the concentration after the resuspension is the same.
Sterile oxford cups were placed at equal intervals on indicator plates (salmonella enteritidis) as described in example 2, and 100 μ L of the above-mentioned resuspension solution was added to the oxford cups. 2 replicates per sample. And (5) culturing the culture dish added with the sample at the constant temperature of 30 ℃ for 24h, measuring the diameter of the inhibition zone and taking a picture.
The experimental result is shown in figure 10, and the result shows that the bacteriostatic effect of the supernatant of the fermentation liquor of the BD3736 strain is optimal after the supernatant is settled by ammonium sulfate with the saturation of 80%.
It should be understood that after reading the above description of the present invention, various changes or modifications can be made by those skilled in the art to the relevant conditions of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Claims (17)
1. A preparation method of antibacterial Paenibacillus sp fermentation liquor extract is characterized by comprising the following steps: fermenting Paenibacillus polymyxa (Paenibacillus polymyxa) CGMCC No.10062 in cow milk, and collecting supernatant of the fermentation liquid.
2. The method of claim 1, wherein the milk is selected from whole milk and/or skim milk.
3. The method of claim 1, wherein said milk has a solids content of 2-15% by mass of said solids as a mass-volume percentage of the volume of said milk.
4. The method of claim 1, wherein the fermentation temperature is 25-35 ℃; and/or the fermentation time is 1-7 days; and/or the fermentation is shaking culture.
5. The method according to claim 4, wherein the rotation speed of the shaking culture is 180 rpm.
6. The method of claim 1, wherein collecting the supernatant of the fermentation broth comprises the steps of: inactivating the paenibacillus polymyxa CGMCC No.10062 in the fermentation liquor, centrifuging and collecting supernatant.
7. The method of claim 6, wherein the inactivation is carried out in a water bath at 50-80 ℃ for 5-15min and cooling.
8. The method of claim 7, wherein the cooling is to room temperature.
9. The method of any one of claims 6-8, wherein the centrifugation is a 9000r/min centrifugation for 25 min.
10. The method of claim 6, further comprising the steps of: adding salt into the supernatant for precipitation and centrifugation, and collecting the precipitate.
11. The method of claim 10, wherein the salt is ammonium sulfate.
12. The method of claim 10, wherein the precipitation time is 4 to 24 hours.
13. The method of any one of claims 10 to 12, wherein the final saturation level of the salt is 40 to 90%, said percentage being the mass volume percentage of the mass of the salt to the total volume of the supernatant and salt.
14. The method of claim 10, further comprising the steps of: purifying the precipitate, said purifying being performed after said collecting the precipitate.
15. The method of claim 14, wherein the purifying comprises the steps of: resuspending the pellet, dialyzing, and passing through gel filtration chromatography.
16. The method according to claim 15, wherein the resuspension is resuspension using ultrapure water; and/or the dialysis bag has a molecular weight cut-off of 3500 daltons and 1000 daltons; and/or the screening molecular weight range of the gel filtration chromatographic column is 100-1500 daltons.
17. The method of claim 1, wherein the bacteriostatic paenibacillus fermentation broth extract comprises proteins with molecular weights between 25 kilodaltons and 37 kilodaltons and less than 10 kilodaltons; and/or, the bacteriostatic paenibacillus fermentation broth extract inhibits gram-negative bacteria and gram-positive bacteria; and/or the bacteriostatic activity of the bacteriostatic paenibacillus fermentation liquor extract is not influenced by temperature; and/or, independent of pH; and/or, is affected by pepsin or proteinase K, and not by lipase or trypsin.
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