CN103013893A - Lactobacillus plantarum CCL67 and application of same - Google Patents
Lactobacillus plantarum CCL67 and application of same Download PDFInfo
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Abstract
The invention relates to lactobacillus plantarum CCL67 with the preservation number of CGMCC No.6996. The lactobacillus plantarum CCL67 provided by the invention also restrains growth of colon bacillus in a chicken intestinal canal and promotes growth of chickens. According to the invention, lactobacillus plantarum CCL67 separated from caecum of a laying hen grows exuberant as the amount of culture bacteria for 10 hours can be up to 4.7*109 cfmu /ml and has a bacteriostatic action as antimicrobial proteins can be generated by utilizing a culture medium to restrain growth of colon bacillus in the chicken intestinal canal. By using the lactobacillus plantarum CCL67 to prepare a bacterial preparation to feed chickens, the growth of chickens can be improved; and the bacterial strain is of great social significance and economical value in the aspect of animal feeding and in particular in the aspect of development of poultry novel feed additives as well as research on antibiotic succedaneums.
Description
Technical field
The invention belongs to the animal microorganism field, relate to lactobacillus plantarum CCL67 and an application thereof, particularly strain plant lactobacillus CCL67 and the application thereof of from the chicken enteron aisle, separating.
Background technology
Milk-acid bacteria is the important probiotic bacterium of a class, is the important dominant microflora of a class in the animal intestinal.Lactic acid bacteria formulation is as a kind of novel green animal probiotics, nontoxic because of it, without resistance, noresidue, having no side effect enjoys the extensive concern of feed circle.
Adopt milk-acid bacteria as the probiotic bacterium feeding animals, except because milk-acid bacteria has certain trophism and the adhesive attraction to enteron aisle, be that mainly milk-acid bacteria is inhibited for some spoilage organism and harmful bacteria.The first, milk-acid bacteria can produce lactic acid, creates sour environment and suppresses harmful bacteria and the growth of acid nonfast spoilage organism; The second, milk-acid bacteria produces H
2O
2, activate catalase-thiocyanic acid system, suppress and kill Gram-negative bacteria, catalase positive bacteria etc.; The 3rd, the part milk-acid bacteria can produce tiny protein or the peptide class of biocidal property, is called bacteriocin, and the pathogenic bacterium such as intestinal bacteria are had antagonistic action.And, the milk-acid bacteria that produces antibacterial albumen has been arranged, just can produce bacteriocin, can substitute antibiotics, it is safer that animal is produced.The milk-acid bacteria that separation screening can produce bacteriocin becomes the study hotspot that present probiotic bacterium is developed.
The originating in lactic acid bacterium that produces bacteriocin is a lot, and the industrial strain of purchase and reference culture have much also can produce bacteriocin, can the separating lactic acid bacterium in environment, soil, pickles, the sour milk.Yet probiotic bacterium is wanted feeding animals at last, and with respect to the bacterial strain in other sources, animal is undoubtedly the preferably source of milk-acid bacteria so.Milk-acid bacteria from animal intestinal can adapt to the animal intestinal environment, and the hydrochloric acid in gastric juice of tolerance animal and the digestion of cholate are undoubtedly best probiotic bacterium source.As seen, will be the important research direction that animal is screened with probiotic bacterium by the milk-acid bacteria that can produce bacteriocin from the animal intestinal Isolation and screening.
Summary of the invention
The purpose of this invention is to provide a lactobacillus plantarum (Lactobacillus plantarum) CCL67, its deposit number is CGMCC No.6996.
Second purpose of the present invention provides above-mentioned plant lactobacillus CCL67 and has the effect that suppresses Escherichia coli Growth in the chicken enteron aisle, promotes chick growth.
The present invention is achieved through the following technical solutions:
One, a lactobacillus plantarum (Lactobacillus plantarum) CCL67, its deposit number is CGMCC No.6996.
Plant lactobacillus CCL67 is Gram-positive among the present invention, and form is shaft-like, is the lactic bacilli strains that separates from the adult laying hen cecal content of raising in cages, the aimed strain that obtains through producing the characteristic measurement screenings such as antibacterial albumen.
The concrete steps of screening are as follows:
1, milk-acid bacteria separation and purification:
Adopt aseptic technique, with the slide glass scraping laying hen cecal content 1g that grows up that raises in cages, place the glass test tube that fills the 9mL stroke-physiological saline solution, fully concussion shakes up, then draw the 0.5mL mixed solution in the test tube that fills the 4.5mL stroke-physiological saline solution, this extent of dilution is 10
-1, repeat above process and make 10 doubling dilutions, to 10
-6Weaker concn selects 10
-4, 10
-5, 10
-6Three extent of dilution are drawn 0.lmL bacterium drop on the MRS culture medium flat plate, adopt methods,anaerobic (5%CO after the dull and stereotyped coating
2) culture dish that coats is put 37 ℃ of incubators cultivation 48h.Adopt four sectional streak methods, with the different bacterium colony of transfering loop picking form in the MRS nutrient agar separation and Culture of ruling, after 48h cultivates, the bacterium colony of picking good separating effect is inoculated in the MRS slant medium with transfering loop and does pure culture in four subregions, pure culture 3 times that repeat to go down to posterity, and it is for subsequent use to be placed on 4 ℃ of Refrigerator stores.
2, the observation of thalli morphology
Get clean slide, get a ring sterilized water on slide with transfering loop, then a small amount of bacterium of picking smoothens, to be dried after, fixing.Gramstaining: with ammonium oxalate crystal violet dye liquor dyeing 1min, the water flushing then drips Lushi's iodine liquid and covers 1min first, the water flushing, then drip 95% ethanol and when ethanol does not present purple, do not stop about 0.5min, redye 1min with the sarranine dye liquor at last, washing.Oil mirror microscopy (16 * 100) chooses that gramstaining is positive, form is that shaft-like or spherical bacterial strain is done the reserve bacterial strain and continued to employ.
Wherein, gramstaining reagent preparation:
1. the preparation of ammonium oxalate crystal violet staining fluid
A liquid Viola crystallina 2g95% alcohol 20mL
B liquid ammonium oxalate 0.8g distilled water 80mL
Mix A, B liquid, leave standstill 48h and use afterwards.
2. Lushi's iodine liquid
Crystalline flake of iodine 1g potassiumiodide 2g distilled water 300mL
Potassiumiodide is dissolved in a small amount of water first, again the crystalline flake of iodine is dissolved in the liquor kalii iodide, after iodine is entirely molten, fill up water and get final product.
3. 95% alcohol
4. sarranine is redyed liquid
Sarranine 2.5g95% alcohol 100mL
Getting the above-mentioned sarranine alcohol 10mL for preparing and 80mL distilled water mixing forms.
3, biocidal property lactic acid screening:
(1) preparation of bacterium liquid: the milk-acid bacteria activation 2-3 that separation and purification is gone out is after generation, is connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), and 37 ℃ of anaerobism cultivation 24h make its bacterial concentration reach 10
8Cfu/mL measures its bacteriostatic activity.Micrococcus flavus is inoculated in the broth culture, cultivated for 2 generations in 37 ℃ of shaking tables, make 10 doubling dilutions with sterilized water, make its bacterial concentration reach 10
5Cfu/mL, for subsequent use as indicator.
(2) acid producing ability is measured: the milk-acid bacteria that separation and purification is gone out activates 2-3 after generation, be connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), 37 ℃ of anaerobism are cultivated, and measure the pH value of each experimental strain 5mL fermented liquid with acidometer respectively at two time points of 0h, 48h.Each test strain fermented liquid is done 3 repetitions, takes the mean.PH value with two each experimental strain fermented liquids of time point of 0h, 48h changes, and namely △ pH value is weighed the acid producing ability of milk-acid bacteria.Choose the high bacterial strain of acid producing ability and carry out next step mensuration.
(3) bacteriostatic experiment: the milk-acid bacteria of adopting the Oxford agar diffusion method that separation and purification is gone out carries out fungistatic effect and measures.
Get 100 μ l indicator liquid and be added drop-wise to respectively and solidified in the good substratum, with the spreading rod coating evenly.Be placed on the plate with 4 Oxfords of aseptic nipper gripping cup symmetry under aseptic condition, then draw respectively 200 μ L streptococcus acidi lactici fermented solutions in 3 Oxford cups, the MRS nutrient solution that adds equivalent in another Oxford cup compares, in 37 ℃ of lower constant temperature culture 16-18h.Each streptococcus acidi lactici fermented solution is done 2 repetitions, surveys the size of its antibacterial circle diameter with vernier callipers, takes the mean, and selects the large milk-acid bacteria of antibacterial ring.
In order to filter out as early as possible required bacterial classification, preliminary screening has adopted antibacterial and 2 basic indexs of acid producing ability add and concentration.In every index, what rank the first adds 100 minutes, occupies second add 99 minutes, by that analogy, comes last add 1 minute.2 results of Primary Screening Test are summed up separately and sort, select the higher top 10 bacterial strain of total points and carry out next step research.
4, the screening of bacteriocin-producing lactic acid bacteria
The milk-acid bacteria that screens in the step 2 is carried out liquid culture, in order to reduce the fungistatic effect of acid, nutrient solution is adjusted pH value to 6.8 with 1mol/L NaOH solution, then carry out the Oxford agar diffusion method and measure antibacterial ring, method is the same, selects the maximum milk-acid bacteria of antibacterial ring.After carrying out liquid culture again, add 7 ℃ of effects of protease 3 2h of 0.1mg/L in nutrient solution, get bacterium liquid and do bacteriostatic test, screening does not have the bacterial strain of antibacterial ring, for producing the target milk-acid bacteria of antibacterial albumen.
In above-mentioned screening method, substratum is the MRS substratum:
Solid plate or slant medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, agar 20g, distilled water 1000mL, pH6.8.
Liquid nutrient medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, distilled water 1000mL, pH6.8.
Two, above-mentioned plant lactobacillus CCL67 has the effect that suppresses Escherichia coli Growth in the chicken enteron aisle, promotes chick growth.
Adopt the positively effect of technique scheme: the present invention isolates plant lactobacillus CCL67 from the laying hen caecum, this lactobacillus growth is vigorous, and breeding bacteria just can reach 4.7 * 10 in 10 hours
9Cfu/mL, and have bacteriostatic action, can utilize substratum to produce antibacterial albumen, suppress Escherichia coli Growth in the chicken enteron aisle; Prepare the bacteria preparation feeding chickling by this lactobacillus, can improve the growth of chick, so this plant lactobacillus CCL67 have great social effect and economic worth in animal rearing especially poultry novel fodder additive exploitation and Substitutes For Antibiotic research.
Description of drawings
Fig. 1 is the gram stain microscopy result of plant lactobacillus;
Fig. 2 is the fungistatic effect contrast of plant lactobacillus bacterium liquid;
Among the figure, 1,2 is the inhibition zone of purpose bacterial strain, and 3,4 is the inhibition zone of other isolated strains;
Fig. 3 is the fungistatic effect of plant lactobacillus bacterium liquid after the protease treatment;
Among the figure, 1 is untreated bacterium liquid, and 2,3 and 4 are respectively Proteinase K, the bacterium liquid that Trypsin and papoid are processed;
Fig. 4 is the pcr amplification result of plant lactobacillus;
Among the figure, 1Marker, 2 plant lactobacilluss;
Fig. 5 is the growth curve chart of plant lactobacillus.
Plant lactobacillus involved in the present invention (Lactobacillus plantarum) CCL67, carried out the patented procedure preservation on December 17th, 2012 at the preservation center that Patent Office of the People's Republic of China or international monopoly tissue are admitted, depositary institution's full name is China Committee for Culture Collection of Microorganisms common micro-organisms center, referred to as CGMCC, depositary institution address: China. Beijing. Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.6996.
Embodiment
Below in conjunction with embodiment, test example technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
The source of biomaterial among the present invention:
1, the bacterium universal primer is available from Shanghai Bo Ya Bioisystech Co., Ltd.
Present embodiment is used for the screening of explanation plant lactobacillus CCL67.
1, milk-acid bacteria separation and purification:
Adopt aseptic technique, with the slide glass scraping laying hen cecal content 1g that grows up that raises in cages, place the glass test tube that fills the 9mL stroke-physiological saline solution, fully concussion shakes up, then draw the 0.5mL mixed solution in the test tube that fills the 4.5mL stroke-physiological saline solution, this extent of dilution is 10
-1, repeat above process and make 10 doubling dilutions, to 10
-6Weaker concn selects 10
-4, 10
-5, 10
-6Three extent of dilution are drawn 0.lmL bacterium drop on the MRS culture medium flat plate, adopt methods,anaerobic (5%CO after the dull and stereotyped coating
2) culture dish that coats is put 37 ℃ of incubators cultivation 48h.Adopt four sectional streak methods, with the different bacterium colony of transfering loop picking form in the MRS nutrient agar separation and Culture of ruling, after 48h cultivates, the bacterium colony of picking good separating effect is inoculated in the MRS slant medium with transfering loop and does pure culture in four subregions, pure culture 3 times that repeat to go down to posterity, and it is for subsequent use to be placed on 4 ℃ of Refrigerator stores.
2, the observation of thalli morphology
Get clean slide, get a ring sterilized water on slide with transfering loop, then a small amount of bacterium of picking smoothens, to be dried after, fixing.Gramstaining: with ammonium oxalate crystal violet dye liquor dyeing 1min, the water flushing then drips Lushi's iodine liquid and covers 1min first, the water flushing, then drip 95% ethanol and when ethanol does not present purple, do not stop about 0.5min, redye 1min with the sarranine dye liquor at last, washing.Oil mirror microscopy (16 * 100) chooses that gramstaining is positive, form is that shaft-like or spherical bacterial strain is done the reserve bacterial strain and continued to employ.The result as shown in Figure 1.
Wherein, gramstaining reagent preparation:
1. the preparation of ammonium oxalate crystal violet staining fluid
A liquid Viola crystallina 2g95% alcohol 20mL
B liquid ammonium oxalate 0.8g distilled water 80mL
Mix A, B liquid, leave standstill 48h and use afterwards.
2. Lushi's iodine liquid
Crystalline flake of iodine 1g potassiumiodide 2g distilled water 300mL
Potassiumiodide is dissolved in a small amount of water first, again the crystalline flake of iodine is dissolved in the liquor kalii iodide, after iodine is entirely molten, fill up water and get final product.
3. 95% alcohol
4. sarranine is redyed liquid
Sarranine 2.5g95% alcohol 100mL
Getting the above-mentioned sarranine alcohol 10mL for preparing and 80mL distilled water mixing forms.
3, biocidal property lactic acid screening:
(1) preparation of bacterium liquid: the milk-acid bacteria activation 2-3 that separation and purification is gone out is after generation, is connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), and 37 ℃ of anaerobism cultivation 24h make its bacterial concentration reach 10
8Cfu/mL measures its bacteriostatic activity.Micrococcus flavus is inoculated in the broth culture, cultivated for 2 generations in 37 ℃ of shaking tables, make 10 doubling dilutions with sterilized water, make its bacterial concentration reach 10
5Cfu/mL, for subsequent use as indicator.
(2) acid producing ability is measured: the milk-acid bacteria that separation and purification is gone out activates 2-3 after generation, be connected to separately in the fresh MRS liquid nutrient medium by the inoculum size of 2% (v/v), 37 ℃ of anaerobism are cultivated, and measure the pH value of each experimental strain 5mL fermented liquid with acidometer respectively at two time points of 0h, 48h.Each test strain fermented liquid is done 3 repetitions, takes the mean.PH value with two each experimental strain fermented liquids of time point of 0h, 48h changes, and namely △ pH value is weighed the acid producing ability of milk-acid bacteria.Choose the high bacterial strain of acid producing ability and carry out next step mensuration.
(3) bacteriostatic experiment: the milk-acid bacteria of adopting the Oxford agar diffusion method that separation and purification is gone out carries out fungistatic effect and measures.
Get 100 μ l indicator liquid and be added drop-wise to respectively and solidified in the good substratum, with the spreading rod coating evenly.Be placed on the plate with 4 Oxfords of aseptic nipper gripping cup symmetry under aseptic condition, then draw respectively 200 μ L streptococcus acidi lactici fermented solutions in 3 Oxford cups, the MRS nutrient solution that adds equivalent in another Oxford cup compares, in 37 ℃ of lower constant temperature culture 16-18h.Each streptococcus acidi lactici fermented solution is done 2 repetitions, surveys the size of its antibacterial circle diameter with vernier callipers, takes the mean, and selects the large milk-acid bacteria of antibacterial ring.
In order to filter out as early as possible required bacterial classification, preliminary screening has adopted antibacterial and 2 basic indexs of acid producing ability add and concentration.In every index, what rank the first adds 100 minutes, occupies second add 99 minutes, by that analogy, comes last add 1 minute.2 results of Primary Screening Test are summed up separately and sort, select the higher top 10 bacterial strain of total points and carry out next step research.
4, the screening of bacteriocin-producing lactic acid bacteria
The milk-acid bacteria that screens in the step 2 is carried out liquid culture, in order to reduce the fungistatic effect of acid, nutrient solution is adjusted pH value to 6.8 with 1mol/L NaOH solution, then carry out the Oxford agar diffusion method and measure antibacterial ring, method is the same, selects the maximum milk-acid bacteria of antibacterial ring.The result as shown in Figure 2.After carrying out liquid culture again, add 7 ℃ of effects of protease 3 2h of 0.1mg/L in nutrient solution, get bacterium liquid and do bacteriostatic test, screening does not have the bacterial strain of antibacterial ring, for producing the target milk-acid bacteria of antibacterial albumen.The result as shown in Figure 3.
In above-mentioned screening method, substratum is the MRS substratum:
Solid plate or slant medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, agar 20g, distilled water 1000mL, pH6.8.
Liquid nutrient medium: Tryptones 10g, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, potassium primary phosphate 2g, sodium acetate 5g, tween-80 1g, sal epsom 0.5g, manganous sulfate 0.2g, distilled water 1000mL, pH6.8.
Present embodiment is used for the evaluation of explanation plant lactobacillus CCL67.
1, bacterial strain Identification
Catalase test: with 3%H
2O
2Directly be added on the lawn on inclined-plane, observe whether alveolate generation.If then positive reaction of Bubble formation is arranged.
Nitrate reduction test: microbionation to substratum, is cultivated 48h for 37 ℃, add first liquid and second liquid along tube wall, observe.Present at once redness, orange red positive reaction.
Gelatin liquification test: microbionation to substratum, is cultivated 48h for 20 ℃, observe.The inoculated tube positive reaction of liquefying.
Indole test: inoculated bacteria is cultivated 48h for 37 ℃ in the MRS nutrient solution.Add dimethylbenzene 2-3mL in nutrient solution, shake up, leave standstill moments later, add indoles reagent 2mL along tube wall, dimethylbenzene lower floor liquid becomes the positive reaction of rose person.
Hydrogen sulfide production test: microbionation behind the MRS nutrient solution, is hung in the inoculated tube with aseptic tweezers gripping one lead acetate paper slip.The lower end does not contact liquid level near media surface.The upper end is filled in tampon.Place 37 ℃ to cultivate 48h, observe.The positive reaction of paper slip blackening.
By catalase test, nitrate reduction test, gelatin liquification test, indole test and hydrogen sulfide production test, the result shows all negative, illustrates that plant lactobacillus CCL67 is lactobacillus genus.
2, the evaluation of bacterial strain kind
Glucose produces sour aerogenesis, gluconate fermentation and other kinds sugar fermentating test: the glucose, gluconate, fructose, ribose, lactose, semi-lactosi, wood sugar, sucrose, maltose, the trehalose that add respectively 0.5% or 1% ratio in the PY basic medium, inoculation is cultivated 48h for 37 ℃.Make indicator with BTB-MR.Substratum indicating agent flavescence represents to produce acid; Produce bubble in the test tube and represent aerogenesis.Identify in the biochemical tube in amygdaloside, pectinose, cellobiose, Vitamin C2, N.F,USP MANNITOL, melizitose, melibiose, raffinose, saligenin, sorbitol fermentation trace with a small amount of microbionation of inoculating needle difference picking, cultivate 48h for 37 ℃.The purple yellowing represents to produce acid, positive reaction in the biochemical tube.
15 ℃ of growth tests: whether a few ring microbionations of picking are cultivated 48h for 15 ℃ on the MRS slant medium, observe bacterium and grow on the inclined-plane.
Arginine produces ammonia test: inoculated bacteria is cultivated 48h for 37 ℃ in containing arginic substratum.Add Nessler's reagent number droplet in the nutrient solution, when producing ammonia, occur orange or the tawny precipitation, be positive reaction.
In above-mentioned screening method, biochemical medium and related reagent are formulated as follows:
Nitrate reduction test substratum: saltpetre 1g; Peptone 20g; Sodium phosphate dibasic 2g; Agar 1g; Glucose 1g; Distilled water 1000mL.With each composition dissolving, adjust pH is 7.2,121 ℃ of autoclaving 20min.
Gelatin-based basal culture medium: peptone 1g; Yeast extract 1g; Glucose 0.1g; Salts solution 4.0mL, distilled water 100mL.pH7.0。To intend the in vitro adding gelatin 0.6g/ pipe of packing, the substratum packing after more above-mentioned preparation being boiled is the 5mL pipe wherein.115 ℃ of autoclaving 20min.
Hydrogen sulfide production test substratum: tryptone 10g; Extractum carnis 3g; Yeast extract 5g; NaCl5g; Halfcystine 0.4g; Glucose 2g; Distilled water 1000mL.Adjust pH is in 7.2,115 ℃ of autoclaving 20min.
PY basic medium: peptone 5g; Tryptones 5g; Yeast extract 10g; Glucose 10g; Anhydrous CaCl
20.2g; MgSO
47H
2O0.48g; K
2HPO
41g; NaHCO
310g; NaCl2g, distilled water 1000mL.After above-mentioned substratum boiled dissolving, 115 ℃ of autoclaving 20min.
Sugar-fermenting substratum: glucose, gluconate, fructose, ribose, lactose, semi-lactosi, wood sugar, sucrose, maltose, trehalose.Ratio in 0.5% or 1% adds sugar respectively in the PY basic medium, makes indicator with BTB-MR reagent.The packing test tube, 115 ℃ of autoclaving 15min.Amygdaloside, pectinose, cellobiose, Vitamin C2, N.F,USP MANNITOL, melizitose, melibiose, raffinose, saligenin, sorbitol fermentation trace are identified biochemical tube, are rich biological purchase the in sea.
Salts solution: anhydrous CaCl
20.2g; MgSO
47H
2O0.48g; K
2HPO
41.0g; NaHCO
310g; NaCl2.0g.With CaCl
2And MgSO
47H
2The O mixed dissolution adds 500mL water again in 300mL distilled water, slowly add other salts while stir.Continue to stir until all dissolvings add 200mL distilled water, stock after the mixing in 4 ℃.
Catalase reagent: 3% hydrogen peroxide.
Nitrate reduction test reagent
First liquid: the 0.8g Sulphanilic Acid is dissolved in 100mL5mol/L acetic acid.
Second liquid: the 0.5g alpha-naphthylamine is blown slowly heating for dissolving behind 100mL5mol/L acetic acid, filter with absorbent cotton.
Indoles reagent: Paradimethylaminobenzaldehyde 1g, dehydrated alcohol 95mL, concentrated hydrochloric acid 20mL.
BTB-MR reagent: dibromothymolsulfonphthalein (BTB) 0.2g, methyl red (MR) 0.1g, 95% ethanol 300mL, distilled water 200mL.
Arginine liquid: L-arginine 1.5g, halfcystine (1g/mL H
2O) 0.05mL, distilled water 10mL transfers pH to 7.0, adds 3 after the sterilization and drops in the PY basic culture solution.Nessler's reagent: 20g KI is dissolved in 50mL distilled water, and in this solution, adds HgI
2Small-particle, to solution reach saturated till (about 32g), and then add 460mL water and 134g KOH.Be stored in the shaded bottle supernatant liquor for subsequent use.
Experimental result such as the table 1 of sugar-fermenting:
Table 1 sugar fermentating test result
Produce sour aerogenesis and other kinds sugar fermentating test and arginine by glucose and produce ammonia test, result and " common bacteria system identification handbook and " classification of lactic-acid-bacterium is identified " and experimental technique contrast, identify that the result shows that plant lactobacillus CCL67 is plant lactobacillus.
3,16S rDNA order-checking is identified
Employing well known to a person skilled in the art method, extracts bacterial genomes DNA with test kit, and reverse transcription is cDNA, and the conservative region according to the 16S rDNA gene order of lactobacillus adopts the bacterium universal primer, carries out the conventional pcr amplification of gene, wherein,
Upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3'
Downstream primer 5'-ACGGCTACCTTGTTACGACTT3'
The amplification electrophoresis detection, the electrophoresis showed band is clear single, illustrates and increases successfully, result such as Fig. 4, band is purified simultaneously, the TA-clone technology, after the order-checking, carry out sequence alignment at the BLAST of Gene bank, be defined as plant lactobacillus (Lactobacillus plantarum).
Test example 1
This test example is used for the effect of explanation plant lactobacillus CCL67.
1, the full bacterium liquid formulation preparation of milk-acid bacteria
(1) lactobacillus-fermented timing: the Bacterium lacticum after will activating is as fermentation seed liquid 1%(v/v) be inoculated in the MRS liquid nutrient medium, 37 ℃, the 170r/min shaking table is cultivated, at front 24h every 2h, behind the 24h the 36th and 48h sampling, after carrying out gradient dilution by 10 times of methods with 0.9% physiological saline, get 10
-4-10
-7Extent of dilution 100 μ l diluents evenly are coated with at the MRS nutrient agar, and each gradient is done 3 repetitions, and plate is at 37 ℃, 5%CO
2Cultivate 24h under the condition.Get colony number and calculate bacterial concentration in the extent of dilution value of 30-300, simultaneously sampling adopts the Oxford agar diffusion method to measure the fungistatic effect of each time point bacterium liquid, determines the suitableeest fermentation time.
As shown in Figure 5, X-coordinate represents the time among the figure, and ordinate zou represents viable count, plant lactobacillus viable count rising in the 2h after inoculation, and through behind the 2h, viable count is from 10
7Rise rapidly, reach maximum to 10h viable bacteria concentration, but from 36h, viable count begins slow decreasing to 10
8Below the order of magnitude, major cause is that substratum nutritive substance concentration and pH value sharply descend, and the reasons such as harmful meta-bolites increase cause the dead increase of thalline.Can find out that from growth curve the best harvesting time of plant lactobacillus is to be 10～12h after cultivation.At this section period results thalline, microbial activity is relatively stable, and can reduce the cost that obtains the unit viable bacteria.
(2) inoculum size full bacterium solution preparation: test strain is activated 2-3 after generation, by 1%(v/v) is connected in the liquid nutrient medium of optimization 37 ℃, the 170r/min shaking table is cultivated 10h, 4 ℃, the centrifugal 30min of 3500rpm, with the supernatant liquor suspension thalline of certain volume, 4 ℃ save backup.
2, lactic acid bacteria formulation is at the effect of breeding chickling
(1) test materials: above-mentioned full bacterium liquid formulation, viable count is 4.7 * 10
9Cfu/mL(bacterium number); Microbiotic is 4% bambermycin.
(2) experimental animal and test design: the AA meat cock of selecting 240 1 age in days health, mean body weight 36.74 ± 0.45g, be divided at random 4 groups, I group (full bacterium liquid formulation group, the full bacterium liquid formulation of basal diet+0.05%), II group (full bacterium liquid formulation group, the full bacterium liquid formulation of basal diet+0.1%), III (microbiotic group, basal diet+0.01% bambermycin), IV group (control group, basal diet), every group of 5 repetitions, each repeats 12 chickens, 28 days trial periods.
(3) testing index:
A, production performance index: each repetition of weighing on an empty stomach heavy (fasting 12h) during respectively at test 8:00 the 1st day, 15 days, 29 days morning, and accurate recording respectively repeats food consumption.Calculate the average daily gain (ADG) of respectively organizing chicken, average daily ingestion amount (ADFI) and feed conversion ratio (FCR).Result such as table 2:
Table 2 lactic acid bacteria formulation is on the impact of chick day weight gain and material anharmonic ratio
Annotate: the colleague relatively, different lowercase alphabet differentials different significantly (p＜0.05), different capitalizations represent difference extremely significantly (p＜0.01), unmarked or alphabetical identical person represents difference not significantly (p〉0.05).
Test-results shows, at whole brood time 1～4w, the day weight gain of 0.1% full bacterium liquid group is apparently higher than control group, and difference extremely remarkable (p＜0.01).The day weight gain of 3-4w microbiotic group significantly (p＜0.05) is higher than control group, and 1～4 all utmost points are significantly higher than control group.In each vegetative period, lactic acid bacteria formulation group and microbiotic group day weight gain are without significant difference (p〉0.05).
At whole brood time 1-4w, to compare with control group, full bacterium liquid group can significantly reduce feedstuff-meat ratio (p＜0.05), and wherein 0.1% full bacterium liquid group can extremely significantly reduce feedstuff-meat ratio (p＜0.01).The microbiotic group is compared with control group, can be extremely during 1-4w significantly (p＜0.01) reduce feedstuff-meat ratio.Lactic acid bacteria formulation group and microbiotic group at the feedstuff-meat ratio in each vegetative period all without significant difference (p〉0.05).
B, caecum Microflora: in test the 15th, 29 day, respectively repeating to get one near the examination chicken of mean body weight at random, the jugular vein sacrificed by exsanguination, dissect immediately, with taking out behind the sterile cotton toe-in bundle caecum, take by weighing cecal content 0.5g under the aseptic condition, 10 times of dilutions, spread plate is measured milk-acid bacteria and intestinal bacteria quantity in the caecum.Result such as table 3:
Table 3 lactic acid bacteria formulation is on the impact (log of milk-acid bacteria in the chick caecum and intestinal bacteria quantity
10Cfu/g)
Annotate: the colleague relatively, different lowercase alphabet differentials different significantly (p＜0.05), different capitalizations represent difference extremely significantly (p＜0.01), unmarked or alphabetical identical person represents difference not significantly (p〉0.05).
Compare with the microbiotic group, in daily ration of broiler, add significantly lactic acid bacterium number in (p＜0.05) raising caecum of 0.05% and 0.1% full bacterium liquid formulation; Compare with control group, without significant difference (p〉0.05).During 4w, the lactic acid bacterium number in the 0.1% full bacterium liquid group caecum extremely significantly (p＜0.01) is higher than the microbiotic group, and 0.1% full bacterium liquid group significantly (p＜0.05) is higher than control group.Compare with control group, during 2w, 0.05% full bacterium liquid formulation group significantly (p＜0.05) reduces intestinal bacteria quantity in the caecum, and 0.1% full bacterium liquid group then can reach extremely significantly (p＜0.01) reduction effect; During 4w, 0.05% full bacterium liquid formulation group significantly (p＜0.05) reduces intestinal bacteria quantity in the caecum, and wherein 0.1% full bacterium liquid group reduces effect extremely significantly (p＜0.01).Compare with the microbiotic group, during 2w, each experimental group on intestinal bacteria quantity impact in the caecum without significant difference (p〉0.05); During 4w, 0.1% full bacterium liquid group can (p＜0.01) extremely significantly reduce colibacillary quantity in the caecum.
To sum up, this bacterial strain can significantly suppress the intestinal bacteria quantity in the chicken enteron aisle, promotes simultaneously chick growth, and alternative traditional microbiotic reduces the application of microbiotic in animal rearing, has greatly improved biological safety.
Claims (2)
1. a lactobacillus plantarum (Lactobacillus plantarum) CCL67, its deposit number is CGMCC No.6996.
2. plant lactobacillus CCL67 claimed in claim 1 has the effect that suppresses Escherichia coli Growth in the chicken enteron aisle, promotes chick growth.
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| CN109628354A (en) * | 2019-01-30 | 2019-04-16 | 江南大学 | The lactobacillus plantarum of one plant height bacteriostatic activity and application |
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