CN104431646A - High water content alfalfa silage bacterial agent active component preparation method - Google Patents
High water content alfalfa silage bacterial agent active component preparation method Download PDFInfo
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Abstract
The present invention discloses a preparation method for an alfalfa silage bacterial agent Lactobacillus plantarum. The preparation method is characterized by comprising: (1) strain separation, (2) strain purification, (3) a yeast inhibition test, (4) a drug sensitive test of the strain, and (5) strain identification. According to the present invention, the Lactobacillus plantarum of the present invention can produce bacteriocin having inhibition effects on a variety of pathogenic bacteria and putrefying bacteria, wherein the bacteriocin has good acid stability and good thermal stability; when the bacterial agent made from the active component prepared in the present invention is adopted to perform silage on the high water content alfalfa, the water content of the high water content alfalfa can up to 81.7%, and the silage time achieves 240 days; and the bacterial inhibition effect of the silage agent is improved while the hidden danger that the drug resistance enters the food chain through the silage alfalfa so as to spread is reduced.
Description
Technical field
The present invention relates to a kind of preparation method of microbe additive, particularly relate to a kind of preparation method of alfalfa ensilage microbial inoculum Lactobacillus plantarum.
Background technology
Lactobacillus plantarum, Lactobacillus plantarum is the one of lactic acid bacteria, and optimum growth temperature is 30 ~ 35, anaerobism or amphimicrobian, bacterial classification be straight or curved shaft-like, single, sometimes in pairs or become chain, optimal pH about 6. 5, belongs to homofermentative lactic bacteria.This bacterium and the difference of other lactic acid bacteria are that the viable count of this bacterium is higher, and product acid that can be a large amount of, makes the pH value in water stablize and do not raise, and the acid mass-energy degraded heavy metal of its output.Because this bacterium is anaerobic bacteria (facultative aerobic), the distinctive lactobacillin of energy output in reproductive process, lactobacillin is the anticorrisive agent of a kind of bion.In the cultivation middle and later periods, due to ight soil and the increase of residual bait of animal, can sink to the bottom in pond, and rot, grow a lot of germ, generate a large amount of ammonia nitrogens and nitrite, dead phenomenon is serious steathily to make bottom.If Long-Time Service lactobacillus plantarum, just can well suppress bottom ight soil and residual bait rot, also just reduce the increase of ammonia nitrogen and nitrite, substantially reduce the number the consumption of chemical industry degradation element, aquaculture cost is reduced.
Along with the development of aquaculture and the soaring of provision price, develop best in quality, that the inexpensive forage feed of safety has become the transformation aquaculture style of economic increase domestic demand.Clover is the forage grass of plant-eating animal high-quality, its major product is hay, but this production is drenched with rain often, fall leaves, expensive drying plant and consume the puzzlement of the problems such as mass energy, alfalfa ensilage not only from above puzzlement, also can reduce nutrient loss to keep the nutritive peculiarity of green forage.
Therefore, a lot of technology discloses the method for alfalfa ensilage, but because the nutritional character of clover self and colony characteristics cause the effect of alfalfa ensilage in existing disclosed technology or method undesirable, subject matter is: one, during a part of technical requirement ensiling, need the water content of below clover wilting or airing to 65%, the requirement of this water content cannot carry out ensiling by causing the clover in rainy season facing results; Two, clover self is with a large amount of saccharomycete and a small amount of lactic acid bacteria, clover needs to use fast, the antibacterial strong lactic bacteria additive of breeding in ensilage, but the alfalfa ensilage product adding agent of lactic acid bacteria will enter food chain when feeding animals, reduce the risk of antibiotic resistance, need evaluate the antibiotic resistance of agent of lactic acid bacteria, namely agent of lactic acid bacteria is to antibiotic sensitivity tests.
In view of feature when above-mentioned alfalfa ensilage lactic bacteria additive Problems existing and alfalfa ensilage, prior art can not complete simultaneously carries out ensiling to high moisture content clover, the problem of the hidden danger that the fungistatic effect of the agent of raising ensiling simultaneously and reduction drug resistance enter food chain by Alfalfa Silage and propagate.
Summary of the invention
For solving deficiency of the prior art, the object of the invention is the preparation method providing a kind of high-moisture alfalfa ensilage microbial inoculum active component.The invention provides a kind of preparation method of alfalfa ensilage microbial inoculum Lactobacillus plantarum, it is characterized in that, the preparation method of described Lactobacillus plantarum is as follows:
(1) separation of bacterial strain: the leachate collecting mud, the ratio of 2:10 joins in sterilized water and mixes by volume, serial dilution, is coated in MRS solid medium respectively, 37 DEG C of Anaerobic culturel 45h by each dilution bacterium liquid 100 microlitre; Single bacterium colony that picking transparent circle is larger, on MRS solid medium, line obtains pure single bacterium colony for 4 times continuously; Picking list bacterium colony is in the MRS fluid nutrient medium not adding calcium carbonate, and observe whether aerogenesis, aerogenesis bacterial strain is heterofermentative lactic bacteria, and anaerogen strain is homofermentative lactic bacteria;
(2) purifying of bacterial strain: in access liquid LB culture medium, under the condition of 14-26 DEG C, shaken cultivation is to exponential phase of growth, the bacterium liquid being in exponential phase of growth is transferred in liquid fermentation medium, 14-26 DEG C of shaken cultivation is to initial stage stationary phase, zymotic fluid is centrifugal, get supernatant, Filter paper filtering, obtain thick bacterium liquid; Be that the film bag of 12KDa is by extremely original for the volume concentration of thick bacterium liquid 1/10 with molecular cut off; Bacterium liquid after concentrated adds ammonium sulfate to 65-70% saturation degree, centrifuging and taking supernatant, continues to add ammonium sulfate to 85-100%, and centrifuging and taking precipitates, in the KPB buffer solution dialysis of pH=7, place 24h; By centrifugal for the bacterium liquid after dialysis, get supernatant, be splined on the CM-Sepharose prepacked column that the KPB buffer solution of pH=7 balances, wash-out, collects activated part, is Lactobacillus plantarum, and-20 DEG C frozen;
(3) press down Yeast assay: the lactic acid bacteria preserved under taking out-20 DEG C of conditions, get on 10 microlitres injection MRS solid mediums and be coated with, under the condition of temperature 37 DEG C, cultivate 15h; With the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 20ml test tube of 10mlMRS culture medium, under the condition of temperature 37 DEG C, cultivate 17h, obtain the lactic acid bacteria activated; By the inoculum concentration of 1%, by the access of the lactic acid bacteria of activation not containing in the 250ml triangular flask of calcium carbonate MRS fluid nutrient medium, liquid amount is 100ml, cultivates 17h for 37 DEG C, now, at least containing 0.993 × 10 in every milliliter of MRS fluid nutrient medium
10cfu lactic acid bacteria; 5000 leave heart 5min, collect supernatant, obtain bateriostatics one; Saccharomycete 28 DEG C of static gas wave refrigerator 48h in YPD fluid nutrient medium are obtained saccharomycete nutrient solution; Double-deck agar plate punch method measures the bacteriostasis of above-mentioned bateriostatics one;
(4) drug sensitive test of bacterial strain: the antibiotic filter paper preparing variable concentrations, cultivates lactic acid bacteria, adopts disc diffusion method to measure the drug sensitivity of lactic acid bacteria, obtains 8 strains to antibiotic sensitive and the bacterial strain good to saccharomycete inhibition;
(5) qualification of bacterial strain: above-mentioned steps is screened the bacterial strain obtained and carry out biological characteristics and the qualification of 16S-23S rDNA spacer sequence.
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, it is characterized in that, described step (1) or (3) middle MRS fluid nutrient medium are: peptone 12g, beef extract 8g, yeast extract 6g, L-light Cystine hydrochloride 0.08 g, hydrogen citrate three ammonium 3g, glucose 25g, Tween 80 mL, sodium acetate 6g, vitamin K
23g, dipotassium hydrogen phosphate 3g, magnesium sulfate 0.8g, calcium carbonate 12 g, manganese sulfate 0.3g, agar 20g, distilled water 1000mL, pH value 6-7;
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, it is characterized in that, the component that described step (1) or (3) middle MRS solid medium often rise and content are: add mass percent 1.5% agar powder in MRS fluid nutrient medium, pH value is 6.5,121 DEG C of sterilizing 20min.
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, it is characterized in that, consisting of of fermentation medium in described step (2): dusty yeast 0.3g/L, glucose 1.2g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 1.5g/L, potassium chloride 0.8g/L, ferrous sulfate 0.03g/L, magnesium sulfate 0.8g/L, pH=8-9.
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, is characterized in that, in described step (3), double-deck agar plate punch method measures and refers to, the MRS solid medium of 30ml is injected the culture dish of diameter 25cm, cooling 1h; Get 10mL saccharomycete nutrient solution to be added to 150ml and to be cooled in the YPD semisolid culturemedium of 45 DEG C, shake up, and be poured on rapidly on bottom MRS culture medium; On bottom MRS culture medium, equidistance place, interval places 3 by the lml pipettor Tip head cut short; This is 1cm by the height cutting short Tip head, and diameter is 0.8cm; The YPD semisolid culturemedium that cooling is injected, the time is 1h, now, makes Double-Medium; Taking out Tip head, making the hole for placing bateriostatics; Get the above-mentioned lactic acid bacteria bateriostatics one of same concentrations, volume number is 150 microlitres, adds wherein in holes; Remain one in contrast, add the sterilizing Russia water of 150 microlitres; Cultivate 48h for 37 DEG C, measure and record lactic acid bacteria to saccharomycetic antibacterial circle diameter;
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, it is characterized in that, the antibiotic filter paper preparing variable concentrations in described step (4) refers to, by streptomysin, Ciprofloxacin and gentamicin are all configured to 85 μ g/ml, three concentration gradients of 350 μ g/ml and 1200 μ g/ml, Amoxicillin, erythromycin and Cefradine are all configured to 0.650 μ g/ml, three concentration gradients of 20 μ g/ml and 80 μ g/ml, the circular filter paper sheet that diameter is 0.8cm is prepared with filter paper, they are put into above-mentioned antibiotic solution, obtain the antibiotic filter paper of variable concentrations.
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, it is characterized in that, cultivate lactic acid bacteria in described step (4) to refer to, the lactic acid bacteria preserved under taking out-20 DEG C of conditions, getting 5 microlitres injects on MRS solid medium, coating, 15h is cultivated under the condition of temperature 37 DEG C, with the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 10ml test tube of 5mlMRS culture medium, the lactic acid bacteria that 17h obtains activating is cultivated under the condition of temperature 37 DEG C, by the lactic acid bacteria of activation by 1% inoculum concentration access be equipped with in the 250ml triangular flask of MRS fluid nutrient medium, liquid amount is 100ml, cultivate 17h for 37 DEG C, obtain lactobacillus suspension, now, at least containing 0.993 × 10 in every milliliter of MRS fluid nutrient medium
10cfu lactic acid bacteria, the bacterial concentration diluting every milliliter of lactic acid bacteria with sterilized water is 1.5 × 10
8cfu.
The preparation method of above-mentioned high-moisture alfalfa ensilage microbial inoculum active component, it is characterized in that, in described step (5), the Sequence Identification of 16S-23S rDNA spacer region refers to, the nucleotide sequence of the 16S-23S rDNA spacer region of bacterial strain WN5 and Lactobacillus plantarum WCFS1(AL935263) homology the highest, its homology is 98%.
The preparation method of above-mentioned high-moisture alfalfa ensilage microbial inoculum active component, is characterized in that the Identification of Biological Characteristics of bacterial strain in described step (5) refers to, the stability of Lactobacillus plantarum to heat and the stability to acid.
The preparation method of above-mentioned a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum, is characterized in that, the component that described YPD fluid nutrient medium often rises and content are: dusty yeast 10 grams, peptone 20 grams, glucose 20 grams; The component that described YPD semisolid culturemedium often rises and content are: dusty yeast 10 grams, peptone 20 grams, glucose 20 grams, the agar powder of 0.7%.
Beneficial effect of the present invention is:
Lactobacillus plantarum of the present invention can produce multiple pathogen and the inhibited bacteriocin of spoilage organisms, this bacteriocin has good absolute acid stability and heat endurance, under 121 DEG C of high temperature, process does not affect its bacteriostatic activity in 30 minutes, the advantages such as antimicrobial spectrum is wide.When the microbial inoculum manufactured by the active component of above-mentioned manufacture is to high-moisture alfalfa ensilage, high-moisture clover moisture is up to 81.7%, the ensiling time reaches 240 days, the cauline leaf of the clover after ensiling is complete to be stored on plant, water content with differ before ensiling seldom, protein content also increases; The fungistatic effect simultaneously improving ensiling agent and the hidden danger reducing drug resistance to enter food chain by Alfalfa Silage and propagate.The antiseptics for natural food and feed addictive with broad prospect of application can be applied as.
Detailed description of the invention
In following embodiment, if no special instructions, conventional method is; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels; Described percentage composition or concentration, be mass percent if no special instructions.
Culture medium used in following embodiment is as follows: MRS fluid nutrient medium is: peptone 12g, beef extract 8g, yeast extract 6g, L-light Cystine hydrochloride 0.08 g, hydrogen citrate three ammonium 3g, glucose 25g, Tween 80 mL, sodium acetate 6g, vitamin K
23g, dipotassium hydrogen phosphate 3g, magnesium sulfate 0.8g, calcium carbonate 12 g, manganese sulfate 0.3g, agar 20g, distilled water 1000mL, pH value 6-7; The component that MRS solid medium often rises and content are: add mass percent 1.5% agar powder in MRS fluid nutrient medium, and pH value is 6.5,121 DEG C of sterilizing 20min; Consisting of of fermentation medium: dusty yeast 0.3g/L, glucose 1.2g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 1.5g/L, potassium chloride 0.8g/L, ferrous sulfate 0.03g/L, magnesium sulfate 0.8g/L, pH=8-9; The component that YPD fluid nutrient medium often rises and content are: dusty yeast 10 grams, peptone 20 grams, glucose 20 grams; The component that described YPD semisolid culturemedium often rises and content are: dusty yeast 10 grams, peptone 20 grams, glucose 20 grams, the agar powder of 0.7%.
embodiment 1
1, the separation of bacterial strain:
The ratio of collecting the leachate 2:10 by volume of mud near Qingdao triumph bridge joins mixing in sterilized water and carries out serial dilution, is coated in MRS solid medium respectively, 37 DEG C of Anaerobic culturel 45h by each dilution bacterium liquid 100 microlitre; Single bacterium colony that picking transparent circle is larger, on MRS solid medium, line obtains pure single bacterium colony for 4 times continuously; Picking list bacterium colony is in the MRS fluid nutrient medium not adding calcium carbonate, and observe whether aerogenesis, aerogenesis bacterial strain is heterofermentative lactic bacteria, and anaerogen strain is homofermentative lactic bacteria;
2, the purifying of bacterial strain: in access liquid LB culture medium, under the condition of 14-26 DEG C, shaken cultivation is to exponential phase of growth, the bacterium liquid being in exponential phase of growth is transferred in liquid fermentation medium, 20 DEG C of shaken cultivation are to initial stage stationary phase, zymotic fluid is centrifugal, get supernatant, Filter paper filtering, obtain thick bacterium liquid; Be that the film bag of 12KDa is by extremely original for the volume concentration of thick bacterium liquid 1/10 with molecular cut off; Bacterium liquid after concentrated adds ammonium sulfate to 65% saturation degree, centrifuging and taking supernatant, continues to add ammonium sulfate to 85%, and centrifuging and taking precipitates, in the KPB buffer solution dialysis of pH=7, place 24h; By centrifugal for the bacterium liquid after dialysis, get supernatant, be splined on the CM-Sepharose prepacked column that the KPB buffer solution of pH=7 balances, wash-out, collects activated part, is Lactobacillus plantarum, and-20 DEG C frozen;
3, Yeast assay is pressed down:
The activation of lactic acid bacteria: the lactic acid bacteria preserved under taking out-20 DEG C of conditions, gets on 10 microlitres injection MRS solid mediums and is coated with, under the condition of temperature 37 DEG C, cultivate 15h; With the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 20ml test tube of 10mlMRS culture medium, under the condition of temperature 37 DEG C, cultivate 17h, obtain the lactic acid bacteria activated; By the inoculum concentration of 1%, by the access of the lactic acid bacteria of activation not containing in the 250ml triangular flask of calcium carbonate MRS fluid nutrient medium, liquid amount is 100ml, cultivates 17h for 37 DEG C, now, at least containing 0.993 × 10 in every milliliter of MRS fluid nutrient medium
10cfu lactic acid bacteria; 5000 leave heart 5min, collect supernatant, obtain bateriostatics one; Saccharomycete 28 DEG C of static gas wave refrigerator 48h in YPD fluid nutrient medium are obtained saccharomycete nutrient solution;
Double-deck agar plate punch method measures the bacteriostasis of above-mentioned bateriostatics one:
The MRS solid medium of 30ml is injected the culture dish of diameter 25cm, cooling 1h; Get 10mL saccharomycete nutrient solution to be added to 150ml and to be cooled in the YPD semisolid culturemedium of 45 DEG C, shake up, and be poured on rapidly on bottom MRS culture medium; On bottom MRS culture medium, equidistance place, interval places 3 by the lml pipettor Tip head cut short; This is 1cm by the height cutting short Tip head, and diameter is 0.8cm; The YPD semisolid culturemedium that cooling is injected, the time is 1h, now, makes Double-Medium; Taking out Tip head, making the hole for placing bateriostatics; Get the above-mentioned lactic acid bacteria bateriostatics one of same concentrations, volume number is 150 microlitres, adds wherein in holes; Remain one in contrast, add the sterilizing Russia water of 150 microlitres; Cultivate 48h for 37 DEG C, measure and record lactic acid bacteria to saccharomycetic antibacterial circle diameter;
4, the drug sensitive test of bacterial strain:
The antibiotic filter paper preparing variable concentrations refers to, streptomysin, Ciprofloxacin and gentamicin are all configured to three concentration gradients of 85 μ g/ml, 350 μ g/ml and 1200 μ g/ml, Amoxicillin, erythromycin and Cefradine are all configured to three concentration gradients of 0.650 μ g/ml, 20 μ g/ml and 80 μ g/ml, the circular filter paper sheet that diameter is 0.8cm is prepared with filter paper, they are put into above-mentioned antibiotic solution, obtains the antibiotic filter paper of variable concentrations;
The cultivation of lactic acid bacteria: the lactic acid bacteria preserved under taking out-20 DEG C of conditions, getting 5 microlitres injects on MRS solid medium, coating, 15h is cultivated under the condition of temperature 37 DEG C, with the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 10ml test tube of 5mlMRS culture medium, the lactic acid bacteria that 17h obtains activating is cultivated under the condition of temperature 37 DEG C, by the lactic acid bacteria of activation by 1% inoculum concentration access be equipped with in the 250ml triangular flask of MRS fluid nutrient medium, liquid amount is 100ml, cultivate 17h for 37 DEG C, obtain lactobacillus suspension, now, at least containing 0.993 × 10 in every milliliter of MRS fluid nutrient medium
10cfu lactic acid bacteria, the bacterial concentration diluting every milliliter of lactic acid bacteria with sterilized water is 1.5 × 10
8cfu.
5, disc diffusion method measures the drug sensitivity of above-mentioned lactic acid bacteria: the culture dish MRS solid medium not adding calcium carbonate of 20ml being injected diameter 25cm, cooling 1h; The every ml concentration drawing 100 microlitres is 1.5 × 10
8the lactobacillus suspension of cfu is central in culture medium, coated plate; Get 4 filter papers, these 4 filter papers are respectively 3 antibiotic of the same race but the different antibiotic filter paper of concentration, and the 4th filter paper is not containing antibiotic contrast; Absorb after lactic acid bacteria until agar surface, paste evenly moderate above-mentioned 4 filter papers in interval in the agar surface of each culture dish; Leave standstill after 5 minutes, upset plate, 37 DEG C of static gas wave refrigerator 24h, measure and record antibiotic to the antibacterial circle diameter of lactic acid bacteria.
Result obtains 8 strains to antibiotic sensitive and the bacterial strain good to saccharomycete inhibition.
6, the qualification of bacterial strain
(1) biological characteristics of bacterial strain:
A, Lactobacillus plantarum are to the stability of heat
The Lactobacillus plantarum list colony inoculation of picking activation, in MRS fluid nutrient medium, seals, 30 DEG C of quiescent culture 24h, zymotic fluid is with 12000rpm, and 4 DEG C of centrifugal 15min, collect supernatant, the supernatant sterilised membrane filter in 0.22 μm of aperture filters, removing thalline and other impurity.Obtain the cell free fermentation supernatant of Lactobacillus plantarum.
The pH6.0 cell free fermentation supernatant of Lactobacillus plantarum is placed on respectively 60 DEG C, 80 DEG C, 100 DEG C and 121 DEG C of process 15min and 30min, according to the Oxford cup double-layer agar technique described in step one, under 30 DEG C of conditions, do bacteriostatic test, after determining treatment of different temperature, bacteriocin is suppressed to the impact of micrococcus luteus activity.
Result is as shown in table 5, and result shows that Lactobacillus plantarum is to thermally-stabilised, processes 30 minutes, still keep very high bacteriostatic activity under 121 DEG C of high temperature, in the food that can be applied to high-temperature sterilization or feed.
Table 5. treatment of different temperature suppresses the impact of micrococcus luteus (Micrococcus luteus) 28001 activity to bacteriocin
Note: antibacterial circle diameter comprises Oxford cup external diameter (7.8mm)
B, Lactobacillus plantarum are to the stability of acid
Its pH value is adjusted to be 2.0 ~ 10.0 with 1mol/L HCL and 1mol/L NaOH respectively Lactobacillus plantarum (Lactobacillus plantarum) No. 3 cell free fermentation supernatants (obtaining according to the method for step 1), incubation 2h at 37 DEG C, adjust pH value to be 6.0, then do bacteriostatic test according to the Oxford cup double-layer agar technique described in step one and survey bacteriostatic activity.Under equal conditions, respectively with 1mol/L HCL and 1mol/L NaOH adjust sterilized liquid MRS culture medium pH value be 2.0 ~ 10.0,37 DEG C at incubation 2h, adjustment pH value is 6.0, contrasts equally according to the Oxford cup double-layer agar technique described in step one.
Table 6. Lactobacillus plantarum (Lactobacillus plantarum) No. 3 bacteriocins are to the stability of acid
Note: antibacterial circle diameter comprises Oxford cup external diameter (7.8mm), and "-" represents antibacterial circle diameter lower than 8.0mm
(2) sequence of 16S-23S rDNA spacer region: the nucleotide sequence of the 16S-23S rDNA spacer region of bacterial strain WN5 and Lactobacillus plantarum WCFS1(AL935263) homology the highest, its homology is 98%.
Based on above feature, the bacterial strain WN5 above-mentioned screening obtained is accredited as Lactobacillus plantarum, in No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 04 16th, 2013, the preserving number of Lactobacillus plantarum has been CGMCC NO. 7469.
embodiment 2
Lactobacillus plantarum WN5 CGMCC NO. 7469 is utilized to make the microbial bacterial agent of high-moisture alfalfa ensilage and the application in high-moisture alfalfa ensilage.
The activation of lactic acid bacteria: the above-mentioned example 1 preserved under taking out-20 DEG C of conditions screens the Lactobacillus plantarum WN5 CGMCC NO. 7469 obtained and gets on 5 microlitres injection MRS solid mediums, and coating, cultivates 15h under the condition of temperature 37 DEG C; With the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 10ml test tube of 5mlMRS culture medium, under the condition of temperature 37 DEG C, cultivate the lactic acid bacteria that 17h obtains activating, now, at least containing 0.993 × 10 in every mlMRS fluid nutrient medium
10cfu lactic acid bacteria.
The microbe additive of high-moisture alfalfa ensilage makes: the bacterium liquid of the Lactobacillus plantarum WN5 CGMCC NO. 7469 of above-mentioned cultivation is carried out being mixed to get the seed liquor of carrying out co-incubation in cfu than the ratio for 1:1, by the inoculum concentration of 10%, mixed above-mentioned seed liquor is inoculated in MRS fluid nutrient medium not calciferous, 37 DEG C of static gas wave refrigerator 15h, make cell density in every ml fluid nutrient medium reach 0.993 × 10
10cfu ~ 0.832 × 10
11cfu, during ensiling, per kilogram adds 30ml ~ 35ml microbe additive.
The application of microbe additive in high-moisture alfalfa ensilage: in high-moisture clover (water content is 79.7%-80.6%) in per kilogram clover in add 30ml ~ 35ml microbe additive ratio add mentioned microorganism additive; Before adding microbial bacterial agent, it is long that high-moisture clover is cut into 5cm ~ 6cm; After adding microbial bacterial agent, turn microbial inoculum and clover mixes to make them; Then, the clover of adding microbial inoculum is encased in ensiling device; Room temperature is placed; Measure Alfalfa Silage material interval moisture (%), pH value, crude protein (%), water soluble carbohydrates (%), ammoniacal nitrogen (%), free amino acid (%) and reducing sugar (%) contained in 2 days, 5 days, 9 days, 15 days, 30 days and 90 days respectively.3 repetitions are established in test, and the mean value repeated for 3 times is end value.
The silage effect of high-moisture alfalfa ensilage material: open ensiling device in different time sections, evenly accurately takes 20 g samples; Add 180 mL distilled water, stir, add preservative film; Lixiviate 24 h at 4 DEG C, filtering gained liquid for twice through four layers of gauze and qualitative filter paper is leaching liquor.Leaching liquor is used for measuring alfalfa ensilage material pH value and ammoniacal nitrogen (NH
3-N).PH value uses thunder magnetic PHS-25 type pH instrument to measure, and ammonia nitrogen content adopts phenol-clorox colorimetric method for determining.Water content is the weight weightless after dry 48 h in 65 DEG C of baking ovens of sample, water soluble carbohydrates adopts anthrone-Sulphuric acid Colorimetry, crude protein adopts Kjeldahl nitrogen determination (FOSS company full-automatic Kjeldahl determination device), reducing sugar adopts 3,5-dinitrosalicylic acid (3,5-Dinitrosalicylic acid, DNS) colorimetric method for determining, free amino acid adopts ninhydrin colorimetry to measure.Measurement result is as shown in table 2.
In table 2 example 4, microbial bacterial agent is to the silage effect of high-moisture clover
As seen from Table 2, with the clover of microbe additive process in example 4, the different ensiling time periods of 2 days ~ 90 days, the indices of ensiling all reached good effect.PH value remains in the different ensiling time level being less than 5, the content of protein, water soluble carbohydrates and reducing sugar does not all have because the prolongation of ensiling time shows obvious minimizing, and the content of water content, ammoniacal nitrogen and free amino acid does not all have because the prolongation of ensiling time shows obvious increase.
Embodiment recited above is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; do not departing under the present invention designs spiritual prerequisite; the various distortion that the common engineers and technicians in this area make technical solution of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.
Claims (9)
1. a preparation method for alfalfa ensilage microbial inoculum Lactobacillus plantarum, is characterized in that, the preparation method of described Lactobacillus plantarum is as follows:
(1) separation of bacterial strain: the leachate collecting mud, the ratio of 2:10 joins in sterilized water and mixes by volume, serial dilution, is coated in MRS solid medium respectively, 37 DEG C of Anaerobic culturel 45h by each dilution bacterium liquid 100 microlitre; Single bacterium colony that picking transparent circle is larger, on MRS solid medium, line obtains pure single bacterium colony for 4 times continuously; Picking list bacterium colony is in the MRS fluid nutrient medium not adding calcium carbonate, and observe whether aerogenesis, aerogenesis bacterial strain is heterofermentative lactic bacteria, and anaerogen strain is homofermentative lactic bacteria;
(2) purifying of bacterial strain: in access liquid LB culture medium, under the condition of 14-26 DEG C, shaken cultivation is to exponential phase of growth, the bacterium liquid being in exponential phase of growth is transferred in liquid fermentation medium, 14-26 DEG C of shaken cultivation is to initial stage stationary phase, zymotic fluid is centrifugal, get supernatant, Filter paper filtering, obtain thick bacterium liquid; Be that the film bag of 12KDa is by extremely original for the volume concentration of thick bacterium liquid 1/10 with molecular cut off; Bacterium liquid after concentrated adds ammonium sulfate to 65-70% saturation degree, centrifuging and taking supernatant, continues to add ammonium sulfate to 85-100%, and centrifuging and taking precipitates, in the KPB buffer solution dialysis of pH=7, place 24h; By centrifugal for the bacterium liquid after dialysis, get supernatant, be splined on the CM-Sepharose prepacked column that the KPB buffer solution of pH=7 balances, wash-out, collects activated part, is Lactobacillus plantarum, and-20 DEG C frozen;
(3) press down Yeast assay: the lactic acid bacteria preserved under taking out-20 DEG C of conditions, get on 10 microlitres injection MRS solid mediums and be coated with, under the condition of temperature 37 DEG C, cultivate 15h; With the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 20ml test tube of 10mlMRS culture medium, under the condition of temperature 37 DEG C, cultivate 17h, obtain the lactic acid bacteria activated; By the inoculum concentration of 1%, by the access of the lactic acid bacteria of activation not containing in the 250ml triangular flask of calcium carbonate MRS fluid nutrient medium, liquid amount is 100ml, cultivates 17h for 37 DEG C, now, at least containing 0.993 × 10 in every milliliter of MRS fluid nutrient medium
10cfu lactic acid bacteria; 5000 leave heart 5min, collect supernatant, obtain bateriostatics one; Saccharomycete 28 DEG C of static gas wave refrigerator 48h in YPD fluid nutrient medium are obtained saccharomycete nutrient solution; Double-deck agar plate punch method measures the bacteriostasis of above-mentioned bateriostatics one;
(4) drug sensitive test of bacterial strain: the antibiotic filter paper preparing variable concentrations, cultivates lactic acid bacteria, adopts disc diffusion method to measure the drug sensitivity of lactic acid bacteria, obtains 8 strains to antibiotic sensitive and the bacterial strain good to saccharomycete inhibition;
(5) qualification of bacterial strain: above-mentioned steps is screened the bacterial strain obtained and carry out biological characteristics and the qualification of 16S-23S rDNA spacer sequence.
2. the preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, it is characterized in that, described step (1) or (3) middle MRS fluid nutrient medium are: peptone 12g, beef extract 8g, yeast extract 6g, L-light Cystine hydrochloride 0.08 g, hydrogen citrate three ammonium 3g, glucose 25g, Tween 80 mL, sodium acetate 6g, vitamin K
23g, dipotassium hydrogen phosphate 3g, magnesium sulfate 0.8g, calcium carbonate 12 g, manganese sulfate 0.3g, agar 20g, distilled water 1000mL, pH value 6-7.
3. the preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, it is characterized in that, the component that described step (1) or (3) middle MRS solid medium often rise and content are: add mass percent 1.5% agar powder in MRS fluid nutrient medium, pH value is 6.5,121 DEG C of sterilizing 20min.
4. the preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, it is characterized in that, consisting of of fermentation medium in described step (2): dusty yeast 0.3g/L, glucose 1.2g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 1.5g/L, potassium chloride 0.8g/L, ferrous sulfate 0.03g/L, magnesium sulfate 0.8g/L, pH=8-9.
5. the preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, it is characterized in that, in described step (3), double-deck agar plate punch method measures and refers to, the MRS solid medium of 30ml is injected the culture dish of diameter 25cm, cooling 1h; Get 10mL saccharomycete nutrient solution to be added to 150ml and to be cooled in the YPD semisolid culturemedium of 45 DEG C, shake up, and be poured on rapidly on bottom MRS culture medium; On bottom MRS culture medium, equidistance place, interval places 3 by the lml pipettor Tip head cut short; This is 1cm by the height cutting short Tip head, and diameter is 0.8cm; The YPD semisolid culturemedium that cooling is injected, the time is 1h, now, makes Double-Medium; Taking out Tip head, making the hole for placing bateriostatics; Get the above-mentioned lactic acid bacteria bateriostatics one of same concentrations, volume number is 150 microlitres, adds wherein in holes; Remain one in contrast, add the sterilizing Russia water of 150 microlitres; Cultivate 48h for 37 DEG C, measure and record lactic acid bacteria to saccharomycetic antibacterial circle diameter;
The preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, it is characterized in that, the antibiotic filter paper preparing variable concentrations in described step (4) refers to, by streptomysin, Ciprofloxacin and gentamicin are all configured to 85 μ g/ml, three concentration gradients of 350 μ g/ml and 1200 μ g/ml, Amoxicillin, erythromycin and Cefradine are all configured to 0.650 μ g/ml, three concentration gradients of 20 μ g/ml and 80 μ g/ml, the circular filter paper sheet that diameter is 0.8cm is prepared with filter paper, they are put into above-mentioned antibiotic solution, obtain the antibiotic filter paper of variable concentrations.
6. the preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, it is characterized in that, cultivate lactic acid bacteria in described step (4) to refer to, the lactic acid bacteria preserved under taking out-20 DEG C of conditions, getting 5 microlitres injects on MRS solid medium, coating, 15h is cultivated under the condition of temperature 37 DEG C, with the single bacterium colony on oese picking MRS solid medium, be inoculated in and be equipped with in the 10ml test tube of 5mlMRS culture medium, the lactic acid bacteria that 17h obtains activating is cultivated under the condition of temperature 37 DEG C, by the lactic acid bacteria of activation by 1% inoculum concentration access be equipped with in the 250ml triangular flask of MRS fluid nutrient medium, liquid amount is 100ml, cultivate 17h for 37 DEG C, obtain lactobacillus suspension, now, at least containing 0.993 × 10 in every milliliter of MRS fluid nutrient medium
10cfu lactic acid bacteria, the bacterial concentration diluting every milliliter of lactic acid bacteria with sterilized water is 1.5 × 10
8cfu.
7. the preparation method of high-moisture alfalfa ensilage microbial inoculum active component according to claim 2, it is characterized in that, in described step (5), the Sequence Identification of 16S-23S rDNA spacer region refers to, the nucleotide sequence of the 16S-23S rDNA spacer region of bacterial strain WN5 and Lactobacillus plantarum WCFS1(AL935263) homology the highest, its homology is 98%.
8. the preparation method of high-moisture alfalfa ensilage microbial inoculum active component according to claim 2, is characterized in that the Identification of Biological Characteristics of bacterial strain in described step (5) refers to, the stability of Lactobacillus plantarum to heat and the stability to acid.
9. the preparation method of a kind of alfalfa ensilage microbial inoculum Lactobacillus plantarum according to claim 1, is characterized in that, the component that described YPD fluid nutrient medium often rises and content are: dusty yeast 10 grams, peptone 20 grams, glucose 20 grams; The component that described YPD semisolid culturemedium often rises and content are: dusty yeast 10 grams, peptone 20 grams, glucose 20 grams, the agar powder of 0.7%.
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| CN105104909A (en) * | 2015-09-24 | 2015-12-02 | 广州金水动物保健品有限公司 | Silage and preparation method and application thereof |
| CN109609410A (en) * | 2019-01-08 | 2019-04-12 | 天津市畜牧兽医研究所 | A kind of preparation method of alfalfa ensilage leavening and alfalfa ensilage |
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| CN105104909A (en) * | 2015-09-24 | 2015-12-02 | 广州金水动物保健品有限公司 | Silage and preparation method and application thereof |
| CN109609410A (en) * | 2019-01-08 | 2019-04-12 | 天津市畜牧兽医研究所 | A kind of preparation method of alfalfa ensilage leavening and alfalfa ensilage |
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