CN111484958B - Lactobacillus plantarum with ETEC (ethylene-tetra-ethyl-carbonate) inhibition effect, fermentation product and application thereof - Google Patents
Lactobacillus plantarum with ETEC (ethylene-tetra-ethyl-carbonate) inhibition effect, fermentation product and application thereof Download PDFInfo
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- CN111484958B CN111484958B CN202010296394.5A CN202010296394A CN111484958B CN 111484958 B CN111484958 B CN 111484958B CN 202010296394 A CN202010296394 A CN 202010296394A CN 111484958 B CN111484958 B CN 111484958B
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- lactobacillus plantarum
- dmslp1210
- bacteriocin
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Abstract
The invention provides lactobacillus plantarum with an inhibiting effect on ETEC (ethylene-tetra-ethyl-carbonate), a fermentation product and application thereof, belonging to the technical field of preparation of probiotics and feed additives. The Lactobacillus plantarum (Lactobacillus plantarum) provided by the invention is named as DMSLP1210, the preservation number is GDMCC No.60717, the Lactobacillus plantarum has the function of inhibiting ETEC, the antibacterial activity of the produced bacteriocin is still kept by more than 80% after heat treatment at 121 ℃ for 30min, and the produced bacteriocin has tolerance to high temperature; the bacteriostatic activity is more than 80% of that of the feed probiotic additive at the pH value of 2-10, and the bacteriostatic activity of the feed probiotic additive is more than 90% of that of the feed probiotic additive after the feed probiotic additive is treated by pepsin, trypsin and proteinase K, so that the feed probiotic additive has good tolerance to intestinal environment, reduces diarrhea rate, improves feed conversion efficiency, has safe and reliable effect, and is suitable for being developed into feed probiotic additives.
Description
Technical Field
The invention relates to the technical field of preparation of probiotics and feed additives, and particularly relates to lactobacillus plantarum with an inhibiting effect on ETEC (enterobacter coli) and a fermentation product and application thereof.
Background
Escherichia coli, commonly known as Escherichia coli, belongs to the genus Escherichia and is a gram-negative bacterium. Most of the E.coli strains living in the intestine are harmless, but some strains with pathogenicity can cause diarrhea in piglets. Pathogenic E.coli can be classified into 6 types according to their biological properties: enteropathogenic E.coli (EIEC), Enteropathogenic E.coli (EPEC), Enterohemorrhagic E.coli (EHEC), Enterotoxigenic E.coli (ETEC), enteroadhesive E.coli (EAEC), and Diffusible Adhesive E.coli (DAEC). At present, enterotoxigenic escherichia coli (ETEC) is a main infection source causing diarrhea of piglets, often causes yellow scour and white scour of piglets, and is a main factor causing death of pigs in the global pig industry.
Antibiotics are still the most widely used antibiotics for treating piglet diarrhea at present, however, the problem of antibiotic residues is increasingly serious, and the development and spread of antibiotic-resistant strains also limit the use of antibiotics. Development of antibiotic substitutes and establishment of alternative therapeutic strategies are not easy. Lactic acid bacteria have long-term history of safe eating, have positive health influence on hosts, lactic acid, hydrogen peroxide, bacteriocin and the like generated by the lactic acid bacteria can effectively inhibit intestinal pathogenic microorganisms, and multiple strains of lactic acid bacteria are proved to be capable of inhibiting pathogenic escherichia coli. In recent years, a number of probiotic products have been used to treat diarrhoea in piglets, including drugs containing live probiotic microorganisms, and in addition some bacterial peptides with antibacterial potential are considered as suitable antibiotic substitutes, of which lactein has become one of the most interesting hotspots.
The lactobacillin is a protein or protein compound with biological activity which is generated through a ribosome synthesis mechanism and extracellularly secreted in the metabolic process of lactic acid bacteria, has the advantages of high safety, good stability, difficult residue, no drug resistance and the like, can inhibit gram-positive bacteria with close relationship with the lactic acid bacteria and non-lactic acid bacteria, thereby cutting off the harm of pathogenic microorganisms to animal intestinal tracts, and has important application value. The lactobacillus bacteriocin is reported to be almost only applied to the field of food at present, but also has wide application prospect in the fields of feed, organic fertilizer, environmental protection, personal care products and the like. At present, Nisin (Nisin) is the only bacteriocin which is approved by the U.S. food and drug administration, and Nisin with high purity (99%) is expensive and difficult to buy in the market, so that Nisin with low development cost has high commercial attraction. In recent years, the research on the lactobacillin mainly focuses on the aspects of bacteriostatic activity, heat stability, pH tolerance and the like, and the intensive research on the biological properties of the lactobacillin is crucial to the processing application of the lactobacillin. The antibacterial property of the lactobacillin on other bacteria is closely related to that of the producing strain, and research shows that the strains such as lactobacillus plantarum, lactobacillus acidophilus, enterococcus faecium and the like can produce the lactobacillin with an inhibiting effect on pathogenic escherichia coli. However, the synthesis amount of lactobacilli is affected by various factors such as high-yielding strains, pH, temperature, medium composition and growth conditions of the strains, and therefore, the isolation of high-efficiency lactobacillus strains and the optimization of bacteriocin synthesis conditions of lactic acid bacteria are of great importance.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum with an inhibiting effect on ETEC and a fermentation product thereof, and bacteriocin generated by the lactobacillus plantarum is high-temperature resistant, acid resistant and high in protease resistance, can be used for preparing a feed additive, and is used for reducing diarrhea rate and improving feed conversion efficiency.
In order to realize the purpose of the invention, the Lactobacillus plantarum DMSLP1210 is obtained by screening 164 strains of lactic acid bacteria separated from pig manure in the breeding base of the three-flower pigs in the city of the northeast university of agriculture through layer upon layer. The strain DMSLP1210 is Lactobacillus plantarum (Lactobacillus plantarum). The strain is preserved in Guangdong province microorganism strain collection center (GDMCC for short, address: No. 59 building No. 5 building of Miyaolu No. 100, Miyaolu, Guangzhou city, microbiological research institute, Guangdong province, zip code 510070) in 7-8 days in 2019, and is classified and named as Lactobacillus plantarum with the collection number of GDMCC No. 60717.
The microbiological characteristics of the Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 provided by the invention are as follows: gram-positive bacteria, the cell morphology is Brevibacterium, irregular shape. The colony is white and round, and the surface is smooth and non-transparent.
Physiological and biochemical tests show that nitrate reduction reaction and catalase tests are negative, gelatin is not liquefied, indole and H2S are not produced, the motility is not caused, cellobiose, heptaphylline, fructose, sorbitol, melibiose, glucose, lactose, maltose, mannitol, mannose, salicin and sucrose can be utilized, and arabinose, raffinose and xylose cannot be utilized.
The strain DMSLP1210 is identified as Lactobacillus plantarum (Lactobacillus plantarum) by combining a physiological and biochemical identification method and a 16S rDNA sequence analysis method.
Carrying out an antibacterial experiment by using cell-free fermentation supernatant of DMSLP1210, simultaneously determining the antibacterial effect of the cell-free fermentation supernatant (pH is adjusted to 6.5) excluding organic acid and the cell-free fermentation broth excluding hydrogen peroxide of the strain, taking ETEC CVCC 1518 as an indicator bacterium, wherein the experimental result shows that the diameter of an antibacterial ring of the cell-free fermentation supernatant is 18.87 +/-0.18 (mm), the diameter of the antibacterial ring of the cell-free fermentation supernatant excluding organic acid is 14.89 +/-0.32 mm, and the diameter of the antibacterial ring of the cell-free fermentation supernatant excluding hydrogen peroxide is 14.87 +/-0.22 mm, which indicates that the fermentation product of H12-10 has ETEC inhibitory activity, and the experiment proves that the fermentation product of the strain retains more than 80% of the antibacterial activity to ETEC when the pH value is 2.0-10.0, and does not generate hemolytic hazard.
The fermentation product of lactobacillus plantarum DMSLP1210 belongs to the protection scope of the present invention.
The invention provides a microbial inoculum containing the lactobacillus plantarum DMSLP1210 or a fermentation product thereof.
The invention also provides an animal feed additive containing the lactobacillus plantarum DMSLP 1210. The invention also provides an animal feed containing the lactobacillus plantarum DMSLP 1210.
The probiotic effect of the lactobacillus plantarum DMSLP1210 is identified by an in vitro method, and the result shows that the lactobacillus plantarum DMSLP1210 can resist acid and resist the internal environment of the gastrointestinal tract and has the potential of probiotics.
The invention also finds that the bacteriocin produced by the lactobacillus plantarum DMSLP1210 has high-temperature tolerance, the bacteriostatic activity is kept above 80% after heat treatment for 30min at 121 ℃, and the bacteriostatic activity of the bacteriocin on ETEC is kept above 90% after the bacteriocin in the fermentation broth of the lactobacillus plantarum DMSLP0315 is respectively treated by pepsin, proteinase K and trypsin, so that the invention provides a medicament containing the lactobacillus plantarum DMSLP1210 for inhibiting ETEC and application of the lactobacillus plantarum DMSLP1210 in reducing ETEC pollution in food or feed.
The invention also provides application of Lactobacillus plantarum DMSLP1210 or a fermentation product thereof in preparing a feed additive. Preferably, the feed additive can inhibit ETEC and has low sensitivity to pepsin, trypsin and proteinase K.
The invention provides application of Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 or a fermentation product thereof in ETEC inhibition.
In the embodiment of the invention, the lactobacillus plantarum DMSLP1210 and the lactobacillus plantarum DMSLP0315 with the preservation number of GDMCC No.60716 are taken as compound strains, and the effective viable count is 1:1, so that the synergistic bacteriostatic effect is achieved. Lactobacillus plantarum DMSLP0315 has been deposited in 2019, 7/8 at Guangdong province microorganism culture collection center (GDMCC for short, address: No. 59 building 5 of Mirabilitum 100, Guangzhou, Japan microbiological research institute, zip code 510070), and is classified and named Lactobacillus plantarum with the number of GDMCC No. 60716. The embodiment of the invention also obtains a cheap natural culture medium for producing bacteriocin by fermenting the compound strain, and the components of the cheap natural culture medium are 4 percent of corn steep liquor powder, 2 percent of yeast extract and 2 percent of glucose; the optimal fermentation conditions of the culture medium are that the fermentation temperature is 32 ℃, the fermentation pH is 5.5, the inoculation amount is 2%, and after the fermentation is carried out for 22 hours according to the optimal process parameters, spray drying is carried out according to the following conditions: the concentration of the material is 30 percent, the air inlet temperature is 160 ℃, the air outlet temperature is 80 ℃, and the titer of bacteriocin in prepared by the method reaches 2394.76 AU/g.
Therefore, the invention further provides a complex microbial inoculum, which is prepared by compounding Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 and Lactobacillus plantarum (Lactobacillus plantarum) DMSLP0315 with the preservation number of GDMCC No. 60716. Preferably, the composition is compounded by 1:1 effective viable count.
Further, the invention also provides application of Lactobacillus plantarum DMSLP1210 or a fermentation product thereof in improving feed conversion rate.
The invention also provides application of Lactobacillus plantarum DMSLP1210 or a fermentation product thereof in promoting animal growth or increasing animal weight.
The invention has the following beneficial effects:
1. lactic acid bacteria can produce various bacteriostatic substances such as organic acid, diacetyl, hydrogen peroxide, bacteriocin and the like in the metabolic process, and the bacteriostatic substances are various and very difficult to remove one by one. The standard for determining bacteriocin accepted in the prior art is that after organic acid and hydrogen peroxide in the fermentation supernatant of the lactic acid bacteria are removed, the bacteriocin still has bacteriostatic activity, namely the existence of the bacteriocin in the fermentation supernatant of the lactic acid bacteria can be preliminarily determined, and further intensive research is needed for the evidence of specific substances. Generally, the interference of organic acid is eliminated by adopting a neutralization acid method, and the interference of hydrogen peroxide is eliminated by treating fermentation liquor by adopting catalase. The fermentation supernatant of the Lactobacillus plantarum DMSLP1210 can still keep obvious bacteriostatic activity after organic acid and hydrogen peroxide are removed, so bacteriocin is considered to exist in the fermentation supernatant of the Lactobacillus plantarum DMSLP 1210. The bacteria are non-hemolytic, resistant to streptomycin and vancomycin, and sensitive to tetracycline and ampicillin.
2. Bacteriocin produced by the lactobacillus plantarum DMSLP1210 has stronger stress resistance, and the high temperature resistance of the bacteriocin makes the bacteriocin not easy to be damaged in the hot processing process; the antibacterial agent has good acid stability and acid-base tolerance, shows good acid stability between pH 2-6, and has antibacterial activity obviously higher than that of pH 8-10 within the range of pH 2-4. The bacteriocin produced by the lactobacillus plantarum DMSLP1210 has low sensitivity to protease, better tolerance to intestinal environment, tolerance to low acid environment in gastrointestinal tract, stronger bacteriostatic action in the gastrointestinal tract and potential for developing the bacteriocin for treating diarrhea.
3. The lactobacillus plantarum DMSLP1210 has remarkable probiotics, can improve the utilization rate of feed, can reduce the disease rate of animals, the diarrhea rate of the animals caused by diet antibiotic additives and the death and culling rate, and more importantly can enhance the immune function of the animals, increase the daily feed intake of the animals, improve the conversion rate of the feed, reduce the feed conversion ratio, enhance the production performance of the animals, improve the steady state of the internal digestive tract environment of the animals, and has the effects of promoting the absorption and utilization of nutrition, promoting the growth of the animals and the like, thereby saving the cost, improving the economic benefit and having good application prospect.
4. The lactobacillus plantarum DMSLP1210 and the lactobacillus plantarum DMSLP0315 with the preservation number of GDMCC No.60716 are compounded in equal proportion, and fermentation is carried out on an improved corn starch culture medium (4 percent of corn starch, 2 percent of yeast extract and 2 percent of glucose) according to the following conditions: the fermentation temperature is 32 ℃, the fermentation pH is 5.5, the inoculation amount is 2%, and the fermentation time is 22 h; after the fermentation was completed, spray drying was performed under the following conditions: the concentration of the material is 30 percent, the air inlet temperature is 160 ℃, the air outlet temperature is 80 ℃, and the fermentation product with the bacteriocin titer of 2394.76AU/g is prepared and can be used as a feed additive.
5. The fermentation product of the lactobacillus plantarum DMSLP0315 is used as a probiotic and a feed additive component, so that the problems of drug resistance and residue of antibiotic feed additives do not exist, and the application is safe.
Drawings
FIG. 1 shows the standard titer curve of bacteriocin produced by DMSLP 0315.
FIG. 2 shows a standard titer curve of bacteriocin produced by DMSLP 1210.
FIG. 3 is a graph showing the growth of DMSLP 0315.
FIG. 4 is a growth graph of DMSLP 1210.
FIG. 5 is a diagram showing the effect of culture time on the bacteriostatic activity of DMSLP 1210.
FIG. 6 is a diagram showing the effect of culture temperature on the bacteriostatic activity of DMSLP 1210.
FIG. 7 is a graph showing the sensitivity of bacteriocin produced by DMSLP1210 to temperature.
FIG. 8 is a diagram showing the pH sensitivity of bacteriocin produced by DMSLP 1210.
FIG. 9 is a diagram showing the sensitivity of bacteriocin produced by DMSLP1210 to enzymes.
FIG. 10 is a schematic diagram of the bacteriostatic activity of metabolites of strains of 7 formulation modes.
FIG. 11 is a schematic diagram showing the changes in the bacteriostatic activity of the supernatant in four culture modes.
FIG. 12 is a graph showing the minimum inhibitory concentration of the fermentation dry product obtained in the fermentation at constant pH 5.5.
FIG. 13 shows the minimum inhibitory concentration of the fermentation product obtained in the non-constant fermentation.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The embodiment of the invention adopts a double-layer flat plate perforation method to detect the bacteriostatic activity of the bacteriocin. Enterotoxigenic Escherichia coli (Enterotoxigenic Escherichia coli) ETEC CVCC 1518: purchased from the China veterinary culture collection center.
MRS culture medium: 10g of tryptone, 2g of diamine hydrogen citrate, 5g of peptone, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 5g of yeast powder, 5g of Tween-801 g, 5g of beef extract, 20g of glucose, 0.25g of manganese sulfate, 0.58g of magnesium sulfate and 1L of distilled water, adding 2% agar into a solid culture medium, adjusting the pH value to 5.8, and sterilizing at 121 ℃ for 15 min.
LB culture medium: 10g of peptone, 10g of sodium chloride, 5g of yeast powder and 1L of distilled water, adding 2% agar into a solid culture medium, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 15 min.
Example 1 isolation and characterization of Lactobacillus plantarum (Lactobacillus plantarum) DMSLP0315
1. 164 strains of lactic acid bacteria isolated from pig manure of the breeding base of the three-flower pig in Archeng, northeast university of agriculture and 168 strains of lactic acid bacteria preserved in the laboratory.
After 85 pig manure samples are subjected to gradient dilution and culture, 164 suspected lactobacillus strains with calcium-dissolving rings are selected, named by letter H, and after the separated strains are cultured in an MRS solid culture medium for 48 hours, the colony morphology is mostly milky round, slightly raised in the middle, opaque or semitransparent and regular in edge. Gram staining is positive, the thallus is in a short rod, a long rod or a round sphere shape, lactobacillus and lactococcus which are frozen and stored in a refrigerator at the temperature of-20 ℃ are respectively inoculated in MRS and M17 liquid culture media in the inoculation amount of 2 percent, and are cultured for 16h at the temperature of 37 ℃ and are continuously activated and passaged for 3 times for standby. Freezing and storing the separated pig source lactic acid bacteria in a refrigerator at the temperature of 20 ℃ below zero, inoculating the frozen and stored strain in an MRS liquid culture medium by 2 percent of inoculation amount before use, culturing for 16h at the temperature of 37 ℃, and continuously activating and passaging for 3 times for later use.
Enterotoxigenic Escherichia coli (Enterotoxic Escherichia coli, ETEC CVCC 1518) was inoculated in LB liquid medium at an inoculum size of 2%, cultured at 37 ℃ for 16 hours, and continuously activated for 3 passages for use.
2. Determination of the bacteriostatic Activity
2.1 preparation of cell-free fermentation supernatant the test strain was inoculated in MRS liquid medium at 2% inoculum size, incubated at 37 ℃ for 20h, centrifuged at 12000r/min for 15min, the supernatant was collected, pH was adjusted to 6.5, interference of organic acids was excluded; dissolving catalase in 0.02mol/L sodium acetate buffer solution, adding the solution into the supernatant, controlling the final mass concentration of catalase to be 5.0mg/mL, and incubating at 37 ℃ for 2h to eliminate the interference of hydrogen peroxide; filtering with 0.22 μm filter membrane to remove thallus and other impurities; the cell-free fermentation supernatant was concentrated 10-fold by freeze-drying and reconstituted with sterile water.
2.2 the diameter of the bacteriostatic circle is measured by adopting a double-layer flat plate punching method. Pouring soft agar containing 1.2 percent into a sterile plate according to 10mL per plate, and airing; inoculating ETEC CVCC 1518 into LB culture medium (about 50 ℃) containing 0.7% agar with the inoculation amount of 0.6%, pouring 6mL of LB agar culture medium containing ETEC CVCC 1518 on the bottom layer agar; after air drying, a hole is punched by a puncher, 100 mu L of cell-free fermentation supernatant is added into the hole, after diffusion for 3 hours in a biological clean bench, the cell-free fermentation supernatant is cultured for 9 hours at 37 ℃, and the diameter of a bacteriostatic circle is measured by a vernier caliper.
Bacteriostatic experiments were performed on cell-free fermentation supernatants of 168 strains of lactic acid bacteria preserved in the laboratory and 164 strains of lactic acid bacteria isolated from swine waste, and the bacteriostatic effects of cell-free fermentation supernatants excluding organic acids (pH adjusted to 6.5) and cell-free fermentation broth excluding hydrogen peroxide of the test strains were determined, using ETEC CVCC 1518 as an indicator, and the test results are shown in Table 1 (partial negative test results are not shown).
TABLE 1 inhibitory Effect of lactic acid bacteria on ETEC CVCC 1518
As can be seen from table 1, four strains of bacteria having bacteriostatic activity were still found after the interference of organic acid was eliminated, wherein 1 strain was DMSLP0315, which was selected from 168 strains of lactic acid bacteria deposited in the laboratory; 3 strains were numbered H3-5, H7-6 and DMSLP1210 and were isolated from 164 strains of lactic acid bacteria of porcine origin. The diameters of the inhibition zones of the supernatant obtained by cell-free fermentation of the four lactic acid bacteria are basically unchanged before and after the treatment by catalase. According to the test results, it can be concluded that the cell-free fermentation supernatant of four lactic acid bacteria may have bacteriocin which has an inhibitory effect on ETEC CVCC 1518 besides organic acid and hydrogen peroxide, and the four bacteria are preliminarily determined to be capable of producing the bacteriocin which inhibits ETEC CVCC 1518.
2.3 determination of bacteriocin potency
AU/mL is used for expressing bacteriocin titer, and the specific determination method comprises the steps of taking a time period sample with the largest diameter of an inhibition zone as a standard substance, after redissolving the standard substance, diluting the time period sample by 1, 2, 4, 8 and 16 times with a sterilized fresh MRS liquid culture medium, determining the diameter of the inhibition zone by adopting a double-layer flat plate punching method, marking the dilution degree of the inhibition zone which can obviously appear in the dilution process as a dilution factor D, drawing a standard curve according to the relationship between the diameter of the inhibition zone and the dilution factor, and calculating the bacteriocin titer according to a formula.
The relationship between the diameter of the inhibition zone and the dilution factor is as follows: r ═ a + b log (d)
The formula for calculating the bacteriocin titer is as follows:
r-diameter of inhibition zone (mm), a-intercept, b-slope, D-dilution factor, D-concentration per hole, namely sample loading/D.
Drawing a bacteriocin standard titer curve (see fig. 1 and fig. 2) according to the size of the inhibition zone corresponding to each dilution, and obtaining that the regression equation of the lactobacillus plantarum DMSLP0315 is that y is 7.42x +0.776, and the correlation coefficient R2 is 0.9870; the regression equation of the lactobacillus plantarum DMSLP1210 is 6.4x +1.7273, and the correlation coefficient R2 is 0.9860. x represents the logarithmic value of the concentration (d) per well, and y represents the zone diameter (mm). The regression equations of the above bacteria all meet the requirements of further tests.
2.4 determination of growth Curve
Lactobacillus plantarum DMSLP0315 and Lactobacillus plantarum DMSLP1210 are inoculated in MRS culture medium at 2%, cultured at 37 deg.C for 36h, and viable count is used as determination index to draw growth curve shown in FIG. 3 and FIG. 4. As can be seen from the figure, the growth lag phase of the lactobacillus plantarum DMSLP0315 is about initial to 6 hours of culture, then the lactobacillus plantarum enters the logarithmic growth phase, the number of viable bacteria is exponentially multiplied in the growth lag phase, the viable bacteria number reaches the highest value when the lactobacillus plantarum is cultured for 20 hours, 16 to 24 hours are the stable phase of the lactobacillus plantarum DMSLP0315, the bacteria grow slowly, and the lactobacillus plantarum gradually enters the decay phase after 24 hours; the growth lag phase of the lactobacillus plantarum DMSLP1210 is about initial to 6 hours, then the lactobacillus plantarum DMSLP1210 enters a logarithmic growth phase, the number of living bacteria is exponentially multiplied in the logarithmic growth phase, the number of the living bacteria reaches the highest value when the lactobacillus plantarum DMSLP1210 is cultured for 22 hours, 16 to 24 hours are the stable phase of the lactobacillus plantarum DMSLP1210, the bacteria grow slowly, and the bacteria gradually enter a decay phase after 24 hours.
3. Identification of lactic acid bacteria
3.1 observing colony and thallus morphology, streaking the screened bacteriocin-producing lactic acid bacteria strains on an MRS solid culture medium respectively, culturing for two days at 37 ℃, and observing colony color, shape, size and other characteristics and the like; meanwhile, single bacterial colony is picked by using an inoculating loop for gram staining, and the staining condition and the morphology of the thallus are observed under a microscope.
3.2 physiological and Biochemical assays
Performing physiological and biochemical identification on the screened bacteriocin-producing lactobacillus strain, including catalase test, gelatin liquefaction test, indole test, nitrate reduction test and H2S production test, motility test, and sugar fermentation test (including arabinose, cellobiose, heptaphylline, fructose, sorbitol, galactose, glucose, lactose, maltose, mannitol, mannose, melibiose, raffinose, salicin, xylose, sucrose).
According to Bergey's Manual of identification of bacteria and Classification and Experimental methods of lactic acid bacteria, strain DMSLP0315 and strain DMSLP1210 can be preliminarily identified as Lactobacillus.
3.316S rDNA sequence analysis
Inoculating the screened bacteriocin-producing lactic acid bacteria strain into an MRS liquid culture medium, culturing at 37 ℃ for 20h, centrifuging and collecting thalli, and extracting genomic DNA according to the instruction of a rhizobacteria genomic DNA extraction kit. And (3) subpackaging the extracted DNA into PCR tubes, and storing in a refrigerator at the temperature of-20 ℃ for later use. The extracted genomic DNA was detected by 1% agarose gel electrophoresis using 5. mu.L of the sample. The target gene is amplified by using universal primers 27F and 1492R, the sequence after PCR amplification is sequenced by Jilin province Kuume biotechnology limited, homology analysis is carried out in a gene bank by BLAST, a strain with the highest similarity with the target gene sequence is searched, a phylogenetic tree is constructed on the strain with the highest similarity by MEGA5.0 software and an adjacency method, and thus, DMSLP0315 and DMSLP1210 are located in the same evolutionary branch as Lactobacillus plantarium _ JCM _1149, Lactobacillus plantarium _ NWAFU1186 and Lactobacillus plantarium _ BCH 930.
The strain DMSLP0315 and strain DMSLP1210 were identified as Lactobacillus plantarum (Lactobacillus plantarum) by combining the results of morphological, physiological, biochemical and 16S rDNA sequence analyses.
The strain DMSLP1210 has been deposited in Guangdong province microorganism culture collection (GDMCC for short, address: No. 59 building 5 of Miyaolu No. 100 of Miyaolu, Guangzhou, and microbiological research institute of Guangdong province, zip code 510070) 7/8 days in 2019, and is classified and named as Lactobacillus plantarum with the deposition number GDMCC No. 60717.
Example 2 Effect of culture conditions on bacteriostatic Activity of Lactobacillus plantarum DMSLP1210
(1) Influence of culture time on bacteriostatic activity of target strain
Inoculating the screened target strain DMSLP1210 into an MRS liquid culture medium in an inoculation amount of 1%, standing and culturing at 37 ℃ for 32h, sampling every two hours from 16h, taking 10mL to prepare cell-free supernatant, determining the antibacterial activity by taking ETEC CVCC 1518 as an indicator bacterium, and determining the optimal culture time. The effect of the culture time on the bacteriostatic activity of lactobacillus plantarum DMSLP1210 is shown in fig. 5. As can be seen from FIG. 5, the bacteriostatic activity of the strain is gradually enhanced from 16h to 24h along with the increase of the culture time, the bacteriostatic activity reaches the maximum at 24h, and the bacteriostatic activity of the strain tends to be stable at 26h to 30 h. The bacteriostatic activity of the strain is not obviously different between 16 and 30 hours (P is more than 0.05).
(2) Influence of culture temperature on bacteriostatic activity of target strain
Inoculating the selected target strain DMSLP1210 in MRS liquid culture medium at an inoculum size of 1%, culturing at 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, and 36 deg.C for 20 hr, collecting 10mL to prepare cell-free supernatant, measuring antibacterial activity with ETEC CVCC 1518 as indicator bacteria, and determining the optimum culture temperature. The effect of the culture time on the bacteriostatic activity of lactobacillus plantarum DMSLP1210 is shown in fig. 6. As can be seen from FIG. 6, the bacteriostatic activity of the strain is highest at a culture temperature of 30 deg.C, and gradually decreases with the increase of the culture temperature at a temperature of 30-36 deg.C. The difference of the bacteriostatic activity of the strains under different culture temperatures is not significant (P > 0.05).
Example 3 evaluation of safety of Lactobacillus plantarum DMSLP1210
1. Hemolytic test
Hemolytic tests were performed with streptococcus pyogenes as a control. After the streptococcus pyogenes is inoculated on a columbian blood agar plate for culture, a transparent hemolysis ring appears around a bacterial colony, and the hemolysis is type B (beta hemolysis); after the lactobacillus plantarum DMSLP0315 and the lactobacillus plantarum DMSLP1210 are inoculated to a Columbia blood agar plate for culture, no hemolytic ring appears around an observed colony, so that the two strains can be judged to have no hemolysis and cannot generate hemolysis harm.
2. Sensitivity to antibiotics
According to the evaluation criteria of the American society for clinical and laboratory standards CLSI, the results of the sensitivity of two strains to 7 common antibiotics were obtained (Table 2), and it can be seen from the table that Lactobacillus plantarum DMSLP0315 and Lactobacillus plantarum DMSLP1210 both have resistance to streptomycin and vancomycin and are sensitive to tetracycline and ampicillin. Wherein, the lactobacillus plantarum DMSLP0315 and the lactobacillus plantarum DMSLP1210 also have resistance to gentamicin and are sensitive to chloramphenicol and erythromycin.
TABLE 2 sensitivity of four lactic acid bacteria to antibiotics
Note: "R" represents drug resistance; "I" represents an intermediary; "S" represents sensitivity.
Example 4 stress resistance assay of bacteriocins produced by Lactobacillus plantarum DMSLP1210
1. Sensitivity of bacteriocins to temperature
The cell-free fermentation supernatant is kept at 60 ℃, 80 ℃, 100 ℃ and 121 ℃ for 10min and 30min respectively, and a bacteriostasis test is carried out after ice cooling to determine the influence of temperature on the bacteriostasis activity of the bacteriocin.
The influence of temperature on bacteriocin in the lactobacillus plantarum DMSLP1210 fermentation broth is shown in fig. 7, and it can be seen from the figure that the bacteriostatic activity of the bacteriocin produced by DMSLP1210 decreases with the increase of the heating temperature and the extension of the heating time, the heating temperature significantly influences the bacteriostatic activity of the bacteriocin (p <0.05), and the heating for 10min and 30min does not significantly influence the bacteriostatic activity of the bacteriocin (p > 0.05); the bacteriocin produced by the lactobacillus plantarum DMSLP0315 keeps more than 90% of bacteriostatic activity after heat treatment at 121 ℃ for 30min, and has the characteristic of high temperature resistance.
2. Sensitivity of bacteriocins to pH
Adjusting pH of the cell-free fermentation supernatant to 2-10 with 3mol/L HCL and NaOH, and performing bacteriostatic experiment after incubating at 37 deg.C for 2h to determine the influence of pH on bacteriocin bacteriostatic activity.
The influence of pH on bacteriocin in the lactobacillus plantarum DMSLP1210 fermentation broth is shown in FIG. 8, and it can be seen from the figure that the bacteriocin produced by the strain has bacteriostatic activity in the range of pH 2-10, the bacteriostatic activity is in a descending trend along with the gradual increase of pH, but the bacteriocin is all kept above 80%, and good acid-base tolerance is shown; the bacteriocin produced by the bacterium has insignificant difference of bacteriostatic activity between pH 2 and pH 4 (p is more than 0.05), and shows better acid stability; the bacteriostatic activity of the bacteriocin in the pH range of 2-4 is obviously higher than that of the bacteriocin in the pH range of 8-10, and the bacteriostatic activity of the bacteriocin reaches the highest when the pH is 2, which indicates that the bacteriocin produced by the bacterium has the best effect in an acidic environment and the bacteriostatic activity is reduced in an alkaline environment.
3. Sensitivity of bacteriocins to enzymes
Adjusting pH of cell-free fermentation supernatant to optimum pH of pepsin, trypsin, proteinase K and catalase with HCL and NaOH. Bacteriostatic tests were performed after 2h in 37 ℃ aqueous solution to determine the effect of the enzyme on the bacteriostatic activity of bacteriocins.
The effect of enzyme treatment on bacteriocin in the fermentation broth of lactobacillus plantarum DMSLP1210 is shown in fig. 9, which shows that the bacteriocin produced by lactobacillus plantarum DMSLP1210 retains more than 90% of bacteriostatic activity after being treated by catalase, pepsin, proteinase K and trypsin, and is insensitive to the effects of four enzymes.
Example 5 Effect of Strain Complex Co-culture on bacteriocin Synthesis amount
Combining biological characteristic results of bacteriocins produced by lactobacillus plantarum DMSLP0315 and lactobacillus plantarum DMSLP1210, selecting lactobacillus plantarum DMSLP0315, lactobacillus plantarum H7-6 (clinical isolates in the invention are identified as lactobacillus plantarum) and lactobacillus plantarum DMSLP1210 with good characteristics, performing compound co-culture according to inoculation viable count 1:1, and then determining the bacteriostatic activity of metabolites (figure 10). As can be seen from the figure, after the lactobacillus plantarum DMSLP0315+ DMSLP1210 is compounded, the bacteriostatic activity gradually increases and then tends to be stable along with the increase of the culture time, and the bacteriostatic activity is highest and the bacteriocin synthesis amount is maximum when the lactobacillus plantarum DMSLP0315 and the lactobacillus plantarum DMSLP1210 are compounded, so that the optimal compounding mode is determined.
The bacteriostatic activity of lactobacillus plantarum DMSLP0315 and lactobacillus plantarum DMSLP1210 after co-culture (inoculum size 1:1) is shown in FIG. 11. As can be seen from the figure: compared with a control group (pure culture of lactobacillus plantarum DMSLP0315, pure culture of lactobacillus plantarum DMSLP1210, equal ratio supernatant of pure culture respectively), the antibacterial activity of bacteriocin is remarkably increased (p is less than 0.05) after the lactobacillus plantarum DMSLP0315 and the lactobacillus plantarum DMSLP1210 are co-cultured, and the synergistic effect is achieved.
Taking lactobacillus plantarum DMSLP0315 and lactobacillus plantarum DMSLP1210 as compound strains to obtain a cheap natural culture medium for producing bacteriocin by fermenting the compound strains, wherein the components of the natural culture medium are 4% of corn steep liquor powder, 2% of yeast extract and 2% of glucose; the optimal fermentation conditions of the culture medium are that the fermentation temperature is 32 ℃, the fermentation pH is 5.5, the inoculation amount is 2%, and after the fermentation is carried out for 22 hours according to the optimal process parameters, spray drying is carried out according to the following conditions: the concentration of the material is 30 percent, the air inlet temperature is 160 ℃, the air outlet temperature is 80 ℃, the prepared lactein is brown yellow powder, and the titer of the bacteriocin reaches 2394.76 AU/g.
Example 6 determination of minimum inhibitory concentrations of Lactobacillus plantarum DMSLP0315 and DMSLP1210 equi
1. Fermentation scale: 10L fermentation tank
2. Culture medium: the corn starch culture medium is prepared as follows: 4% of corn steep liquor powder, 2% of yeast extract, 2% of glucose and 2% of tween 80.
3. Fermentation conditions are as follows: inoculating strain DMSLP0315 and strain DMSLP1210 (effective viable count 1:1) at 2% inoculum concentration, fermenting at 32 deg.C and pH5.5 for 22h without adjusting pH.
4. And (3) freeze drying conditions: freeze-drying with vacuum freeze-drying machine under the following conditions: the fermentation product is pre-frozen in a refrigerator at-20 deg.C for 12h, and then freeze-dried at-60 deg.C for 24 h.
5. Method for measuring bacteriocin titer
The bacteriocin titer was calculated with reference to the formula of example 1.
6. Results
(1) Fermentation at constant pH5.5
According to the conditions, the fermentation is carried out by maintaining the constant pH value of 5.5, the pH value is adjusted by adopting sodium hydroxide, the antibacterial activity of the final freeze-dried fermentation product reaches 2394.76AU/g, the lactic acid content is 19.368mg/g, and the antibacterial activity of the final freeze-dried fermentation product after dilution by sterile water is found to be 1836.25AU/mL when the prepared dried fermentation product is diluted by 11 times, and no obvious antibacterial ring appears when the prepared dried fermentation product is diluted by 21 times. After the pH value of the final fermentation product is adjusted to 6.5, the final fermentation product is subjected to freeze drying, the bacteriostatic activity is found to reach 2265.80AU/g, the dilution multiple of the dried product with an obvious bacteriostatic zone can be detected to be 11 times (figure 12), and the content of the lactic acid measured at the time is 12.845 mg/g.
(2) Natural fermentation with non-constant pH
According to the conditions, non-constant pH fermentation is adopted, the antibacterial activity of the final freeze-dried fermented product is measured to reach 3538.67AU/g, the lactic acid content is 46.364mg/g, the antibacterial activity after dilution with sterile water is shown in figure 13, the antibacterial activity of the prepared dried fermented product is 1359.18AU/mL when the prepared dried fermented product is diluted by 51 times, and no obvious antibacterial ring appears after the dilution times are increased. After the pH value of the final fermentation product is adjusted to 6.5, spray drying is carried out, the dilution multiple of the dried product with an obvious antibacterial zone is 11 times, and the measured lactic acid content in the dried product is as follows: 11.485 mg/g.
According to the results, the non-constant pH fermentation is recommended, the fermentation is finished after 22 hours, and the obvious inhibition zone can still be obtained after the dried product is diluted by 51 times, wherein the lactic acid content is 46.364 mg/g.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. Lactobacillus plantarum DMSLP1210, deposited with GDMCC No. 60717.
2. A fermentation product of Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 according to claim 1.
3. A microbial agent or animal feed comprising Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 or a fermentation product thereof according to claim 1.
4. A pharmaceutical agent comprising Lactobacillus plantarum (DMSLP 1210) or a fermentation product thereof according to claim 1.
5. The agent of claim 4, which is an ETEC-inhibiting agent.
6. An animal health product comprising Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 according to claim 1 or a fermentation product thereof.
7. Use of Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 or its fermentation product according to claim 1 for the preparation of a feed additive.
8. Use of Lactobacillus plantarum (Lactobacillus plantarum) DMSLP1210 or its fermentation product according to claim 1 for the preparation of a medicament or health product inhibiting ETEC.
9. Use of Lactobacillus plantarum DMSLP1210 or its fermentation product according to claim 1 for increasing feed conversion ratio.
10. Use of Lactobacillus plantarum DMSLP1210 or its fermentation product according to claim 1 for promoting animal growth or increasing animal weight.
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Non-Patent Citations (4)
| Title |
|---|
| Lactobacillus plantarum BSGP201683 Isolated from Giant Panda Feces Attenuated Inflammation and Improved Gut Microflora in Mice Challenged with Enterotoxigenic Escherichia coli;Qian Liu等;《Front Microbiol》;20170926;1885 * |
| Probiotic Lactobacillus plantarum Promotes Intestinal Barrier Function by Strengthening the Epithelium and Modulating Gut Microbiota;Jing Wang等;《Front Microbiol》;20180831;1953 * |
| 植物乳杆菌对大肠杆菌致猪肠上皮细胞损伤的保护作用研究;曾燕霞等;《中国优秀硕士学位论文全文数据库》;20180215;D050-88 * |
| 植物乳杆菌对感染大肠杆菌K88幼龄仔猪肠黏膜先天免疫反应的影响;杨宽民等;《第七届中国饲料营养学术研讨会论文集》;20141016;214 * |
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