CN117568240A - Lactobacillus plantarum for inhibiting aeromonas hydrophila, and preparation method and application of cell-free supernatant of lactobacillus plantarum - Google Patents
Lactobacillus plantarum for inhibiting aeromonas hydrophila, and preparation method and application of cell-free supernatant of lactobacillus plantarum Download PDFInfo
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses lactobacillus plantarum for inhibiting aeromonas hydrophila and a preparation method and application of a cell-free supernatant thereof, and belongs to the technical field of biology. The lactobacillus plantarum is preserved in China general microbiological culture Collection center (CGMCC) at the 12 th month of 2022 and the preservation number is 26280. The cell-free supernatant has the effect of inhibiting aeromonas hydrophila which causes the quality safety problem of the aquatic products, is a natural thermal green bacteriostatic agent, and has positive effects of delaying the quality degradation speed of the aquatic products, reducing the food-borne diseases caused by the aeromonas hydrophila and preventing the generation of drug resistance of the aeromonas hydrophila. Is hopeful to replace the common chemical inhibition means and meets the requirements of consumers on green natural products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to lactobacillus plantarum for inhibiting aeromonas hydrophila, and a preparation method and application of cell-free supernatant thereof.
Background
Aeromonas hydrophila (Aeromonas hydrophila) is a genus of Aeromonas in the family of Vibrionaceae, is a gram-negative Brevibacterium, and is commonly found in seawater and various environments and foods. Can cause food poisoning reaction such as gastroenteritis, and can also cause food spoilage, and is common food-borne pathogenic and decay-causing microorganism in aquatic products.
At present, the quality and safety problems of aquatic products brought by controlling microorganisms mainly adopt physical or chemical means. Physical means include physical bacteriostasis means such as low-temperature storage, modified atmosphere packaging, low-temperature plasma and the like, but the treatment process is relatively complicated. The chemical method mainly uses chemical bacteriostats, but long-term use of the chemical bacteriostats causes environmental pollution, causes mutation of microorganisms and drug resistance, and meanwhile, does not meet the increasingly pursued 'green and natural' foods of consumers. Therefore, the quality and safety problems of aquatic products caused by the control of microorganisms by using biological methods are receiving more and more attention.
Lactobacillus plantarum has been recognized as a safe microorganism by the us FDA as one of the lactic acid bacteria. Lactobacillus plantarum supernatant is a metabolite of lactobacillus plantarum excluding the microbial body, including secreted proteins, enzymes, short fatty acids, organic acids, amino acids, polypeptides, extracellular vesicles, and the like. The biological activity and stability of lactobacillus plantarum supernatant have also been demonstrated to be higher than that of either of the cell itself and the supernatant. Based on the method, the supernatant of the lactobacillus plantarum with the effect of inhibiting aeromonas hydrophila is screened, and has important significance in delaying quality degradation of aquatic products and guaranteeing safety of the aquatic products.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum for inhibiting aeromonas hydrophila and a preparation method and application of a cell-free supernatant thereof, so as to solve the problems in the prior art. The invention provides a lactobacillus plantarum from fermented yoghourt, natural and safe supernatant of the lactobacillus plantarum is utilized to inhibit aeromonas hydrophila, so that the quality safety problem of aquatic products caused by the aeromonas hydrophila is reduced, the common chemical inhibition means is replaced, and the requirements of consumers on green natural products are met.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a Lactobacillus plantarum (Lactobacillus plantarum) MY2 for inhibiting aeromonas hydrophila, wherein the Lactobacillus plantarum MY2 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 26280 in 12 months and 26 days of 2022.
The invention also provides a cell-free supernatant of the lactobacillus plantarum MY 2.
The invention also provides a preparation method of the cell-free supernatant, which comprises the steps of fermenting and culturing the lactobacillus plantarum MY2 in an MRS culture medium, centrifuging and filtering the obtained fermentation culture solution, and collecting the supernatant to obtain the cell-free supernatant of the lactobacillus plantarum MY 2.
Further, the temperature of the fermentation culture is 37 ℃ and the centrifugation is carried out for 48 hours.
Further, the centrifugation condition was 8000 Xg for 5min.
Further, the step of filtering is to pass the supernatant after centrifugation through a 0.22 μm filter membrane.
The invention also provides an application of the lactobacillus plantarum MY2 or the cell-free supernatant in delaying fish spoilage.
Further, the fish meat includes tuna meat.
The invention also provides a method of delaying spoilage of fish meat comprising immersing said fish meat in a cell-free supernatant comprising said fish meat.
Further, the fish meat includes tuna meat.
The invention discloses the following technical effects:
the lactobacillus plantarum MY2 is separated and screened from the self-produced yoghurt by the herd in the sea area of the Hulenbell of the Mongolian autonomous region, the cell-free supernatant has the effect of inhibiting aeromonas hydrophila (Aeromonas hydrophila) which causes the quality safety problem of aquatic products, the minimum antibacterial concentration is 10% (v/v), and the aeromonas hydrophila used by the invention is the dominant saprophytic bacteria screened in tuna in the laboratory. In-situ experiments for inhibiting aeromonas hydrophila are carried out in tuna, and the invention discovers that lactobacillus plantarum supernatant shows obvious inhibiting capability on aeromonas hydrophila.
The lactobacillus plantarum supernatant separated in the invention has good capability of inhibiting aeromonas hydrophila, is a natural thermal green antibacterial agent, and has positive effects of delaying quality degradation speed of aquatic products, reducing food-borne diseases caused by aeromonas hydrophila and preventing the generation of drug resistance of aeromonas hydrophila. Is hopeful to replace the common chemical inhibition means and meets the requirements of consumers on green natural products.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of Aeromonas hydrophila growth;
FIG. 2 is a scanning electron microscope image of Aeromonas hydrophila after treatment of lactobacillus plantarum cell-free supernatant;
FIG. 3 shows the change in the total number of colonies (A) and the number of colonies of Aeromonas hydrophila (B) in tuna after the treatment of lactobacillus plantarum cell-free supernatant.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Examples
1. Isolation of strains and preparation of cell-free supernatant:
screening: taking 25mL of self-fermented yoghurt of herlands of the inner Mongolian autonomous region, mixing uniformly, taking 100 mu L of homogeneous liquid to be coated on an MRS culture medium, culturing for 72 hours at 37 ℃, picking single bacterial colonies for streak purification until the bacterial colonies are detected to be single bacterial strains by a bacterial colony microscope, carrying out gram staining and catalase detection on the bacterial strains, and preserving the bacterial strains with gram positive and negative peroxidation detection on a slant culture medium at 4 ℃.
Cell-free supernatant preparation: inoculating the selected strain into a commercial MRS culture medium, culturing at 37 ℃ for 48 hours, centrifuging the obtained fermentation broth for 5min at 8000 Xg, removing thalli, filtering the supernatant with a 0.22 mu m filter membrane, and collecting the supernatant, namely the lactobacillus plantarum cell-free supernatant.
2. Screening of strains:
inoculating Aeromonas hydrophila strain isolated from tuna into TSB culture medium, culturing at 30deg.C for 18 hr, and diluting the obtained bacterial suspension with PBS to 10 8 CFU/mL is inoculated into a TSA culture medium (v/v) which is sterilized and cooled to 50 ℃, and after shaking and mixing, the TSA culture medium containing bacterial liquid is poured into a sterilization flat plate to prepare the TSA flat plate containing aeromonas hydrophila. After the plates were coagulated, 3 small holes were uniformly punched in each plate using a 6mm punch, 100. Mu.L of the prepared cell-free supernatant was added to the holes, spread at 4℃for 8 hours, transferred to 30℃for cultivation for 72 hours, and then the size of the inhibition zone was observed (sterilized ultrapure water was used as a control), and the strain having the largest inhibition zone was designated as MY2 and used for the subsequent test.
3. Molecular biological identification of strains:
the bacterial 16s rRNA universal primer is adopted to sequence the MY2 strain, the sequencing result is shown as SEQ ID NO.1, the result is compared in NCBI database, the identification result shows that the strain MY2 is lactobacillus plantarum Lactiplantibacillus plantarum, the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at the month 26 of 2022, the preservation address is CGMCC No. 3 national academy of sciences of China, the Beijing area towards the sun, the preservation number is CGMCC No.26280, and the strain is classified and named as lactobacillus plantarum Lactiplantibacillus plantarum.
SEQ ID NO.1：
GTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTA。
4. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of cell-free supernatant of the Strain against Aeromonas hydrophila
Cell-free supernatant of strain MY2 was prepared into 8 dilution gradient solutions with concentrations of 80%, 40%, 20%, 10%, 5%, 2.5%, 1.25% and 0.75% in 96-well plates, respectively, using a 2-fold dilution method, and each well contained 100. Mu.L of cell-free supernatant solution; culturing Aeromonas hydrophila in TSB culture medium at 30deg.C for 16 hr to logarithmic strain phase, and diluting the obtained bacterial solution with sterilized TSB to 10 6 After CFU/mL, the mixture was added to a 96-well plate containing cell-free supernatant, and 100. Mu.L of Aeromonas hydrophila liquid was added to each well to give final concentration of cell-free supernatant in each well40%, 20%, 10%, 5%, 2.5%, 1.25%, 0.75% and 0.325%; after incubation of the 96-well plates at 30℃for 24h, turbidity was observed for each well. The control wells were: does not contain MRS sterilization culture medium and aeromonas hydrophila; the negative control group was: cell-free supernatant + sterilized TSB medium without aeromonas hydrophila. Cell-free supernatant concentration without macroscopic turbidity wells was defined as MIC value; the culture medium in the non-turbid wells was aspirated and spread on TSA plate medium and incubated at 30℃for 48h, and the concentration of cell-free supernatant in the 96-well plates corresponding to the non-turbid colony growth plates was defined as MBC value (Table 1). Preparation of culture tubes for strains MIC and MBC, procedure as described above, but the whole culture system was amplified to 10mL and completed in the tubes, sampling was performed every 2h to determine OD 600nm Values and strain culture curves were plotted as shown in figure 1.
TABLE 1 MIC and MBC of Lactobacillus plantarum cell-free supernatant for Aeromonas hydrophila
5. Strain scanning electron microscope image after strain cell-free supernatant treatment
Taking aeromonas hydrophila bacterial liquid cultured for 4 hours in a test tube in the step 4, centrifuging for 5 minutes at 8000 Xg and 4 ℃, adding 2.5% glutaraldehyde solution into bacterial precipitate after removing supernatant, fixing for 4 hours at 4 ℃, centrifuging again, removing supernatant, washing bacterial liquid with PBS buffer for 3 times, carrying out gradient dehydration (30, 50, 70, 85 and 90%) on bacterial liquid with ethanol, standing for 15 minutes after adding ethanol each time, finally dehydrating with 100% ethanol for 2 times, centrifuging for 15 minutes, removing supernatant, adding isoamyl acetate for replacement for 2 times, carrying out gradient freezing for 12 hours at-20 ℃, 40 ℃ and 80 ℃ below zero on the prepared bacterial precipitate, taking out, freeze-drying the bacterial precipitate, and observing the freeze-dried bacterial precipitate by adopting a scanning electron microscope. As shown in FIG. 2, the bacterial cells of the control group showed a complete rod-like structure, but the surface of the strain was wrinkled after the treatment of the cell-free supernatant of Lactobacillus plantarum, the surface of the strain was wrinkled to an increased extent with the increase of the concentration of the cell-free supernatant, and the cells showed different degrees of collapse and breakage.
6. Application of cell-free supernatant of strain in delaying putrefaction of tuna
Culturing Aeromonas hydrophila in TSB culture medium for 18 hr, collecting bacterial liquid, centrifuging at 10000 Xg and 4deg.C for 2min, discarding supernatant, washing bacterial cells with PBS buffer solution for 2 times, and diluting bacterial liquid with PBS to 10 6 CFU/mL was ready for use. The commercially available ultralow temperature frozen tuna is cut into 2cm x 2cm squares in a sterile super clean bench, and the cut squares are first immersed in 10 6 CFU/mL aeromonas hydrophila liquid (1:5, w/v), airing in an ultra-clean bench for 15min, immersing in cell-free supernatant of lactobacillus plantarum MY2 again (1:5, w/v), airing again for 15min, filling the tuna blocks into a sterile sampling bag, and placing in a 4 ℃ incubator for 8d, and sampling every 2 d. The control tuna blocks were immersed in sterile PBS prior to the sterile MRS medium. 25g of tuna is taken from each bag of each sampling point, added into 225mL of sterile physiological saline, homogenized, and 100 mu L of the homogenized tuna is respectively coated on a TSA flat plate culture medium and an Aeromonas flat plate culture medium (Aeromonas Medium Base, ryan) to measure the colony count and the change of the colony count of the Aeromonas hydrophila. As can be seen from FIG. 3, the total colony count of the control group increased to 6.2log CFU/g after 4 days of storage, but the total colony count of MIC group and 2MIC group was still less than 4.0log CFU/g and 3.8log CFU/g and 3.3log CFU/g; after 4 days of storage, the colony count of the aeromonas hydrophila in the control group is 5.3log CFU/g, and the colony count of the aeromonas hydrophila in the MIC group and the 2MIC group is only 3.1log CFU/g and 2.8log CFU/g. The test result shows that the lactobacillus plantarum cell-free supernatant can effectively inhibit the growth of aeromonas hydrophila, delay the quality degradation speed of tuna and prolong the storage time of the tuna.
In conclusion, the lactobacillus plantarum MY2 cell-free supernatant can effectively inhibit the growth of aeromonas hydrophila and delay the quality degradation speed of tuna.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The Lactobacillus plantarum MY2 for inhibiting aeromonas hydrophila is characterized in that the Lactobacillus plantarum MY2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 26280 in 12 months and 26 days of 2022.
2. A cell-free supernatant of lactobacillus plantarum MY2 as claimed in claim 1.
3. The method for preparing cell-free supernatant according to claim 2, wherein lactobacillus plantarum MY2 is fermented in MRS medium, and the obtained fermented culture solution is centrifuged and filtered, and the supernatant is collected to obtain the cell-free supernatant of lactobacillus plantarum MY 2.
4. A method according to claim 3, wherein the fermentation culture is carried out at a temperature of 37 ℃ for a period of 48 hours.
5. A method according to claim 3, wherein the centrifugation conditions are 8000 x g centrifugation for 5min.
6. A method according to claim 3, wherein the step of filtering is performed by passing the centrifuged supernatant through a 0.22 μm filter.
7. Use of lactobacillus plantarum MY2 as claimed in claim 1 or a cell-free supernatant as claimed in claim 2 for delaying fish spoilage.
8. The use according to claim 7, wherein the fish meat comprises tuna meat.
9. A method of delaying spoilage of fish comprising immersing the fish in a cell-free supernatant of claim 2.
10. The method of claim 9, wherein the fish meat comprises tuna meat.
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