CN104911131B - Enterococcus mundtii and application thereof in low-temperature silage - Google Patents
Enterococcus mundtii and application thereof in low-temperature silage Download PDFInfo
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- CN104911131B CN104911131B CN201510339993.XA CN201510339993A CN104911131B CN 104911131 B CN104911131 B CN 104911131B CN 201510339993 A CN201510339993 A CN 201510339993A CN 104911131 B CN104911131 B CN 104911131B
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- Prior art keywords
- enterococcus mundtii
- enterococcus
- mundtii
- ensilage
- ensiling
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enterococcus mundtii strain and application thereof in low-temperature ensiling. The Enterococcus mundtii provided by the invention is specifically Enterococcus mundtii (Enterococcus mundtii) QH1-255, and the preservation number of the Enterococcus mundtii in China center for type culture Collection is CCTCC NO: m2015253. The Enterococcus mundtii (Enterococcus mundtii) QH1-255 CCTCC NO: m2015253 has stress resistance such as low temperature resistance, salt resistance, acid and alkali resistance, and can rapidly propagate and produce acid to reduce pH in the low temperature ensiling process (5 ℃), effectively inhibit the growth or production of harmful mixed bacteria, effectively retain nutrient components such as crude protein, crude fat, crude fiber, and the like, reduce non-nutrient substances such as crude ash components, and achieve the effect of long-term storage of the ensiling feed.
Description
Technical field
The invention belongs to microorganism field, it is related to one plant of enterococcus mundtii and its application in low temperature ensiling.
Background technology
Ensiling be under the anaerobic digestion of lactic acid bacteria by it is carbohydrate-modifying be organic acid so that pH reduction reach
To the purpose stored for a long time.Lactic acid bacteria and temperature are the deciding factors of ensilage fermentation quality quality.
Oat is that extremely frigid zones Winter-Spring raises the main forage crop mended, and because extremely frigid zones temperature low altitude area is high, is unfavorable for
The growth of lactic acid bacteria, it is difficult to the feed that is best in quality and can storing for a long time that ferments.
Therefore, the stronger bacterial strain of resistance of high-low temperature resistant, salt tolerant, acid and alkali-resistance is filtered out, and is added as leavening
It is added in low temperature ensiling oat feed, it will have huge application value to the silage making of extremely frigid zones.
The content of the invention
First purpose of the present invention is to provide one plant of enterococcus mundtii.
Enterococcus mundtii provided by the present invention is specially enterococcus mundtii (Enterococcus mundtii) QH1-
255, the bacterial strain has been preserved in China typical culture collection center on April 28th, 2015, and (abbreviation CCTCC, address is：Hubei
No. 299 Wuhan Universitys of wuchang, wuhan area Bayi Road of province Wuhan University's collection in the school), its deposit number is CCTCC NO：M
2015253。
Enterococcus mundtii (Enterococcus mundtii) QH1-255 is from Qinghai Province Jianzha County Wild Wheats
Upper isolated, the bacterial strain single bacterium colony is shaped as circle, and Gram-negative does not produce hydrogen peroxide, it is impossible to utilize glucose
Fermentation gas, are homofermentation bacterial strain.Faint, remaining measure bar is grown under conditions of 50 DEG C, 6.5%NaCl and pH value 3 to 4
The equal well-grown of part, with preferable high-low temperature resistant, alkaline-resisting and salt tolerant growth ability.Its 16S rDNA sequences such as sequence table
Shown in middle sequence 1.
Second object of the present invention is to provide a kind of microbial inoculum.
The active component of microbial inoculum provided by the present invention is the enterococcus mundtii (Enterococcus mundtii)
QH1-255CCTCC NO：M 2015253.
The microbial inoculum except comprising as activity into the enterococcus mundtii (Enterococcus mundtii) QH1-
255CCTCC NO：Outside M 2015253, auxiliary material can be also included, such as (solvent is water to MRS solid mediums, and solute and its concentration are such as
Under：Peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/
L, dipotassium hydrogen phosphate 2g/L, ammonium citrate 2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L, agar 17g/L).
Third object of the present invention is to provide a kind of silage additive.
The active component of silage additive provided by the present invention is the enterococcus mundtii (Enterococcus
mundtii)QH1-255CCTCC NO：M 2015253.
Fourth object of the present invention is to provide a kind of ensilage.
Contain the silage additive in ensilage provided by the present invention.
Enterococcus mundtii (Enterococcus mundtii) the QH1-255CCTCC NO：M 2015253 or described bacterium
Agent it is following it is any in application fall within protection scope of the present invention：
(a1) bacterium is suppressed；
(a2) bacterial inhibitor is prepared；
(a3) silage additive is prepared；
(a4) ensilage is prepared.
The application of (a1) is non-diseases diagnoses and treatment application.
In (a1) and (a2), the bacterium concretely micrococcus luteus and/or salmonella.
Wherein, enterococcus mundtii (Enterococcus mundtii) the QH1-255CCTCC NO：M 2015253 or
Suppression of the microbial inoculum to the bacterium is embodied as the suppression under the conditions of 30 DEG C.
Application of the silage additive in the ensilage is prepared falls within protection scope of the present invention.
The 5th purpose of the present invention is to provide a kind of method for preparing the ensilage.
The method provided by the present invention for preparing the ensilage, specifically may include：By ensiling raw material and the Meng Shi
Enterococcus (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 is mixed, and carries out solid anaerobic fermentation, is received
Collect all tunnings, obtain the ensilage；
In the present invention, the ensiling raw material is oat complete stool；Specially the oat complete stool of milk stage, moisture
82.97%, cut into 1-2cm segments.
In the process, the oat complete stool and the enterococcus mundtii (Enterococcus mundtii) QH1-
255CCTCC NO：M 2015253 proportioning can be 100g：105cfu；The fermentation is cold fermentation；The low temperature can be 5
℃；The time of the fermentation can be 30 days.
Enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO provided by the present invention：
M2015253 has the resistance such as high-low temperature resistant, salt tolerant, acid and alkali-resistance, and breeds and produce rapidly during low temperature ensiling (5 DEG C)
PH drops in acid, effectively the growth or generation of the harmful miscellaneous bacteria of suppression, and the nutritional ingredient such as thick protein, crude fat, crude fibre is effectively protected
Stay, non-nutritious matter such as coarse ash constituent reduction, reach the long-term effect for preserving ensilage.
Preservation explanation
Strain name：Enterococcus mundtii
Latin name：(Enterococcus mundtii)
Strain number：QH1-255
Preservation mechanism：China typical culture collection center
Preservation mechanism is referred to as：CCTCC
Address：No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road Wuhan University's collection in the school
Preservation date：On April 28th, 2015
Collection is registered on the books numbering：CCTCC NO：M 2015253
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The separation and identification of embodiment 1, enterococcus mundtii (Enterococcus mundtii) QH1-255
First, bacterial strain QH1-255 separation and screening
Qinghai Province Jianzha County Wild Wheats 10g is taken, 90mL sterilized waters are put into, shaken 10 seconds, 1mL liquid is drawn and is put in
1.5mL centrifuge tubes, dilute 10 successively1, 103, 105Times, take the μ L of diluent liquid 20 to be respectively coated on MRS solid mediums, 30 DEG C
Anaerobic culturel 48 hours, takes single bacterium colony MRS solid mediums to expand culture, is QH1-255 by the wherein one plant bacterium numbering of acquisition.
Wherein, the solvent of the MRS solid mediums is water, and solute and its concentration are as follows：Peptone 10g/L, beef extract
10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, lemon
Sour ammonium 2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L, agar 17g/L.
2nd, bacterial strain QH1-255 identification
Obtained bacterial strain QH1-255 is separated and screened to step one from the following aspects to identify.
1st, Morphological Identification
Step one is separated and screened after obtained bacterial strain QH1-255 single bacterium colonies, Gram's staining, micro- Microscopic observation single bacterium
Fall shape rounded.
2nd, physiological and biochemical property is identified
Step one separates and screened obtained bacterial strain QH1-255 Gram-negatives, does not produce hydrogen peroxide, it is impossible to utilize
Glucose fermentation aerogenesis, is homofermentation bacterial strain.
Fermentation utilization powers of the bacterial strain QH1-255 to different carbon source is detected using API 50CH.As a result it is as shown in table 1.Can
See, bacterial strain QH1-255 can utilize ribose, xylose, mannose, amarogentin, ursin, aesculin, salicin, fiber two
Sugar, lactose, sucrose, N- methyl-glucamines, fructose, glucose, maltose, L-arabinose, trehalose is carried out as carbon source
Fermentation；Glycerine, L- trehaloses, Alpha-Methyl-D-Glucose glycosides, D- turanoses, D-arabitol, melezitose, cotton cannot be utilized
Sub- sugar, glycogen, 2- ketone groups-gluconate, D- lyxoses, rhamnose, D-R, L- xyloses, Beta-methyl-xyloside, chrysanthemum
Powder, sorbose, galactitol, inositol, starch, D- trehaloses, xylitol, antierythrite, gluconate, L- arabites,
Ribitol, 5- ketone groups-gluconate is used as fermenting carbon source；Can with faint utilization galactolipin, mannitol, sorbierite, melibiose, α-
Methyl-Dmannose glycosides, gentiobiose, D-Tag are fermented as carbon source.
Fermentation utilization powers of the bacterial strain QH1-255 of table 1 to different carbon source
Note："+" represents positive, can utilize；"-" represents negative, i.e., can not utilize；" w " represents weakly positive, i.e., faint profit
With.
3rd, 16S rDNA sequence homology analysis
The bacterial strain QH1-255 of the gained of extraction step one genomic DNA, using it as template, is entered using bacterial universal primers
Performing PCR is expanded, and obtains bacterial strain QH1-255 16S rDNA fragments, and carries out sequencing, and its sequence is sequence 1 in sequence table.
Sequence 1 is subjected to BLAST (http in GenBank databases://blast.ncbi.nlm.nih.gov/Blast.cgi) it is same
Source property is compared, and determines strain classification.
According to above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis results, by step one institute
The bacterial strain QH1-255 obtained is accredited as enterococcus mundtii (Enterococcus mundtii), and is preserved on April 28th, 2015
(abbreviation CCTCC, address is China typical culture collection center：No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road
In the school, Wuhan University's collection, postcode 430072), preserving number is CCTCC NO：M 2015253, its Classification And Nomenclature is Meng Shi
Enterococcus (Enterococcus mundtii) QH1-255.
Embodiment 2, enterococcus mundtii (Enterococcus mundtii) QH1-255 are in different temperatures, pH and salinity
Under growth
1st, under different temperatures enterococcus mundtii (Enterococcus mundtii) QH1-255 growth
By enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO of activation：M 2015253 is inoculated with
In MRS fluid nutrient mediums, the incubated 24h 5, at a temperature of 10,45 and 50 DEG C respectively, at spectrophotometric determination 600nm
Absorbance value (OD600), to observe enterococcus mundtii under different temperatures (Enterococcus mundtii) QH1-255 growth
Situation.
Wherein, the solvent of MRS fluid nutrient mediums is water, and solute and its concentration are as follows：Peptone 10g/L, beef extract 10g/
L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate
2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L；pH6.8.
As a result it is as shown in table 2, it is seen that enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
2015253 grow fine at 5-45 DEG C, and 50 DEG C of growths are faint, show that the bacterial strain has good lower temperature resistance, heat-resisting quantity phase
To slightly worse.
2nd, under difference pH enterococcus mundtii (Enterococcus mundtii) QH1-255 growth
The starting pH of MRS fluid nutrient mediums (formula is ibid) is distinguished with 1mol/L hydrochloric acid and 1mol/L sodium hydroxide
It is adjusted to 3.0,3.5,4.0,4.5,5.0,5.5,6.0,9.0 and 10.0,30 DEG C of constant temperature quiescent culture enterococcus mundtiis
(Enterococcus mundtii)QH1-255CCTCC NO：M 2015253, cultivates 24h, not acid adjustment in incubation, with point
Absorbance value (OD600) at light photometric determination 600nm, to observe enterococcus mundtii (Enterococcus under different pH
Mundtii) QH1-255 growing state.
As a result it is as shown in table 2, it is seen that enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
2015253 can grow good in pH4.5-10.0 culture environment, are grown in pH3.0-4.0 faint, show that its is resistance to
Alkali better performances, acid resistance is relatively slightly worse.
3rd, under different salinity enterococcus mundtii (Enterococcus mundtii) QH1-255 growth
By enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO of activation：M 2015253 distinguishes
The MRS fluid nutrient mediums (being formulated ibid, pH6.8) that the weight/mass percentage composition containing NaCl is 3% and 6.5% are inoculated in, in 30
DEG C constant temperature quiescent culture 24h, with absorbance value (OD600) at spectrophotometric determination 600nm, is covered with observing under different salinity
Family name enterococcus (Enterococcus mundtii) QH1-255 growing state.
As a result it is as shown in table 2, it is seen that enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
2015253 under 3% NaCl concentration well-grown, grown under 6.5% NaCl concentration faint, show that it has certain
Salt tolerance.
The growth characteristics of the bacterial strain QH1-255 of table 2 under various circumstances
Note："+" represents well-grown；" w " expression growth is faint, and (OD600 is considered as well-grown more than 0.3, small more than 0.2
In faint to grow equal to 0.3).
According to result above, it is known that enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
2015253 growth adaptation is very capable, is resistant to extreme environment Survival Reproduction.
The Antibacterial Activity of embodiment 3, enterococcus mundtii (Enterococcus mundtii) QH1-255
For examination bacterium：Micrococcus luteus, micrococcus luteus, salmonella and Escherichia coli.
1st, by enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO of activation：M 2015253 connects
Plant in MRS fluid nutrient mediums (being formulated ibid, pH6.8), 30 DEG C of culture 48h, zymotic fluid centrifuges 5min through 10000rpm, taken
Clear liquid is used to determine.
2nd, its bacteriostatic activity is determined using Oxford cup double-layer agar technique, sterilized agar medium is heated to completely to melt
Change, be poured in culture dish, per ware 15ml (lower floor), treat that it solidifies.In addition, the PDA culture medium of thawing is cooled into 50 DEG C or so
It is mixed into for examination bacterium, the culture medium 5ml for being mixed with bacterium is added to (upper strata) to be solidified on the culture medium solidified.Existed with sterile working
Media surface directly vertically puts Oxford cup (internal diameter 6mm, external diameter 8mm, high 10mm circular tubule), gently pressurizes, makes it
Tight is contacted with culture medium, enterococcus mundtii (Enterococcus mundtii) QH1- that step 1 is obtained is added in cup
255CCTCC NO：The zymotic fluids of M 2015253.Fill it up with rearmounted 37 DEG C to cultivate 16-18 hours, observe result, inhibition zone dipstick metering.
Control is used as using MRS fluid nutrient mediums substitution zymotic fluid.Experiment is repeated 3 times, and is as a result represented with average value.
It is as a result as shown in table 3, it is seen that：Under 30 DEG C of condition of culture, enterococcus mundtii (Enterococcus mundtii)
QH1-255CCTCC NO：M 2015253 has good inhibiting effect to micrococcus luteus and salmonella.
The bacterial strain QH1-255 of table 3 bacteriostatic activity
Note：Antibacterial circle diameter includes Oxford cup external diameter (7.8mm)；"-" indicates no inhibition zone.
Embodiment 4, enterococcus mundtii (Enterococcus mundtii) QH1-255 ensiling oats
Ensiling raw material：Milk stage oat complete stool, moisture 82.97% cuts into 1-2cm segments.
First, oat ensilage is prepared
1st, with enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 is used as feed
Additive prepares oat ensilage
(1) enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：The bacteria suspensions of M 2015253
Prepare
Under aseptic condition, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO are taken：M2015253
30 DEG C of overnight incubations in MRS fluid nutrient mediums are inoculated in, enterococcus mundtii (Enterococcus mundtii) QH1- is obtained
255CCTCC NO：Enterococcus mundtii (Enterococcus mundtii) QH1- in the bacteria suspensions of M 2015253, bacteria suspension
255CCTCC NO：M 2015253 content is 106cfu/ml。
Wherein, the solvent of MRS fluid nutrient mediums is water, and solute and its concentration are as follows：Peptone 10g/L, beef extract 10g/
L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate
2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L；pH6.8.
(2) with enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 is used as feeding
Feed additives prepare oat ensilage
1-2cm or so length (moisture 82.97%) is cut into chopper after milk stage oat complete stool is gathered in, 100g is taken
It is put into bag silo, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO that step (1) is obtained：M
The μ l of 2015253 every bag of bacteria suspension 100 are separately added into bag silo, are mixed, using vacuum packing machine sealed after being vacuumized, are put into 5
In DEG C refrigerator-freezer.
2nd, oat ensilage is compareed
1-2cm or so length (moisture 82.97%) is cut into chopper after milk stage oat complete stool is gathered in, 100g is taken
It is put into bag silo, every bag of 100 μ l of sterilized water is separately added into bag silo, mixes, vacuumized using vacuum packing machine rear close
Envelope, is put into 5 DEG C of refrigerator-freezers.
2nd, the microbiology turbidity of oat is determined in low temperature ensilage
Using plate dilution assay method microbiology turbidity.Feed Sample 10g is added in 90ml sterile distilled waters, utilized
Vortex oscillator shakes 30s, and then 10 times of gradient dilutions take 10 respectively-1、10-3With 10-5Each 20 μ l coatings of times sample diluting liquid
(it is used to detect saccharomycete and mould, according to bacterium colony in MRS (being used to detect lactic acid bacteria), BLB (being used to detect Escherichia coli), PDA
Form, naked eyes are distinguished, and mold colony is in villiform, and the color of its spore is presented in cotton-shaped, spider reticulation, and saccharomycete is smaller,
Shaft-like, helical form is spherical, and bacterium colony surface is smooth sticky or dry) NA (for detecting aerobic bacteria) culture medium；By 10-1
With 10-2Times sample diluting liquid 1ml is after 75 DEG C of water-bath water-bath 15min, and taking 20 μ l to be coated on NA respectively (is used to detect gemma bar
Bacterium) and CLO (for detecting clostridium) culture medium (formula of CLO culture mediums：Contain peptone 15g, soybean egg in every liter of culture medium
White peptone 7.5g, yeast extract 7.5g, beef extract 7.5g, ferric citrate 1g, sodium hydrogensulfite 1g, L-cysteine hydrochloride
0.75g, agar 15g, surplus is water) on, these coated culture mediums are inverted and are put in 30 DEG C of constant incubator culture 48h,
Wherein MRS and CLO culture mediums should be positioned over anaerobic culture box, and other culture mediums are positioned over common constant incubator.Culture
48 hours postscript single bacterium colony numbers are n, bacterium colony (logCFU/g)=log10[(n×10)/20×10-3].Experiment is repeated 3 times, knot
Fruit is represented in the form of mean+SD.
It is as a result as shown in table 4, it is seen that：Under 5 DEG C of low temperature environments, ensiling the 1st day, enterococcus mundtii (Enterococcus
mundtii)QH1-255CCTCC NO：The addition group lactic acid bacterias of M 2015253 breed rapidly, and viable lactic acid bacteria quantity is significantly larger than
Control group (P≤0.01)；Ensiling the 3rd day, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
The lactic acid bacterium number pole of 2015253 addition groups is significantly higher than control group (P≤0.01), Escherichia coli, bacillus and mould etc.
The quantity of harmful bacteria is significantly or pole is substantially less than control group (P≤0.05 or P≤0.01), in control group detection have saccharomycete and
Harmful miscellaneous bacteria such as clostridium, in enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 adds
Plus do not detected then in group；In low temperature ensiling the 30th day, pathogenic bacteria Escherichia coli are detected in control group, and in enterococcus mundtii
(Enterococcus mundtii)QH1-255CCTCC NO：Do not detected then in the addition groups of M 2015253.To sum up, low temperature ring
Under border (5 DEG C), enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 is to ensiling oat
In be harmful to miscellaneous bacteria there is effective inhibitory action.
The microbiology turbidity (logCFU/g) of oat during the low temperature ensiling of table 4 (5 DEG C)a
Note：aMean+SD (n=3).Same file contain different capitalizations represent otherness extremely significantly (P≤
0.01)；Same file contains different lowercase letter indication differences significantly (P≤0.05)；ND represents not detect.
3rd, the measure that the pH of oat, free water and dry weight content change in low temperature ensilage
1st, pH assay methods
Feed Sample 10g is added in 90ml sterile distilled waters, 30s is shaken using vortex oscillator, takes liquid to pass through
Filter paper is filtered, and the pH value of filtrate is determined using pH acidometers.Experiment is repeated 3 times, as a result the table in the form of mean+SD
Show.
2nd, free water content and the assay method of dry weight content
65 DEG C, a blank sheet of paper is ordered into a paper bag first, paper bag is weighed on assay balance by 48h seasonings,
W1 is designated as, Feed Sample is put into paper bag, claims gross weight, W2 is designated as, constant temperature is divulged information after 65 DEG C of dry 48h, is positioned over drier
Weighed after middle 30min, be designated as W3.Free moisture (%)=(W2-W3)/(W2-W1) × 100.Dry weight (%)=100%- dissociates
Moisture (%).Experiment is repeated 3 times, and is as a result represented in the form of mean+SD.
It is as a result as shown in table 5, it is seen that：In 5 DEG C of low temperature environments, in ensiling the 3rd day, the 7th day and the 30th day, ensiling oat
Middle addition enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 experimental group is with compareing
Group is significantly reduced (P≤0.01) compared to pH poles.Show enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC
NO：Ensiling oat pH effectively can be reduced to beneficial to ensiling level rapidly by M 2015253 under 5 DEG C of low temperature environments.In green grass or young crops
Store the 1st day, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：The ensiling of the addition groups of M 2015253
Oat dry weight content slightly increases, and moisture is slightly reduced；Ensiling the 3rd day, enterococcus mundtii (Enterococcus
mundtii)QH1-255CCTCC NO：M2015253 addition group oats are compared with control group, and dry weight content significantly increases, moisture
Content significantly reduces (P≤0.05), shows under low temperature ensiling environment, addition enterococcus mundtii (Enterococcus
mundtii)QH1-255CCTCC NO：M 2015253 has on dry weight and free water slightly to be influenceed.
The pH of oat, free water and dry weight content change (%/fresh weight) during the low temperature ensiling of table 5 (5 DEG C)a
Note：aMean+SD (n=3)；" * " represents significant difference (P≤0.05)；" NS " represents that difference is not notable
(p ＞ 0.05).
4th, the neutral detergent fiber of oat and the measure of acid detergent fiber change in low temperature ensilage
1st, the measure of neutral detergent fiber (neutral detergent fiber, NDF)
After plant feed boils processing through neutral detergent, undissolved residue is neutral detergent fiber, predominantly
Cell wall constituent, neutral detergent fiber includes cellulose, this quality and hemicellulose, may be used as quantitative Livestock roughage
Feed intake.
(1) 20min opens crude fiber test instrument device and cooling circulating water in advance.
(2) assay balance weighs about 0.5g (W1) left and right Feed Sample and is positioned in glass pot.
(3) machine on, adds (3g/100ml) lauryl sodium sulfate of 100ml neutral detergents 3% and appropriate octyl alconyl is made
Defoamer, 100 DEG C are heated to boiling, keep 50 DEG C or so heating 70min.
(4) glass pot after suction filtration, is removed, acetone rinsing three times is utilized.
(5) glass pot is put in fume hood and be dried overnight.
(6) later glass pot will be dried to be positioned in baking oven, more than 135 DEG C of constant temperature aeration-drying 2h are put into drying
Cooled down in device after 1h, weigh, be designated as W2.
(7) glass pot is put on electric furnace and is ashed, until cigarette disperses, taking out crucible using crucible tongs is positioned over 530
In DEG C Muffle furnace after heating ashing 2h, taking-up is positioned over 1h in drier and cooled, and weighs, is designated as W3.
NDF (%)=(W2-W3)/W1 × 100.
Experiment is repeated 3 times, and is as a result represented in the form of mean+SD.
2nd, the measure of acid detergent fiber (acid detergent fiber, ADF)
Acid detergent fiber assay method：After being handled using acid detergent, residual residue is acid detergent fiber,
Including lignin and cellulose.Residue of the acid detergent fiber after sulfuric acid treatment is lignin, fine from acidic cleaning
The residue that 72% sulfuric acid treatment is subtracted in dimension value is feed fibre cellulose content.
(1) 20min opens crude fiber test instrument device and cooling circulating water in advance.
(2) assay balance weighs about 0.5g (W1) left and right Feed Sample and is positioned in glass pot.
(3) machine on, adds (2g/100ml) cetyl trimethylammonium bromide of 100ml acid detergents 2% and appropriate fourth
Octanol makees defoamer, and 100 DEG C are heated to boiling, keeps 50 DEG C or so heating 70min.
(4) glass pot after suction filtration, is removed, acetone rinsing three times is utilized.
(5) glass pot is put in fume hood and be dried overnight.
(6) later glass pot will be dried to be positioned in baking oven, more than 135 DEG C of constant temperature aeration-drying 2h are put into drying
Cooled down in device after 1h, weigh, be designated as W2.
(7) glass pot is put on electric furnace and is ashed, until cigarette disperses, taking out crucible using crucible tongs is positioned over 530
In DEG C Muffle furnace after heating ashing 2h, taking-up is positioned over 1h in drier and cooled, and weighs, is designated as W3.
ADF (%)=(W2-W3)/W1 × 100.
Experiment is repeated 3 times, and is as a result represented in the form of mean+SD.
It is as a result as shown in table 6, it is seen that：Enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
2015253 addition groups are compared with control group, ensiling the 1st day, enterococcus mundtii (Enterococcus mundtii) QH1-
255CCTCC NO：The addition group NDF contents of M 2015253 are significantly reduced (P≤0.05)；Ensiling the 3rd day and the 7th day, Meng Shi intestines balls
Bacterium (Enterococcus mundtii) QH1-255CCTCC NO：The addition group NDF contents of M 2015253 are basically unchanged, but right
According to a group continuous decrease；To the 30th day, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
2015253 addition groups and the NDF contents of control group fall below 50.33% and 50.93% similar level respectively.Low temperature ensiling
Cheng Zhong, acid detergent fiber content does not have significant changes (p ＞ 0.05).To sum up, in low temperature (5 DEG C) ensiling environment, 3 before ensiling
My god, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 accelerates NDF contents drop
It is low, but ensiling is finally not significantly different (P ＞ 0.05) with the NDF contents compareed.
Change (the %/fresh of the neutral detergent fiber of oat and acid detergent fiber during the low temperature ensiling of table 6 (5 DEG C)
Weight)a
Note：aMean+SD (n=3)；" NS " represents difference not significantly (P ＞ 0.05)；" * " represents significant difference
(P≤0.05).NDF is neutral detergent fiber；ADF is acid detergent fiber.
5th, the measure that the coarse ash of oat, crude protein and crude fat change in low temperature ensilage
1st, the measure of coarse ash (Crude ash)
(1) porcelain crucible is heated after 2h in 530 DEG C of Muffle furnaces, and taking-up is positioned over 1h in drier and cooled, and weighs, is designated as
W1。
(2) 2g (W2) left and right Feed Sample is weighed in porcelain crucible by assay balance, is positioned on electric furnace and is ashed,
Untill cigarette disperses, take out porcelain crucible with crucible tongs and be positioned in 530 DEG C of Muffle furnaces after heating 2h, taking-up is positioned over drier
Middle 1h is cooled, and is weighed, and is designated as W3.
Coarse ash (%)=(W3-W1)/W2 × 100.
Experiment is repeated 3 times, and is as a result represented in the form of mean+SD.
2nd, the measure of crude protein
Sizing technique is boiled using disappearing and determines crude protein.
(1) about 1g (W1) Feed Sample is weighed by assay balance, pan paper is put into disappear after wrapping and boiled in pipe, added
0.2g copper sulphate and 6.0g potassium sulfates.
(2) the 20ml concentrated sulfuric acids are gently added to boil in pipe in disappearing, gently shakes, sample is fully distributed in concentrated sulfuric acid.
(3) it will disappear to boil pipe connection and be positioned over to disappear and boil on pipe, and prevent the volatilization of sulfuric acid.
(4) circulating water device is opened, will disappear to boil pipe and be placed on to disappear and boil on device, begin heat to 420 DEG C.
(5) wait disappearing and boil liquid in pipe and be changed into after green transparent, continue 420 DEG C and disappear to boil 2h.
(6) disappear and boil after decomposition completes, turn off to disappear and boil device, remove to disappear to boil pipe and be put on thermal insulation board and cool.
(7) after cooling down, digest tube is put into kjeldahl apparatus, starts titration (according to AOAC (Official Methods
of Analysis[M].15th ed.Association of official analytical chemists.Arlington,
VA.1999) method described is analyzed).
Total nitrogen content (%)=n (V1-V2) × M × 0.014/W1 × 100；
Crude protein quality (%)=total nitrogen content (%) × 6.25；
Wherein, V1：Titer ml；V2：Blank titration amount；M：Titrating solution HCl concentration；W1：Example weight.
Experiment is repeated 3 times, and is as a result represented in the form of mean+SD.
3rd, the measure of crude fat
Using aether extraction.Feed Sample 3h extractive distillations crude fat in ether, in addition to neutral fat, phosphate ester
Matter, free fatty, fat-soluble pigment etc. are also included.
(1) fatty bottle, which is positioned in 100 DEG C of baking ovens, dries after 2h, and taking-up, which is placed in drier, cools 1h, weighs, is designated as
W1。
(2) assay balance weighs 1g (W2) left and right Feed Sample and is placed on filter paper, fills in filter paper in fatty bucket after wrapping,
Blocked up with absorbent cotton.
(3) the fatty bucket that will be equipped with Feed Sample is put into fatty withdrawing device, connects fatty bottle, adds about 50ml ether
In fatty bottle bottle, cooling water system is opened, starts more than 3h extractings.
(4) complete after extracting, take out fatty bucket, fatty bottle is placed in 100 DEG C of baking ovens after 3h dryings, taking-up is placed on dry
30m is cooled down in dry device, is weighed, W3 is designated as.
Crude fat (%)=(W3-W1)/W2 × 100.Experiment is repeated 3 times, as a result the table in the form of mean+SD
Show.
It is as a result as shown in table 7, it is seen that：Under the conditions of low temperature (5 DEG C) ensiling, ensiling the 3rd day, enterococcus mundtii
(Enterococcus mundtii)QH1-255CCTCC NO：The crude ash content of the addition group ensiling oats of M 2015253 is notable
Less than control group (P≤0.05), in whole ensilage, enterococcus mundtii (Enterococcus mundtii) QH1-
255CCTCC NO：The crude ash content of the addition group ensiling oats of M 2015253 is slightly below control group, shows under cryogenic conditions,
Enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M 2015253 can be reduced in ensiling oat
Crude ash content.In ensiling the 7th day, enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M
The protein content reduction of 2015253 addition groups, remaining low temperature ensiling ensilage contains to the crude protein and crude fibre in oat
Amount has no significant effect (p ＞ 0.05), shows under cryogenic conditions, enterococcus mundtii (Enterococcus mundtii) QH1-
255CCTCC NO：M 2015253 can preferably retain crude fat and crude protein composition in ensiling oat.
The coarse ash of oat during the low temperature ensiling of table 7 (5 DEG C), crude protein and crude fat content change (%/fresh weight)a
Note：aMean+SD (n=3)；" NS " represents difference not significantly (P ＞ 0.05)；" * " represents significant difference
(P≤0.05)。
In summary, it is seen that enterococcus mundtii (Enterococcus mundtii) QH1-255CCTCC NO：M2015253
It is rapid during low temperature ensiling (5 DEG C) to breed and produce acid drop pH, the effective growth or generation for suppressing harmful miscellaneous bacteria, thick egg
In vain, the nutritional ingredient such as crude fat, crude fibre is effectively retained, non-nutritious matter such as coarse ash constituent reduction, reaches that long-term preservation is blue or green
Store the effect of feed.
Claims (9)
1. enterococcus mundtii（Enterococcus mundtii）QH1-255, its guarantor in China typical culture collection center
It is CCTCC NO to hide numbering：M 2015253.
2. a kind of microbial inoculum, it is characterised in that：The active component of the microbial inoculum is the enterococcus mundtii described in claim 1
（Enterococcus mundtii）QH1-255.
3. a kind of silage additive, it is characterised in that：The active component of the silage additive is claim 1 institute
The enterococcus mundtii stated（Enterococcus mundtii）QH1-255.
4. a kind of ensilage, it is characterised in that：Added in the ensilage containing the ensilage described in claim 3
Agent.
5. ensilage according to claim 4, it is characterised in that：The ensilage is according to comprising the following steps
What method was prepared：
By the enterococcus mundtii described in ensiling raw material and claim 1（Enterococcus mundtii）QH1-255 is mixed, and is entered
Row solid anaerobic fermentation, collects all tunnings, obtains the ensilage；
The ensiling raw material is specially oat complete stool；
The oat complete stool and the enterococcus mundtii（Enterococcus mundtii）QH1-255 proportioning is 100g：
105cfu；
The fermentation is cold fermentation；The low temperature is 5 DEG C；
The time of the fermentation is 30 days.
6. the enterococcus mundtii described in claim 1（Enterococcus mundtii）QH1-255 it is following it is any in should
With：
（a1）Prepare bacterial inhibitor；
（a2）Prepare the silage additive described in claim 3；
（a3）Prepare the ensilage described in claim 4；
Described（a1）In, the bacterium is specially micrococcus luteus or salmonella.
7. application according to claim 6, it is characterised in that：The enterococcus mundtii（Enterococcus mundtii）
The suppression that is suppressed to 30 DEG C under the conditions of of the QH1-255 to the bacterium.
8. application of the silage additive in the ensilage described in claim 4 or 5 is prepared described in claim 3.
9. the method for ensilage described in claim 4 is prepared, including：By the Meng Shi intestines described in ensiling raw material and claim 1
Coccus（Enterococcus mundtii）QH1-255 is mixed, and carries out solid anaerobic fermentation, is collected all tunnings, is obtained
The ensilage；
The ensiling raw material is specially oat complete stool；
The oat complete stool and the enterococcus mundtii（Enterococcus mundtii）QH1-255 proportioning is 100g：
105cfu；
The fermentation is cold fermentation；The low temperature is 5 DEG C；
The time of the fermentation is 30 days.
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