CN104894029B - Leuconostoc mesenteroides and its application in low temperature ensiling - Google Patents
Leuconostoc mesenteroides and its application in low temperature ensiling Download PDFInfo
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Abstract
The invention discloses one plant of Leuconostoc mesenteroides and its application in low temperature ensiling.Leuconostoc mesenteroides provided by the present invention is specially Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1 1135, and it is CCTCC NO in the deposit number of China typical culture collection center：M 2015256.Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1 1135CCTCC NO provided by the present invention：M 2015256 has the resistance such as low temperature resistant, salt tolerant, acid and alkali-resistance, and bred rapidly during low temperature ensiling (5 DEG C) and produce acid drop pH, the effective growth or generation for suppressing harmful miscellaneous bacteria, the nutritional ingredients such as thick protein, crude fat, crude fibre are effectively retained, non-nutritious matter such as coarse ash constituent reduction, reaches the long-term effect for preserving ensilage.
Description
Technical field
The invention belongs to microorganism field, it is related to one plant of Leuconostoc mesenteroides 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 Leuconostoc mesenteroides.
Leuconostoc mesenteroides provided by the present invention is specially Leuconostoc mesenteroides (Leuconostoc
Mesenteroides) QH1-1135, the bacterial strain is preserved in China typical culture collection center (letter on April 28th, 2015
Claim CCTCC, address is：No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road Wuhan University's collection in the school), its
Deposit number is CCTCC NO：M 2015256.
Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135 is from Qinghai Province Gui Dedan
Rosy clouds landforms wheatgrass is isolated, and the bacterial strain single bacterium colony is circle, and Gram-negative does not produce hydrogen peroxide；The bacterial strain energy
Enough glucose fermentation aerogenesis, are heterofermentation.Grown fine at 5 DEG C and 10 DEG C, 45 DEG C of ambient growths are faint, it is good in 50 DEG C of growths
It is good, show that the bacterial strain has good low temperature resistant growth ability.The equal well-grown in 3.00% NaCl concentration, 6.50%
NaCl concentration in can also survive, show that the bacterial strain has certain salt resistance ability.5 DEG C of liquid is cultivated 7-14 days, the bacterial strain
Supernatant pH drop to 3.5 respectively, acid producing ability is preferable.Bacterial strain well-grown in pH3.0-9.0 environment, shows the bacterium
Strain has preferable acid and alkali-resistance growth ability.Its 16S rDNA sequence is as shown in sequence 1 in sequence table.
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 Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：M 2015256.
The microbial inoculum except comprising as activity into the Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：Outside M 2015256, auxiliary material, such as MRS solid mediums (solvent can be also included
For water, solute and its concentration are as follows：Peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, three hydration second
Sour sodium 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) etc..
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 Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：M 2015256.
Fourth object of the present invention is to provide a kind of ensilage.
Contain the silage additive in ensilage provided by the present invention.
Leuconostoc mesenteroides (Leuconostoc mesenteroides) the QH1-1135CCTCC NO：M 2015256
Or the microbial inoculum 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 the application that non-diseases is diagnosed or treated.
In (a1) and (a2), the bacterium concretely micrococcus luteus and/or salmonella.
Wherein, Leuconostoc mesenteroides (Leuconostoc mesenteroides) the QH1-1135CCTCC NO：
Suppression of the M2015256 or described microbial inoculums 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 goldbeater's skin
Leukonid (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M 2015256 is mixed, and is carried out solid and is detested
Aerobe fermentation, collects all tunnings, obtains the ensilage；
In the present invention, the ensiling raw material is oat complete stool；Specially milk stage oat complete stool, moisture
82.97%, cut into 1-2cm segments.
In the process, the oat complete stool and the Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：M 2015256 proportioning can be 100g：105cfu；The fermentation is low temperature
Fermentation；The low temperature can be 5 DEG C；The time of the fermentation can be 30 days.
Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC provided by the present invention
NO：M 2015256 has the resistance such as low temperature resistant, salt tolerant, acid and alkali-resistance, and is bred rapidly simultaneously during low temperature ensiling (5 DEG C)
PH drops in production 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 effective
Retain, non-nutritious matter such as coarse ash constituent reduction, reach the long-term effect for preserving ensilage.
Preservation explanation
Strain name：Leuconostoc mesenteroides
Latin name：(Leuconostoc mesenteroides)
Strain number：QH1-1135
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 2015256
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, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135
First, bacterial strain QH1-1135 separation and screening
Qinghai Province Guide "Danxia" landform wheatgrass 10g is taken, 90mL sterilized waters are put into, shaken 10 seconds, 1mL liquid is drawn and puts
In 1.5mL centrifuge tubes, 10 are diluted successively1, 103, 105Times, take the μ L of diluent liquid 20 to be respectively coated on MRS agar mediums, 30
DEG C Anaerobic culturel 48 hours, takes single bacterium colony MRS solid mediums to expand culture, is QH1- by the wherein one plant bacterium numbering of acquisition
1135。
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 QH1-1135 identification
Obtained bacterial strain QH1-1135 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 QH1-1135 single bacterium colony 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 QH1-1135 Gram-negatives, does not produce hydrogen peroxide；It can send out
Ferment glucose aerogenesis, is heterofermentation.
Fermentation utilization powers of the bacterial strain QH1-1135 to different carbon source is detected using API 50CH.As a result it is as shown in table 1.Can
See, bacterial strain QH1-1135, which can ferment, utilizes galactolipin, glucose, D-MANNOSE, aesculin, cellobiose, sucrose, ribose,
L-arabinose, D- xyloses, Alpha-Methyl-D- glucosides, melibiose, mannitol, maltose, melezitose, gossypose, D-Fructose is done
Carbon source；It cannot ferment and utilize D-R alcohol, glycerine, D-R, Beta-methyl-xyloside, L- xyloses, galactolipin
Alcohol, inosite, gluconate, glycogen, xylitol, 2- ketone groups-gluconate, D- trehaloses, L- arabites, D- lyxoses,
Erythritol, 5- ketone groups-gluconate, ribitol, L- sorboses, lactose, rhamnose, sorbose, starch, amygdalin, black bearberry
Glycosides, Alpha-Methyl-D-MANNOSE, synanthrin, β-gentiobiose, Tagatose, L- trehaloses are cooked carbon source；Bigcatkin willow can be utilized with faint fermentation
Glycosides, trehalose, N-acetyl-glucosamine, D- turanoses do carbon source.
Fermentation utilization powers of the bacterial strain QH1-1135 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 QH1-1135 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 QH1-1135 16S rDNA fragments, and carries out sequencing, and its sequence is sequence in sequence table
1.Sequence 1 is subjected to BLAST (http in GenBank databases://blast.ncbi.nlm.nih.gov/Blast.cgi)
Sequence analysis, 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 QH1-1135 obtained is accredited as Leuconostoc mesenteroides (Leuconostoc mesenteroides), and April 28 in 2015
Day is preserved in China typical culture collection center, and (abbreviation CCTCC, address is：Wuhan City, Hubei Province Wuchang District Bayi Road 299
Wuhan University in the school, Wuhan University's collection, postcode 430072), its deposit number be CCTCC NO：M 2015256, its point
Class is named as Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135.
Embodiment 2, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135 are in different temperatures, pH
And the growth under salinity
1st, under different temperatures Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135 growth
By Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO of activation：
M2015256 is inoculated in MRS fluid nutrient mediums, and the incubated 24h 5, at a temperature of 10,45 and 50 DEG C, uses spectrophotometer respectively
Absorbance value (OD600) at 600nm is determined, to observe Leuconostoc mesenteroides (Leuconostoc under different temperatures
Mesenteroides) QH1-1135 growing state.
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 Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-
1135CCTCC NO：M 2015256 grows fine at 5 DEG C and 10 DEG C, and 45 DEG C of ambient growths are faint, in 50 DEG C of well-growns, table
The bright bacterial strain has good low temperature resistant growth ability.
2nd, under difference pH Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135 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 Leuconostoc mesenteroides
(Leuconostoc mesenteroides)QH1-1135CCTCC NO：M 2015256, cultivates uncomfortable in 24h, incubation
Acid, with absorbance value (OD600) at spectrophotometric determination 600nm, to observe Leuconostoc mesenteroides under different pH
(Leuconostoc mesenteroides) QH1-1135 growing state.
As a result it is as shown in table 2, it is seen that Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-
1135CCTCC NO：M 2015256 can grow well in pH3.0-9.0 culture environment, show its acid-proof alkaline
It is good.
3rd, under different salinity Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135 growth
By Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO of activation：
M2015256 be inoculated in respectively the weight/mass percentage composition containing NaCl be 3% and 6.5% MRS fluid nutrient mediums (formula ibid,
PH6.8), in 30 DEG C of constant temperature quiescent culture 24h, with absorbance value (OD600) at spectrophotometric determination 600nm, to observe not
With Leuconostoc mesenteroides under salinity (Leuconostoc mesenteroides) QH1-1135 growing state.
As a result it is as shown in table 2, it is seen that Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-
1135CCTCC NO：The well-growns under 3% NaCl concentration of M 2015256, can also be deposited in 6.50% NaCl concentration
It is living, show that it has certain salt tolerance.
4th, (5 DEG C) culture results of regular determination pH changes of low temperature
MRS fluid nutrient mediums (are formulated ibid, pH6.8) 1.5ml and are put in 2ml centrifuge tubes, QH1-1135 single bacterium colonies are accessed,
5 DEG C of cultures, respectively at 7 days, 10 days, determine the pH value of filtrate with pH acidometers at 14 days.Experiment is repeated 3 times, as a result with average
The form of value is represented.
As a result it is as shown in table 2, it is seen that cryogenic conditions (5 DEG C) Liquid Culture 7-14 days, Leuconostoc mesenteroides
(Leuconostoc mesenteroides)QH1-1135CCTCC NO：M 2015256 supernatant pH drops to 3.5, table respectively
Its bright acid producing ability is preferable.
The growth characteristics of the bacterial strain QH1-1135 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 Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC
NO：M 2015256 growth adaptation is very capable, is resistant to extreme environment Survival Reproduction.
The Antibacterial Activity of embodiment 3, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135
For examination bacterium：Micrococcus luteus, micrococcus luteus, salmonella and Escherichia coli.
1st, by Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO of activation：M
2015256 are inoculated in MRS fluid nutrient mediums (being formulated ibid, pH6.8), and 30 DEG C of culture 48h, zymotic fluid is centrifuged through 10000rpm
5min, takes supernatant to be 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, the Leuconostoc mesenteroides (Leuconostoc that step 1 is obtained is added in cup
mesenteroides)QH1-1135CCTCC NO：The zymotic fluids of M 2015256.Fill it up with rearmounted 37 DEG C to cultivate 16-18 hours, observation
As a result, inhibition zone dipstick metering.Control is used as using MRS fluid nutrient mediums substitution zymotic fluid.Experiment is repeated 3 times, as a result with average value
Form represent.
It is as a result as shown in table 3, it is seen that：Under 30 DEG C of condition of culture, Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：M 2015256 has to micrococcus luteus and salmonella preferably to be suppressed to make
With.
The bacterial strain QH1-1135 of table 3 bacteriostatic activity
Note：Antibacterial circle diameter includes Oxford cup external diameter (7.8mm)；"-" indicates no inhibition zone.
Embodiment 4, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135 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 Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M 2015256
Oat ensilage is prepared as feed addictive
(1) Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M 2015256
The preparation of bacteria suspension
Under aseptic condition, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO are taken：
M 2015256 is inoculated in 30 DEG C of overnight incubations in MRS fluid nutrient mediums, obtains Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：Leuconostoc mesenteroides in the bacteria suspensions of M 2015256, bacteria suspension
(Leuconostoc mesenteroides)QH1-1135CCTCC NO：M 2015256 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 Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M2015256
Oat ensilage is prepared as feed addictive
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, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1- that step (1) will be obtained
1135CCTCC NO：The μ l of 2015256 every bag of bacteria suspensions of M 100 are separately added into bag silo, are mixed, are taken out very using vacuum packing machine
Seal, be put into 5 DEG C of refrigerator-freezers after sky.
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 the conditions of low temperature (5 DEG C), the bright string of goldbeater's skin is added in ensiling the 1st day and ensiling the 3rd day
Pearl bacterium (Leuconostoc mesenteroides) QH1-1135CCTCC NO：The ensiling oats of M 2015256 compared with the control,
Lactic acid bacterium number increases sharply, and Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC
NO：The addition groups of M 2015256 are in the 1st day no detection bacillus of ensiling, in ensiling the 3rd day no detection mould, saccharomycete
And clostridium, the miscellaneous bacterias such as Escherichia coli are not detected within the 7th day in ensiling, and the harmful miscellaneous bacteria detection value volume and range of product of control group is more.
Consolidated statement 3, shows under the conditions of low temperature ensiling, adds Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-
1135CCTCC NO：The poles of M 2015256 significantly reduce ensilage pH, can effectively suppress Escherichia coli, and bacillus is mould
The growth of harmful miscellaneous bacteria such as bacterium, saccharomycete and clostridium.
The microbiology turbidity (logCFU/g) of oat during the low temperature ensiling of table 4 (5 DEG C)a
Note：aMean+SD (n=3).ND represents not detect.
3rd, the pH of oat, free water, dry weight and the measure of crude ash 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.
3rd, 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.
It is as a result as shown in table 5, it is seen that：In whole low temperature (5 DEG C) ensilage, Leuconostoc mesenteroides is added
(Leuconostoc mesenteroides)QH1-1135CCTCC NO：M 2015256 oat ensilage pH is extremely notable
Lower than without the control group (P≤0.01) of addition microbial inoculum；Add Leuconostoc mesenteroides (Leuconostoc mesenteroides)
QH1-1135CCTCC NO：M 2015256 ensilage, in ensiling the 30th day, pH fell below 4.26 and is very beneficial to green grass or young crops
The level of storage.Show under 5 DEG C of low temperature environments, add Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-
1135CCTCC NO：Ensiling oat pH effectively can be reduced to beneficial to ensiling level rapidly by M 2015256.With control group phase
Than adding Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M 2015256 can be with
Effectively the coarse ash non-nutritive composition crude ash content of reduction ensilage, does not have notable shadow to free moisture and dry weight content
Ring.
The pH of oat, free water, dry weight and crude ash 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 crude fat of oat in low temperature ensilage, crude protein, neutral detergent fiber and acid detergent fiber change
Determine
1st, 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.
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 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.
4th, 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：Under the conditions of low temperature (5 DEG C) ensiling, Leuconostoc mesenteroides (Leuconostoc
mesenteroides)QH1-1135CCTCC NO：M 2015256 does not show to the crude fat and crude protein content of ensiling oat
Influence is write, except the 3rd day, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M
Outside 2015256 addition groups neutral detergent fiber content compared with control group is significantly reduced, in remaining ensiling period, the bright beading of goldbeater's skin
Bacterium (Leuconostoc mesenteroides) QH1-1135CCTCC NO：M 2015256 is fine to the acidic cleaning of ensiling oat
Peacekeeping neutral detergent fiber has no significant effect.In a word, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-
1135CCTCC NO：The addition groups of M 2015256 are in 5 DEG C of low temperature ensilages, crude fat, crude protein, acid detergent fiber and
Neutral detergent fiber composition retains preferably.
The crude fat of oat, crude protein change, neutral detergent fiber and acidic cleaning during the low temperature ensiling of table 6 (5 DEG C)
Fiber (%/fresh weight)a
Note：aMean+SD (n=3)；" NS " represents difference not significantly (p ＞ 0.05)；" * " represents significant difference
(p≤.05).NDF is neutral detergent fiber；ADF is acid detergent fiber.
In summary, it is seen that Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-1135CCTCC NO：
M 2015256 is rapid during low temperature ensiling (5 DEG C) to be bred and produces acid drop pH, the effective growth or production for suppressing harmful miscellaneous bacteria
Raw, the nutritional ingredient such as crude protein, crude fat, crude fibre is effectively retained, non-nutritious matter such as coarse ash constituent reduction, is reached long-term
Preserve the effect of ensilage.
Claims (9)
1. Leuconostoc mesenteroides（Leuconostoc mesenteroides）QH1-1135, it is in China typical culture collection
The deposit number at center is CCTCC NO：M 2015256.
2. a kind of microbial inoculum, it is characterised in that：The active component of the microbial inoculum is the Leuconostoc mesenteroides described in claim 1
（Leuconostoc mesenteroides）QH1-1135.
3. a kind of silage additive, it is characterised in that：The active component of the silage additive is claim 1 institute
The Leuconostoc mesenteroides stated（Leuconostoc mesenteroides）QH1-1135.
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 Leuconostoc mesenteroides described in ensiling raw material and claim 1（Leuconostoc mesenteroides）QH1-
1135 mixing, carry out solid anaerobic fermentation, collect all tunnings, obtain the ensilage；
The ensiling raw material is specially oat complete stool；
The oat complete stool and the Leuconostoc mesenteroides（Leuconostoc mesenteroides）QH1-1135 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 Leuconostoc mesenteroides described in claim 1（Leuconostoc mesenteroides）QH1-1135 is appointing as follows
Application in one：
（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 Leuconostoc mesenteroides（Leuconostoc mesenteroides）The suppression that is suppressed to 30 DEG C under the conditions of of the QH1-1135 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：Ensiling raw material and goldbeater's skin described in claim 1 is bright
Beading bacterium（Leuconostoc mesenteroides）QH1-1135 is mixed, and carries out solid anaerobic fermentation, collects all fermentation productions
Thing, obtains the ensilage；
The ensiling raw material is specially oat complete stool；
The oat complete stool and the Leuconostoc mesenteroides（Leuconostoc mesenteroides）QH1-1135 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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| JP6875041B2 (en) * | 2016-07-15 | 2021-05-19 | シージェイ チェイルジェダン コーポレーションＣｊ Ｃｈｅｉｌｊｅｄａｎｇ Ｃｏｒｐｏｒａｔｉｏｎ | Lyconostock mesenteroides CJLM627 strain with low gas generation and kimchi production method using this strain |
| JP6760677B2 (en) * | 2016-07-15 | 2020-09-23 | シージェイ チェイルジェダン コーポレーションＣｊ Ｃｈｅｉｌｊｅｄａｎｇ Ｃｏｒｐｏｒａｔｉｏｎ | Ryconostock mesenteroides CJLM181 strain with low gas generation and kimchi production method using this strain |
| CN107034159B (en) * | 2017-04-27 | 2021-06-08 | 长春中俄科技园股份有限公司 | Pathogenic bacteria antagonist and application thereof |
| CN109456914A (en) * | 2018-11-19 | 2019-03-12 | 江苏渔多多生物科技有限公司 | One kind low temperature resistant lactic acid bacteria used for aquiculture and its application |
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| CN101886043A (en) * | 2010-06-24 | 2010-11-17 | 郑州大学 | Composite yoghurt starter and application thereof |
| CN101974450A (en) * | 2010-09-13 | 2011-02-16 | 郑州大学 | Leuconostoc mesenteroides and application thereof |
| KR101302465B1 (en) * | 2011-04-20 | 2013-09-02 | 조선대학교산학협력단 | Lactic acid bacterium separated from kimchii and fermented food using the strain |
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| CN101886043A (en) * | 2010-06-24 | 2010-11-17 | 郑州大学 | Composite yoghurt starter and application thereof |
| CN101974450A (en) * | 2010-09-13 | 2011-02-16 | 郑州大学 | Leuconostoc mesenteroides and application thereof |
| KR101302465B1 (en) * | 2011-04-20 | 2013-09-02 | 조선대학교산학협력단 | Lactic acid bacterium separated from kimchii and fermented food using the strain |
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