CN104894030B - One plant of Lactococcus lactis subsp. lactis and its application in low temperature ensiling - Google Patents
One plant of Lactococcus lactis subsp. lactis and its application in low temperature ensiling Download PDFInfo
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Abstract
The invention discloses one plant of Lactococcus lactis subsp. lactis and its application in low temperature ensiling.Lactococcus lactis subsp. lactis provided by the present invention is specially Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1 666, and it is CCTCC NO in the deposit number of China typical culture collection center：M 2015255.Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1 666CCTCC NO provided by the present invention：M 2015255 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, and reach the long-term effect for preserving ensilage.
Description
Technical field
The invention belongs to microorganism field, it is related to one plant of Lactococcus lactis subsp. lactis and its answering in low temperature ensiling
With.
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.
Wheat stalk is that cold district Winter-Spring raises the forage crop mended, because cold district temperature is low, limitation lactic acid bacteria
Growth, 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 wheat stalk 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 Lactococcus lactis subsp. lactis.
Lactococcus lactis subsp. lactis provided by the present invention is specially Lactococcus lactis subsp. lactis (Lactococcus
Lactis subsp.lactis) QH1-666, the bacterial strain is preserved in China typical culture collection on April 28th, 2015
(abbreviation CCTCC, address is the heart：Bayi Road No. 299 Wuhan Universitys in Wuhan City, Hubei Province Wuchang District are in the school in Wuhan University's preservation
The heart), its deposit number is CCTCC NO：M 2015255.
Lactococcus lactis subsp. lactis (the Lactococcus lactis subsp.lactis) QH1-666 is from China
It is isolated that Qinghai Province perceives county town rape, and the bacterial strain single bacterium colony is circle, and Gram-negative does not produce hydrogen peroxide；
The bacterial strain can not glucose fermentation aerogenesis, be homofermentation.Grown fine at 5 DEG C and 10 DEG C, 45 DEG C of ambient growths are faint,
50 DEG C do not grow, and show that the bacterial strain has good low temperature resistant growth ability.The bacterial strain is dense in the NaCl of 3.00% and 6.50%
Equal well-grown is faint in degree, shows that the bacterial strain has general salt resistance ability.5 DEG C of liquid is cultivated 7 days, the supernatant of the bacterial strain
PH drops to 4.0, and culture drops to 3.5 to pH at 10-14 days, shows that its acid producing ability is preferable.Environment of the bacterial strain in pH4.0-9.0
Middle well-grown, shows that the bacterial strain has preferable acid and alkali-resistance growth ability.The institute of sequence 1 in its 16S rDNA sequence such as sequence table
Show.
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 Lactococcus lactis subsp. lactis (Lactococcus
lactis subsp.lactis)QH1-666。
The microbial inoculum except comprising as activity into the Lactococcus lactis subsp. lactis (Lactococcus lactis
Subsp.lactis) outside QH1-666, 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) 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 Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255.
Fourth object of the present invention is to provide a kind of ensilage.
Contain the silage additive in ensilage provided by the present invention.
Lactococcus lactis subsp. lactis (the Lactococcus lactis subsp.lactis) QH1-666CCTCC
NO：M 2015255 or described microbial inoculums 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, Lactococcus lactis subsp. lactis (the Lactococcus lactis subsp.lactis) QH1-
666CCTCC NO：Suppression of the M 2015255 or described microbial inoculums to the bacterium is specially 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 lactic acid
Galactococcus lactic acid subspecies (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO：M 2015255 is mixed,
Solid anaerobic fermentation is carried out, all tunnings is collected, obtains the ensilage；
In the present invention, the ensiling raw material is wheat stalk；Specially milk stage wheat stalk, moisture
73.27%, cut into 1-2cm segments.
In the process, the wheat stalk and the Lactococcus lactis subsp. lactis (Lactococcus lactis
subsp.lactis)QH1-666CCTCC NO：M 2015255 proportioning can be 100g：105cfu；The fermentation is low temperature hair
Ferment；The low temperature can be 5 DEG C；The time of the fermentation can be 30 days.
Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1- provided by the present invention
666CCTCC NO：M 2015255 has the resistance such as low temperature resistant, salt tolerant, acid and alkali-resistance, and fast during low temperature ensiling (5 DEG C)
Speed is bred and produces acid drop pH, the effective growth or generation for suppressing harmful miscellaneous bacteria, the nutrition such as thick protein, crude fat, crude fibre
Composition is effectively retained, and reaches the long-term effect for preserving ensilage.
Preservation explanation
Strain name：Lactococcus lactis subsp. lactis
Latin name：(Lactococcus lactis subsp.lactis)
Strain number：QH1-666
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 2015255
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.
Embodiment 1, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666
Separation and identification
First, bacterial strain QH1-666 separation and screening
Take Qinghai Province to perceive county town rape 10g, be put into 90mL sterilized waters, shake 10 seconds, draw 1mL liquid and be 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 agar mediums, 30 DEG C
Anaerobic culturel 48 hours, takes single bacterium colony MRS solid mediums to expand culture, is QH1-666 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 QH1-666 identification
Obtained bacterial strain QH1-666 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 QH1-666 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 QH1-666 Gram-negatives, does not produce hydrogen peroxide, it is impossible to send out
Ferment glucose aerogenesis, is homofermentation.
Fermentation situations of the bacterial strain QH1-666 to different carbon source is detected using API 50CH.As a result it is as shown in table 1.It can be seen that, bacterium
Strain QH1-666 can utilize amygdalin, ursin, aesculin, salicin, cellobiose, maltose, lactose, glucose, core
Sugar, fructose, L- arabinoses, sucrose, melibiose, trehalose, D- xyloses, galactolipin, mannose, mannitol is cooked carbon source and sent out
Ferment；Can not utilize D- lyxoses, D-R alcohol, glycogen, xylitol, melezitose, sorbierite, D-R, L- I
Uncle's sugar, inosite, L- xyloses, ribitol, sorbose, galactitol, Beta-methyl-xyloside, glycerine, antierythrite, 2- ketone groups-
Gluconate, inulin, glycogen, D- trehaloses, 5- ketone groups-gluconate, rhamnose, L- trehaloses, Alpha-Methyl-D-MANNOSE,
Gossypose, D- turanoses, D-Tag, Alpha-Methyl-D- glucosides do carbon source and fermented；Can be with faint utilization starch, rough gentian
Disaccharides, gluconate does carbon source and fermented.
Fermentation situations of the bacterial strain QH1-666 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 QH1-666 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 QH1-666 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
Bacterial strain QH1-666 be accredited as Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis), and in
It is preserved on April 28th, 2015 and was preserved in China typical culture collection center on April 28 (abbreviation CCTCC, address is：Hubei
No. 299 Wuhan Universitys of wuchang, wuhan area Bayi Road of province in the school, Wuhan University's collection, postcode 430072), its deposit number
For CCTCC NO：M 2015255, its Classification And Nomenclature is Lactococcus lactis subsp. lactis (Lactococcus lactis
subsp.lactis)QH1-666。
Embodiment 2, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666 exist
Growth under different temperatures, pH and salinity
1st, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1- under different temperatures
666 growth
By Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1- of activation
666CCTCC NO：M 2015255 is inoculated in MRS fluid nutrient mediums, incubated 5, at a temperature of 10,45 and 50 DEG C respectively
24h, with absorbance value (OD600) at spectrophotometric determination 600nm, to observe Lactococcus lactis subsp. lactis under different temperatures
(Lactococcus lactis subsp.lactis) QH1-666 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 Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis)
QH1-666CCTCC NO：M 2015255 grows fine at 5 DEG C and 10 DEG C, and 45 DEG C of ambient growths are faint, can not be grown at 50 DEG C,
Show that the bacterial strain has good low temperature resistant growth ability.
2nd, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666 under difference pH
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 Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255, is cultivated in 24h, incubation
Not acid adjustment, it is sub- to observe Lactococcus lactis under different pH with absorbance value (OD600) at spectrophotometric determination 600nm
Plant (Lactococcus lactis subsp.lactis) QH1-666 growing state.
As a result it is as shown in table 2, it is seen that Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis)
QH1-666CCTCC NO：The well-growns in pH4.0-9.0 environment of M 2015255, show that the bacterial strain has preferable acid and alkali-resistance
Growth ability.
3rd, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1- under different salinity
666 growth
By Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1- of activation
666CCTCC NO：M 2015255 is inoculated in the MRS liquid that the weight/mass percentage composition containing NaCl is 3% and 6.5% and trained respectively
Base (being formulated ibid, pH6.8) is supported, in 30 DEG C of constant temperature quiescent culture 24h, with absorbance value at spectrophotometric determination 600nm
(OD600), to observe Lactococcus lactis subsp. lactis under different salinity (Lactococcus lactis subsp.lactis)
QH1-666 growing state.
As a result it is as shown in table 2, it is seen that Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis)
QH1-666CCTCC NO：The equal well-growns in 3.00% and 6.50% NaCl concentration of M 2015255 are faint, show the bacterium
Strain has general salt resistance ability.
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, QH1-666 single bacterium colonies, 5 is accessed
DEG C culture, respectively at 7 days, 10 days, at 14 days with pH acidometers determine filtrate pH value.Experiment is repeated 3 times, as a result with average value
Form represent.
As a result it is as shown in table 2, it is seen that cryogenic conditions (5 DEG C) Liquid Culture 7 days, Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 supernatant pH drops to 4.0,
PH drops to 3.5 when cultivating 10-14 days, shows that its acid producing ability is preferable.
The growth characteristics of the bacterial strain QH1-666 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 Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis)
QH1-666CCTCC NO：M 2015255 growth adaptation is very capable, is resistant to extreme environment Survival Reproduction.
Embodiment 3, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666
Antibacterial Activity
For examination bacterium：Micrococcus luteus, micrococcus luteus, salmonella and Escherichia coli.
1st, by Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1- of activation
666CCTCC NO：M 2015255 is inoculated in MRS fluid nutrient mediums (being formulated ibid, pH6.8), 30 DEG C of culture 48h, zymotic fluid
5min is centrifuged through 10000rpm, 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, Lactococcus lactis subsp. lactis (the Lactococcus lactis that step 1 is obtained are added in cup
subsp.lactis)QH1-666CCTCC NO：The zymotic fluids of M 2015255.Fill it up with rearmounted 37 DEG C to cultivate 16-18 hours, observation knot
Really, 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 is represented.
It is as a result as shown in table 3, it is seen that：Under 30 DEG C of condition of culture, Lactococcus lactis subsp. lactis (Lactococcus
lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 has preferable suppression to micrococcus luteus and salmonella
Make and use.
The bacterial strain QH1-666 of table 3 bacteriostatic activity
Note：Antibacterial circle diameter includes Oxford cup external diameter (7.8mm)；"-" indicates no inhibition zone.
Embodiment 4, Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666 are blue or green
Store wheat stalk
Ensiling raw material：Milk stage wheat stalk, moisture 73.27% cuts into 1-2cm segments.
First, wheat stalk ensilage is prepared
1st, with Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC
NO：M 2015255 prepares wheat stalk ensilage as feed addictive
(1) Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC
NO：The preparation of the bacteria suspensions of M 2015255
Under aseptic condition, extracting lactic acid galactococcus lactic acid subspecies (L.lactis subsp.lactis) QH1-666CCTCC M
2015255 are inoculated in 30 DEG C of overnight incubations in MRS fluid nutrient mediums, obtain Lactococcus lactis subsp. lactis (Lactococcus
lactis subsp.lactis)QH1-666CCTCC NO：Lactococcus lactis is sub- in the bacteria suspensions of M 2015255, bacteria suspension
Plant (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO：M 2015255 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 Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC
NO：M 2015255 prepares wheat stalk ensilage as feed addictive
Fringe will be spent after milk stage small wheat harvesting, chopper is cut into 1-2cm or so length (moisture 73.27%), taken
100g is put into bag silo, Lactococcus lactis subsp. lactis (the Lactococcus lactis that step (1) is obtained
subsp.lactis)QH1-666CCTCC NO：The μ l of 2015255 every bag of bacteria suspensions of M 100 are separately added into bag silo, are mixed, profit
Vacuum packing machine sealed after being vacuumized is used, is put into 5 DEG C of refrigerator-freezers.
2nd, wheat stalk ensilage is compareed
Fringe will be spent after milk stage small wheat harvesting, chopper is cut into 1-2cm or so length (moisture 72.37%), taken
100g is put into bag silo, and every bag of 100 μ l of sterilized water are separately added into bag silo, is mixed, is vacuumized using vacuum packing machine
After seal, be put into 5 DEG C of refrigerator-freezers.
2nd, the microbiology turbidity of wheat stalk 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), Lactococcus lactis subsp. lactis is added in ensiling the 1st day
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 ensiling wheat stalk with it is right
Compared according to group, high 6 orders of magnitude of lactic acid bacterium number；Ensiling the 3rd and the 7th day, add Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 ensiling wheat stalk with it is right
Compared according to group, high 3 orders of magnitude of lactic acid bacterium number；In ensiling the 3rd day and the 7th day, Lactococcus lactis subsp. lactis is added
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：In M 2015255 ensiling wheat stalk
Escherichia coli quantity pole was substantially less than control group (P≤0.01), in ensiling the 30th day, added Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 ensilage is not detected
Escherichia coli；Ensiling the 7th day, addition Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis)
QH1-666CCTCC NO：M 2015255 ensilage does not detect bacillus.In a word, breast is added in ensiling wheat stalk
Yogurt coccus lactic acid subspecies (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO：M 2015255, breast
Yogurt coccus lactic acid subspecies (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO：M 2015255 is fast
The a length of dominant strain of fast-growing, can effectively suppress Escherichia coli, the growth of harmful miscellaneous bacteria such as bacillus.
The microbiology turbidity (logCFU/g) of wheat stalk during the low temperature ensiling of table 4 (5 DEG C)a
Note：aMean+SD (n=3).ND represents not detect.Same file contains different capitalization generations
Difference opposite pole is significantly (P≤0.01)；Same file contains different lowercase letter indication differences significantly (P≤0.05).
3rd, the measure that the pH of wheat stalk, 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 whole low temperature (5 DEG C) ensilage, Lactococcus lactis subsp. lactis is added
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 wheat stalk ensilage
PH poles do not add the control group (P≤0.01) of microbial inoculum substantially less than, add Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 ensilage, in ensiling
PH falls below 3.79 level for being very beneficial to ensiling at 7 days, shows under 5 DEG C of low temperature environments, adds Lactococcus lactis
Subspecies (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO：M 2015255 can be effective rapidly
Ensiling wheat stalk pH is reduced to beneficial to ensiling level.Compared with control group, Lactococcus lactis subsp. lactis is added
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 contains to free moisture and dry weight
Amount has no significant effect, and shows that ensiling wheat stalk remains the fresh and tender succulence of feed.
The pH of wheat stalk, 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 measure that the crude fat of wheat stalk, crude protein and neutral detergent fiber change in low temperature ensilage
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.
It is as a result as shown in table 6, it is seen that：In whole low temperature (5 DEG C) ensilage, Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis)QH1-666CCTCC NO：M 2015255 is to wheat stalk ensilage
Crude protein and neutral detergent fiber content have no significant effect；Ensiling the 1st day and ensiling the 3rd day, ensiling wheat stalk it is thick
Fat content changes big rise and fall, remaining ensiling time addition Lactococcus lactis subsp. lactis (Lactococcus lactis
subsp.lactis)QH1-666CCTCC NO：M 2015255 experimental group no significant changes compared with control group.Exist in a word
In 5 DEG C of low temperature ensilages, crude protein, neutral detergent fiber and crude fat composition retain preferably.
During the low temperature ensiling of table 6 (5 DEG C) crude fat of wheat stalk, crude protein and neutral detergent fiber change (%/
Fresh 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.
In summary, it is seen that Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) QH1-
666CCTCC NO：M 2015255 is rapid during low temperature ensiling (5 DEG C) to be bred and produces acid drop pH, and effective suppression is harmful miscellaneous
The growth or generation of bacterium, the nutritional ingredient such as crude protein, crude fat, crude fibre are effectively retained, and reach the long-term ensilage that preserves
Effect.
Claims (9)
1. Lactococcus lactis subsp. lactis（Lactococcus lactis subsp. lactis）QH1-666, it is in Chinese allusion quotation
Type culture collection deposit number is CCTCC NO：M 2015255.
2. a kind of microbial inoculum, it is characterised in that：The active component of the microbial inoculum is that the Lactococcus lactis described in claim 1 is sub-
Kind（Lactococcus lactis subsp. lactis）QH1-666.
3. a kind of silage additive, it is characterised in that：The active component of the silage additive is claim 1 institute
The Lactococcus lactis subsp. lactis stated（Lactococcus lactis subsp. lactis）QH1-666.
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 Lactococcus lactis subsp. lactis described in ensiling raw material and claim 1（Lactococcus
lactis subsp. lactis）QH1-666 is mixed, and carries out solid anaerobic fermentation, is collected all tunnings, is obtained the green grass or young crops
Store feed；
The ensiling raw material is specially wheat stalk；
The wheat stalk and the Lactococcus lactis subsp. lactis（Lactococcus lactis subsp. lactis）
QH1-666 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 Lactococcus lactis subsp. lactis described in claim 1（Lactococcuslactis subsp. lactis）QH1-
666 or claim 2 described in microbial inoculum it is following it is any in application：
（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 Lactococcus lactis subsp. lactis（Lactococcus
lactis subsp. lactis）The suppression that is suppressed to 30 DEG C under the conditions of of the QH1-666 or described microbial inoculums 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 lactic acid breast described in ensiling raw material and claim 1
Coccus lactic acid subspecies（Lactococcus lactis subsp. lactis）QH1-666 is mixed, and carries out solid anaerobic fermentation, is received
Collect all tunnings, obtain the ensilage；
The ensiling raw material is specially wheat stalk；
The wheat stalk and the Lactococcus lactis subsp. lactis（Lactococcus lactis subsp. lactis）
QH1-666 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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| CN106282053B (en) * | 2016-08-10 | 2019-08-06 | 江南大学 | One plant of acidproof Lactococcus lactis and its application |
| CN112515045A (en) * | 2020-11-30 | 2021-03-19 | 成都理工大学 | Silage additive for pasture silage |
| CN113832072B (en) * | 2021-10-20 | 2023-10-27 | 江南大学 | Lactococcus lactis subspecies lactate with inulin utilization capacity and application thereof |
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