CN111484958A - 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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- CN111484958A CN111484958A CN202010296394.5A CN202010296394A CN111484958A CN 111484958 A CN111484958 A CN 111484958A CN 202010296394 A CN202010296394 A CN 202010296394A CN 111484958 A CN111484958 A CN 111484958A
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- lactobacillus plantarum
- plantarum
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- fermentation
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Abstract
The invention provides lactobacillus plantarum with an inhibiting effect on ETEC (enterobacter coli) and a fermentation product and application thereof, belonging to the technical field of preparation of probiotics and feed additives.A lactobacillus plantarum (L acetobacter plantarum) provided by the invention is named as DMS L P1210 with the preservation number of GDMCC No.60717, has an ETEC inhibiting function, maintains more than 80% of the antibacterial activity after heat treatment at 121 ℃ for 30min, has tolerance to high temperature, maintains more than 80% of the antibacterial activity at the pH value of 2-10, maintains more than 90% of the antibacterial activity after treatment of the bacterium with pepsin, trypsin and proteinase K, has good tolerance to the 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 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 (L actinobacillus plantarum) DMS L P1210 is obtained by screening 164 lactobacillus strains separated from pig manure of a three-flower pig breeding base in Atlantic university, northeast China through a layer upon layer, wherein the strain DMS L P1210 is the lactobacillus plantarum (L actinobacillus plantarum), the strain is preserved in Guangdong microbial cultures collection center (GDMCC, address: No. 59 building 5 of Mitsugaku No. 100, Guangdong institute of microbiology, postal code 510070) in 7, 8 days in 2019, and is classified and named as L actinobacillus plantarum with the preservation number of GDMCC No. 60717.
The Lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 has the microbiological characteristics of gram-positive bacteria, irregular shape and white and round convex colony, and the surface is smooth and opaque.
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 DMS L P1210 is identified as lactobacillus plantarum (L actobacillus 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 DMS L P1210, and 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, wherein an ETEC CVCC1518 is used as an indicator bacterium, the test 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 antibacterial activity on ETEC at the pH value of 2.0-10.0 without hemolytic hazard.
The fermentation product of lactobacillus plantarum DMS L P1210 belongs to the scope of protection of the present invention.
The invention provides a microbial inoculum containing the lactobacillus plantarum DMS L P1210 or a fermentation product thereof.
The invention also provides an animal feed additive containing the lactobacillus plantarum DMS L P1210 and animal feed containing the lactobacillus plantarum DMS L P1210.
The probiotic effect of the lactobacillus plantarum DMS L P1210 is identified by an in vitro method, and the result shows that the lactobacillus plantarum DMS L P1210 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 DMS L P1210 has high-temperature resistance, the antibacterial activity is kept above 80% after heat treatment for 30min at 121 ℃, and the antibacterial activity of the bacteriocin on ETEC is kept above 90% after the bacteriocin in the fermentation broth of the lactobacillus plantarum DMS L P0315 is respectively treated by pepsin, proteinase K and trypsin, so that the invention provides the ETEC inhibiting medicine containing the lactobacillus plantarum DMS L P1210 and the application of the lactobacillus plantarum DMS L P1210 in reducing the ETEC pollution in food or feed.
The invention also provides application of the lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or a fermentation product thereof in preparing a feed additive.
The invention provides application of lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or a fermentation product thereof in ETEC inhibition.
In the embodiment of the invention, lactobacillus plantarum DMS L P1210 and lactobacillus plantarum DMS L P0315 with the preservation number of GDMCC No.60716 are taken as compound strains, the mixture ratio is carried out according to the effective viable count of 1:1, and the synergistic antibacterial effect is achieved, lactobacillus plantarum DMS L P0315 is deposited in the microbial strain preservation center of Guangdong province in 7-8 days 2019 (GDMCC for short, address: 59 th building 5 of Mitsukushou No. 100 Dazhong institute of Guangzhou province, postfix 510070), classified and named as L actinobacillus plantarum, the deposition number is GDMCC No. 60716. the embodiment of the invention also obtains a cheap natural culture medium for producing bacteriocin the fermentation of the compound strains, the components are 4% of corn pulp powder, 2% of yeast extract and 2% of glucose, the fermentation conditions of the fermentation temperature of the culture medium are 32 ℃, the fermentation pH5.5, the inoculation amount of the fermentation is 2%, the fermentation temperature of the fermentation is 22h, the air outlet concentration is 30%, the optimal temperature of the material is 160 ℃, and the optimal temperature of the air outlet temperature is 2394.76 g of the optimal lactobacillus material, and the intake temperature is 2394.76 g of the optimal.
Therefore, the invention further provides a composite microbial inoculum which is prepared by compounding Lactobacillus plantarum (L Acobacillus plantarum) DMS L P1210 and Lactobacillus plantarum (L Acobacillus plantarum) DMS L P0315 with the preservation number of GDMCC No.60716, preferably, the composite microbial inoculum is prepared by compounding the effective viable count of 1: 1.
Further, the invention also provides application of lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or a fermentation product thereof in improving feed conversion rate.
The invention also provides application of the lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or a fermentation product thereof in promoting animal growth or increasing animal weight.
The invention has the following beneficial effects:
1. the lactobacillus plantarum DMS L P1210 fermentation supernatant can still maintain obvious bacteriostatic activity after the organic acid and the hydrogen peroxide are eliminated, so the bacterium is considered to have no vancomycin, resistance to streptomycin and archaebacterium and sensitivity to tetracycline and penicillin.
2. The bacteriocin produced by the lactobacillus plantarum DMS L P1210 has strong stress resistance, high temperature resistance enables the bacteriocin to be not easily damaged in the thermal processing process, good acid stability and acid-base tolerance are achieved, good acid stability is shown between pH 2 and 6, and the bacteriostatic activity in the pH range of 2 to 4 is obviously higher than that in the pH range of 8 to 10.
3. The lactobacillus plantarum DMS L P1210 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 dietary 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 feed conversion rate, 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 DMS L P1210 and the lactobacillus plantarum DMS L P0315 with the preservation number of GDMCC No.60716 are compounded in equal proportion, an improved corn starch culture medium (4% of corn starch, 2% of yeast extract and 2% of glucose) is fermented according to the conditions that the fermentation temperature is 32 ℃, the fermentation pH is 5.5, the inoculation amount is 2% and the fermentation time is 22 hours, and after the fermentation is finished, spray drying is carried out according to the conditions that the material concentration is 30%, the air inlet temperature is 160 ℃, the air outlet temperature is 80 ℃ to prepare a fermentation product with the bacteriocin titer of 2394.76AU/g, and the fermentation product can be used as a feed additive.
5. The fermentation product of the lactobacillus plantarum DMS L P0315 is used as a probiotic and feed additive ingredient, so that the problems of drug resistance and residue of antibiotic feed additives are solved, and the application is safe.
Drawings
FIG. 1 shows the standard titer curve of bacteriocin produced by DMS L P0315.
FIG. 2 is a standard titer curve for bacteriocin produced by DMS L P1210.
FIG. 3 is a graph of the growth of DMS L P0315.
Figure 4 is a graph of the growth of DMS L P1210.
FIG. 5 is a graph showing the effect of incubation time on the bacteriostatic activity of DMS L P1210.
FIG. 6 is a schematic diagram showing the effect of culture temperature on the bacteriostatic activity of DMS L P1210.
FIG. 7 is a graph showing the temperature sensitivity of bacteriocin produced by DMS L P1210.
FIG. 8 is a graph showing the pH sensitivity of bacteriocins produced by DMS L P1210.
FIG. 9 is a graph showing the sensitivity of bacteriocin produced by DMS L P1210 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.
10g of tryptone, 2g of dihydrodiamine citrate, 5g of peptone, 5g of sodium acetate, 2g of dipotassium 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, 2% agar is added into a solid culture medium, the pH value is adjusted to be 5.8, and the solid culture medium is sterilized for 15min at 121 ℃.
L B culture medium comprising peptone 10g, sodium chloride 10g, yeast powder 5g, and distilled water 1L, adding 2% agar into solid culture medium, adjusting pH to 7.2, and sterilizing at 121 deg.C for 15 min.
Example 1 isolation and characterization of Lactobacillus plantarum (L actinobacillus plantarum) DMS L P0315
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 into L B liquid medium at an inoculum size of 2%, cultured at 37 deg.C for 16h, and continuously activated for 3 passages for use.
2. Determination of the bacteriostatic Activity
2.1 preparation of cell-free fermented supernatant the test strain was inoculated in 2% inoculum size in MRS liquid medium, incubated at 37 ℃ for 20h, centrifuged at 12000r/min for 15min, the supernatant was collected, pH adjusted to 6.5 to exclude interference of organic acids, catalase was dissolved in 0.02 mol/L sodium acetate buffer, added to the supernatant, final mass concentration of catalase was controlled at 5.0mg/m L, incubated at 37 ℃ for 2h to exclude interference of hydrogen peroxide, bacteria and other impurities in the supernatant were removed by filtration with 0.22 μm filter, cell-free fermented supernatant was concentrated 10-fold by freeze-drying and reconstituted with sterile water.
2.2 determination of the diameter of the zone of inhibition Using a double-layer plate perforation method, pour 1.2% soft agar into sterile plates at 10m L per plate, air dry, inoculate 0.6% ETEC CVCC1518 into L B medium (about 50 deg.C) containing 0.7% agar, pour 6m L of L B agar medium containing ETEC CVCC1518 onto the bottom agar, perforate after air dry, add 100 μ L cell-free fermentation supernatant into the well, diffuse 3 hours in biological clean bench, incubate at 37 deg.C for 9 hours, determine the diameter of the zone of inhibition using 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 CVCC1518 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 CVCC1518
As can be seen from Table 1, four strains of bacteria still have bacteriostatic activity after the interference of organic acid is eliminated, wherein 1 strain is DMS L P0315, which is screened from 168 strains of lactic acid bacteria preserved in a laboratory, 3 strains are numbered H3-5, H7-6 and DMS L P1210, which are separated from 164 strains of swine-origin lactic acid bacteria, the diameters of bacteriostatic circles of cell-free fermented supernatants of the four strains of lactic acid bacteria are basically unchanged before and after the cell-free fermented supernatants of the four strains of lactic acid bacteria are treated by catalase, and the cell-free fermented supernatants of the four strains of lactic acid bacteria can possibly have bacteriocin which has inhibitory effect on ETEC CVCC1518 besides organic acid and hydrogen peroxide, so that the four strains of bacteria can be primarily determined to be capable of producing the bacteriocin which inhibits ETEC CVCC 1518.
2.3 determination of bacteriocin potency
AU/m L 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, re-dissolving the standard substance, diluting the standard substance 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 at which an obvious inhibition zone can 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.
And (3) drawing a bacteriocin standard titer curve according to the sizes of the inhibition zones corresponding to different dilutions (see fig. 1 and fig. 2), and obtaining a lactobacillus plantarum DMS L P0315 regression equation with y being 7.42x +0.776 and a correlation coefficient R2 being 0.9870, a lactobacillus plantarum DMS L P1210 regression equation with y being 6.4x +1.7273, a correlation coefficient R2 being 0.9860. x represents the logarithm value of the concentration (d) of each hole, and y represents the diameter (mm) of the inhibition zone.
2.4 determination of growth Curve
Lactobacillus plantarum DMS L P0315 and Lactobacillus plantarum DMS L P1210 are inoculated in MRS culture medium in an inoculation amount of 2%, cultured at 37 ℃ for 36h, and a growth curve is drawn by taking the number of living bacteria as a determination index and is shown in figures 3 and 4. from the figures, the growth lag phase of Lactobacillus plantarum DMS L P0315 is about initial to 6h of culture, then the strain enters a logarithmic growth phase, the strain is exponentially multiplied in the logarithmic growth phase, the number of living bacteria reaches the highest value when the strain is cultured for 20h, 16 to 24h are the stable phase of growth of Lactobacillus plantarum DMS L P0315, the strain grows slowly, the strain gradually enters a decay phase after 24h, the growth lag phase of Lactobacillus plantarum DMS L P1210 is about initial to 6h, then the strain enters the logarithmic growth phase, the exponential multiplication in the decay phase, the number of living bacteria reaches the highest value when the strain is cultured for 22h, and 16 to 24h are the stable phase of growth of Lactobacillus plantarum DMS L P1210, the strain grows slowly, and gradually enters the decay phase after 24 h.
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 DMS L P0315 and strain DMS L P1210 can be preliminarily identified as Lactobacillus.
3.316S rDNA sequence analysis
The screened bacteriocin-producing lactic acid bacteria strain is inoculated in MRS liquid culture medium, the strain is cultured for 20 hours at 37 ℃, thalli are collected by centrifugation, genomic DNA is extracted according to the specification of a rhizobacteria genomic DNA extraction kit, the extracted DNA is subpackaged in a PCR tube and is stored in a refrigerator at-20 ℃ for later use, a5 mu L sample is subjected to 1% agarose gel electrophoresis to detect the extracted genomic DNA, a target gene is amplified by adopting general primers 27F and 1492R, the sequence after the PCR amplification is sequenced by Jilin province American biotechnology limited company, homology analysis is carried out in a gene bank by B L AST to search a strain with the highest sequence similarity with the target gene, MEGA5.0 software is adopted to construct a phylogenetic tree by utilizing an adjacency method on the strain with the highest similarity, and L P0315 and L P1210 are the same as the BCH of L actinobacillus planum strain JCM _1149, L actinobacillus planus _ WA _ BCH _ C6 and L strain BCH _ BCH 930.
The strain DMS L P0315 and strain DMS L P1210 were identified as Lactobacillus plantarum (L actinobacillus plantarum) by combining the results of morphological, physiological, biochemical and 16S rDNA sequence analyses.
The strain DMS L P1210 has been deposited in Guangdong province microorganism culture collection center (GDMCC for short, address: No. 59 building 5 of Mirabilitum 100, Guangzhou city, and postal code 510070) 7/8.2019, and is classified and named as L actinobacillus plantarum with the accession number of GDMCC No. 60717.
Example 2 Effect of culture conditions on the bacteriostatic Activity of Lactobacillus plantarum DMS L P1210
(1) Influence of culture time on bacteriostatic activity of target strain
The screened target strain DMS L P1210 is inoculated in an MRS liquid culture medium in an inoculation amount of 1%, the mixture is statically cultured for 32 hours at 37 ℃, samples are taken every two hours from 16 hours, 10m L is taken to prepare cell-free supernatant, ETEC CVCC1518 is taken as indicator bacteria to measure the bacteriostatic activity, and the optimum culture time is determined.
(2) Influence of culture temperature on bacteriostatic activity of target strain
The screened target strain DMS L P1210 is inoculated in an MRS liquid culture medium in an inoculation amount of 1%, the constant temperature culture is carried out for 20 hours at the temperature of 28 ℃, 30 ℃, 32 ℃, 34 ℃ and 36 ℃, 10m L is taken to prepare cell-free supernatant, the ETEC CVCC1518 is taken as an indicator bacterium to measure the bacteriostatic activity, the optimum culture temperature is determined, the influence of the culture time on the bacteriostatic activity of the lactobacillus plantarum DMS L P1210 is shown in figure 6, the bacteriostatic activity of the strain is the highest when the culture temperature is 30 ℃, the bacteriostatic activity of the strain gradually decreases along with the increase of the culture temperature when the culture temperature is 30 ℃ as can be seen from figure 6, and the bacteriostatic activity difference of the strain is not significant (P >0.05) at different culture temperatures.
Example 3 safety evaluation of Lactobacillus plantarum DMS L P1210
1. Hemolytic test
A hemolytic test is carried out by taking streptococcus pyogenes as a control, wherein transparent hemolytic rings appear around colonies after the streptococcus pyogenes are inoculated on a Columbia blood agar plate for culture, and are type B hemolysis (β hemolysis), and after lactobacillus plantarum DMS L P0315 and lactobacillus plantarum DMS L P1210 are inoculated on the Columbia blood agar plate for culture, no hemolytic rings appear around the colonies, so that the two strains can be judged to have no hemolysis and no hemolytic hazard.
2. Sensitivity to antibiotics
According to the evaluation standard of the American society for clinical and laboratory standards C L SI, sensitivity results of two strains to 7 common antibiotics are obtained (Table 2), and from the table, it can be seen that the Lactobacillus plantarum DMS L P0315 and the Lactobacillus plantarum DMS L P1210 both have resistance to streptomycin and vancomycin and are sensitive to tetracycline and ampicillin, wherein the Lactobacillus plantarum DMS L P0315 and the Lactobacillus plantarum DMS L P1210 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 DMS L P1210
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 fermentation broth of lactobacillus plantarum DMS L P1210 is shown in FIG. 7, which shows that the bacteriostatic activity of the bacteriocin produced by DMS L P1210 is in a descending trend along with the increase of heating temperature and the extension of heating time, the bacteriostatic activity of the bacteriocin is significantly influenced by the heating temperature (P <0.05), the bacteriostatic activity of the bacteriocin is not significantly influenced by heating for 10min and 30min (P >0.05), and the bacteriostatic activity of the bacteriocin produced by lactobacillus plantarum DMS L P0315 is maintained by more than 90% after heat treatment at 121 ℃ and 30min, so that the lactobacillus plantarum DMS L P1210 has the characteristic of high temperature resistance.
2. Sensitivity of bacteriocins to pH
Adjusting the pH value of the cell-free fermentation supernatant to 2-10 by using 3 mol/L HC L and NaOH, and performing a bacteriostatic experiment after incubating for 2h at 37 ℃ to determine the influence of the pH on the bacteriostatic activity of the bacteriocin.
The influence of pH on bacteriocin in fermentation broth of Lactobacillus plantarum DMS L P1210 is shown in FIG. 8, which shows that the bacteriocin produced by the strain has bacteriostatic activity within the range of pH 2-10, shows a descending trend along with the gradual increase of pH, but keeps more than 80% of the bacteriostatic activity, shows good acid-base tolerance, shows no significant difference of the bacteriostatic activity of the bacteriocin the range of pH 2-4 (P is greater than 0.05), shows good acid stability, shows that the bacteriostatic activity of the bacteriocin within the range of pH 2-4 is obviously higher than that of the bacteriocin at pH 8-10, and reaches the highest bacteriostatic activity at pH 2, which indicates that the bacteriocin produced by the strain has the best effect in an acidic environment and the bacteriostatic activity of the bacteriocin an alkaline environment is reduced.
3. Sensitivity of bacteriocins to enzymes
Adjusting the pH value of the supernatant after cell-free fermentation to the optimum pH value of pepsin, trypsin, proteinase K and catalase by using HC L and NaOH, and performing a bacteriostasis test after 2 hours of aqueous solution at 37 ℃ to determine the influence of the enzyme on the bacteriostatic activity of the bacteriocin.
The effect of enzyme treatment on bacteriocin in the fermentation broth of lactobacillus plantarum DMS L P1210 is shown in FIG. 9, which shows that the bacteriocin produced by lactobacillus plantarum DMS L P1210 retains over 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
By integrating the biological characteristic results of bacteriocins produced by lactobacillus plantarum DMS L P0315 and lactobacillus plantarum DMS L P1210, lactobacillus plantarum DMS L P0315, lactobacillus plantarum H7-6 (the clinical isolate in the invention is identified as lactobacillus plantarum) and lactobacillus plantarum DMS L P1210 with good characteristics are selected and are subjected to compound co-culture according to the number of inoculated live bacteria 1:1, and then the bacteriostatic activity of metabolites is determined (figure 10). As can be seen from the figure, after lactobacillus plantarum DMS L P0315+ DMS L P1210 is compounded, the bacteriostatic activity gradually increases and then tends to be stable along with the increase of culture time, the bacteriostatic activity is the highest when the lactobacillus plantarum DMS is cultured for 22H, and the bacteriocin synthesis amount is the largest, so that the compound of lactobacillus plantarum DMS L P0315 and lactobacillus plantarum DMS L P1210 is determined to be the optimal compound mode.
The bacteriostatic activity of lactobacillus plantarum DMS L P0315 and lactobacillus plantarum DMS L P1210 after co-culture (inoculum size 1:1) is shown in figure 11. from the figure, compared with the control group (lactobacillus plantarum DMS L P0315 pure culture, lactobacillus plantarum DMS L P1210 pure culture, equal ratio supernatant of pure culture respectively), the bacteriostatic activity of bacteriocin after co-culture of lactobacillus plantarum DMS L P0315 and lactobacillus plantarum DMS L P1210 is obviously increased (P is less than 0.05), and the synergistic effect is achieved.
The lactobacillus plantarum DMS L P0315 and the lactobacillus plantarum DMS L P1210 are used as compound strains to obtain a cheap natural culture medium for producing bacteriocin by fermenting the compound strains, the components of the culture medium are 4% of corn pulp powder, 2% of yeast extract and 2% of glucose, the optimal fermentation conditions of the culture medium are fermentation temperature 32 ℃, fermentation pH5.5 and inoculation amount 2%, after fermentation is carried out for 22 hours by optimal process parameters, spray drying is carried out according to the conditions that the material concentration is 30%, the air inlet temperature is 160 ℃, the air outlet temperature is 80 ℃, the prepared lactein is brown yellow powder, and the bacteriocin titer reaches 2394.76 AU/g.
Example 6 determination of minimum inhibitory concentration of Lactobacillus plantarum DMS L P0315 and DMS L P1210 geometric co-culture fermentates
1. Fermentation Scale 10L fermenter
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. The fermentation conditions comprise inoculating strain DMS L P0315 and strain DMS L P1210 (effective viable count 1:1) at 2% inoculum concentration, fermenting at 32 deg.C and pH5.5 at constant pH, and fermenting 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, fermentation is carried out by maintaining constant pH value of 5.5, pH is adjusted by sodium hydroxide, the antibacterial activity of the final freeze-dried fermented product is 2394.76AU/g, the lactic acid content is 19.368mg/g, the antibacterial activity after dilution by sterile water is found to be 1836.25AU/m L when the prepared dried fermented product is diluted by 11 times, no obvious antibacterial ring appears when the dried fermented product is diluted by 21 times, freeze-drying is carried out after the pH of the final fermentation product is adjusted to 6.5, the antibacterial activity is found to be 2265.80AU/g, the dilution multiple of the dried product with the obvious antibacterial ring can be detected to be 11 times (figure 12), and the measured lactic acid content 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/m L when the dried fermented product is diluted by 51 times, no obvious antibacterial zone appears after the dilution multiple is increased, the pH of the final fermentation product is adjusted to 6.5 and then spray drying is carried out, the dilution multiple of the dried product with the obvious antibacterial zone is 11 times, and the measured lactic acid content in the dried product is 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 (L Acobacterium plantrum) DMS L P1210, characterized in that it has accession number GDMCC No. 60717.
2. A fermentation product of Lactobacillus plantarum (L Acobacillus plantarum) DMS L P1210 according to claim 1.
3. A microbial agent or animal feed containing Lactobacillus plantarum (L Acobacillus plantarum) DMS L P1210 or a fermentation product thereof according to claim 1.
4. A medicament comprising Lactobacillus plantarum (L Acobacillus plantarum) DMS L P1210 or its fermentation product according to claim 1.
5. The agent of claim 4, which is an ETEC-inhibiting agent.
6. The medicament according to claim 4 or 5, which is an animal health product.
7. Use of lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or its fermentation product according to claim 1 for the preparation of feed additives.
8. Use of lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or its fermentation product according to claim 1 for inhibiting ETEC.
9. Use of lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or its fermentation product according to claim 1 for increasing feed conversion ratio.
10. Use of lactobacillus plantarum (L actinobacillus plantarum) DMS L P1210 or its fermentation product according to claim 1 for promoting animal growth or increasing animal weight.
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