CN104894030A - Lactococcus lactis subsp.lactis and application of lactococcus lactis subsp.lactis in low-temperature silage - Google Patents
Lactococcus lactis subsp.lactis and application of lactococcus lactis subsp.lactis in low-temperature silage Download PDFInfo
- Publication number
- CN104894030A CN104894030A CN201510340152.0A CN201510340152A CN104894030A CN 104894030 A CN104894030 A CN 104894030A CN 201510340152 A CN201510340152 A CN 201510340152A CN 104894030 A CN104894030 A CN 104894030A
- Authority
- CN
- China
- Prior art keywords
- lactis
- lactococcus lactis
- lactis subsp
- subsp
- silage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses lactococcus lactis subsp.lactis and an application of the lactococcus lactis subsp.lactis in low-temperature silage. The lactococcus lactis subsp.lactis is specifically lactococcus lactis subsp.lactis QH1-666, and the collection number of the lactococcus lactis subsp.lactis in the CCTCC (China center for type culture collection) is CCTCC No: M 2015255. The lactococcus lactis subsp.lactis QH1-666 with the CCTCC NO: M 2015255 has stress resistance such as low-temperature resistance, salt resistance, acid-base resistance and the like, can quickly proliferate at the temperature of 5 DEG C in the low-temperature silage process and produce acid to reduce pH (potential of hydrogen), growth or generation of harmful infectious microbes is effectively inhibited, nutritional ingredients such as crude protein, crude fat, crude fiber and the like are effectively retained, and the effect of long-term storage of silage fodder is realized.
Description
Technical field
The invention belongs to microorganism field, relate to a strain Lactococcus lactis subsp.lactis and the application in low temperature ensiling thereof.
Background technology
Ensiling be under the anaerobic digestion of milk-acid bacteria by carbohydrate-modifying for organic acid, thus make pH reduce the object reaching long storage periods.Milk-acid bacteria and temperature are the deciding factors of silage fermatation quality quality.
Wheat stalk is the fodder crop that benefit is raised in Winter-Spring, cold district, because cold district temperature is low, the growth of restriction milk-acid bacteria, very difficult ferment best in quality and can the feed of long storage periods.
Therefore, filter out the stronger bacterial strain of resistance of high-low temperature resistant, salt tolerant, acid and alkali-resistance, and it can be used as starter to add in low temperature ensiling wheat stalk feed, huge using value will be had to the silage making of extremely frigid zones.
Summary of the invention
First object of the present invention is to provide a strain 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, this bacterial strain is preserved in China typical culture collection center on April 28th, 2015 and (is called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys Wuhan University's preservation center in the school), its deposit number is CCTCC NO:M 2015255.
Described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 perceives county town rape and is separated and obtains from Qinghai Province, this bacterial strain list bacterium colony be circle, Gram-negative, does not produce hydrogen peroxide; This bacterial strain can not glucose fermentation aerogenesis, is homofermentation.Grow fine 5 DEG C and 10 DEG C, 45 DEG C of ambient growth are faint, do not grow at 50 DEG C, show that this bacterial strain all has good low temperature resistant energy for growth.This bacterial strain equal well-grown in the NaCl concentration of 3.00% and 6.50% is faint, shows that this bacterial strain has general salt resistance ability.Liquid 5 DEG C is cultivated 7 days, and the supernatant liquor pH of this bacterial strain drops to 4.0, cultivates and drops to 3.5 to pH during 10-14 days, show that its acid producing ability is better.This bacterial strain is well-grown in the environment of pH4.0-9.0, shows that this bacterial strain has good acid and alkali-resistance energy for growth.Its 16S rDNA sequence is as shown in sequence in sequence table 1.
Second object of the present invention is to provide a kind of microbial inoculum.
The activeconstituents of microbial inoculum provided by the present invention is described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666.
Described microbial inoculum is except comprising described Lactococcus lactis subsp.lactis (the Lactococcus lactis subsp.lactis) QH1-666 as activity one-tenth, also auxiliary material can be comprised, as MRS solid medium, (solvent is water, solute and concentration as follows: peptone 10g/L, extractum carnis 10g/L, yeast extract paste 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, manganous sulfate 0.25g/L, agar 17g/L) etc.
3rd object of the present invention is to provide a kind of silage additive.
The activeconstituents of silage additive provided by the present invention is described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255.
4th object of the present invention is to provide a kind of silage.
Containing described silage additive in silage provided by the present invention.
The application in arbitrary as follows of described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 or described microbial inoculum also belongs to protection scope of the present invention:
(a1) anti-bacteria;
(a2) bacterial inhibitor is prepared;
(a3) described silage additive is prepared;
(a4) described silage is prepared.
The application being applied as non-diseases diagnosis or treatment of described (a1).
In described (a1) and described (a2), described bacterium specifically can be micrococcus luteus and/or Salmonellas.
Wherein, described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 or the suppression of described microbial inoculum to described bacterium are specially the suppression under 30 DEG C of conditions.
The application of described silage additive in the described silage of preparation also belongs to protection scope of the present invention.
5th object of the present invention is to provide a kind of method preparing described silage.
The method of the described silage of preparation provided by the present invention, specifically can comprise: ensiling raw material is mixed with described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255, carry out solid anaerobic fermentation, collect all tunnings, obtain described silage;
In the present invention, described ensiling raw material is wheat stalk; Be specially milk stage wheat stalk, moisture content 73.27%, cut into 1-2cm segment.
In the process, the proportioning of described wheat stalk and described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 can be 100g:10
5cfu; Described fermentation is low temperature fermentation; Described low temperature can be 5 DEG C; The time of described fermentation can be 30 days.
Lactococcus lactis subsp.lactis provided by the present invention (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 has low temperature resistant, the resistance such as salt tolerant, acid and alkali-resistance, and rapidly breed and produce acid in low temperature ensiling (5 DEG C) process and fall pH, effective suppression is harmful to growth or the generation of miscellaneous bacteria, the nutritive ingredients such as crude protein, crude fat, robust fibre effectively retain, and reach long-term effect of preserving silage.
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 called for short: CCTCC
Address: Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys Wuhan University's preservation center in the school
Preservation date: on April 28th, 2015
Register on the books numbering: CCTCC NO:M 2015255 at preservation center
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The Isolation and ldentification of embodiment 1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666
One, the isolation and screening of bacterial strain QH1-666
Get Qinghai Province and perceive county town rape 10g, put into 90mL sterilized water, shake 10 seconds, draw 1mL liquid and be put in 1.5mL centrifuge tube, dilute 10 successively
1, 10
3, 10
5doubly, get diluted liquid 20 μ L and be applied on MRS nutrient agar respectively, 30 DEG C of Anaerobic culturel 48 hours, get single bacterium colony MRS solid medium enlarged culturing, the wherein strain bacterium obtained are numbered QH1-666.
Wherein, the solvent of described MRS solid medium is water, solute and concentration as follows: peptone 10g/L, extractum carnis 10g/L, yeast extract paste 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, manganous sulfate 0.25g/L, agar 17g/L.
Two, the qualification of bacterial strain QH1-666
From the following aspects step one is separated and screens the bacterial strain QH1-666 obtained and identify.
1, Morphological Identification
Step one is separated and after screening the mono-bacterium colony gramstaining of the bacterial strain QH1-666 obtained, basis of microscopic observation list colony shape is rounded.
2, physiological and biochemical property qualification
Step one is separated and screens the bacterial strain QH1-666 Gram-negative obtained, and does not produce hydrogen peroxide, can not glucose fermentation aerogenesis, is homofermentation.
API 50CH is utilized to detect bacterial strain QH1-666 to the fermentation situation of different carbon source.Result is as shown in table 1.Visible, bacterial strain QH1-666 can utilize Vitamin B17, arbutin, polychrom, saligenin, cellobiose, maltose, lactose, glucose, ribose, fructose, L-arabinose, sucrose, melibiose, trehalose, D-wood sugar, semi-lactosi, seminose, N.F,USP MANNITOL is cooked carbon source and ferments; D-lyxose can not be utilized, D-R alcohol, glycogen, Xylitol, melizitose, sorbyl alcohol, D-R, L-arabinose, fibres sugars, L-wood sugar, ribitol, sorbose, melampyrum, Beta-methyl-xyloside, glycerine, erythritol, 2-ketone group-gluconate, inulin, glycogen, D-trehalose, 5-ketone group-gluconate, rhamnosyl, L-trehalose, Alpha-Methyl-D-MANNOSE, raffinose, D-turanose, D-Tag, Alpha-Methyl-D-glucoside does carbon source and ferments; Faintly can utilize starch, gentiobiose, gluconate does carbon source and ferments.
Table 1 bacterial strain QH1-666 is to the fermentation situation of different carbon source
Note: "+" represents positive, can utilize; "-" represents negative, namely can not utilize; " w " represents the weak positive, i.e. faint utilization.
3,16S rDNA sequence homology analysis
The genomic dna of the bacterial strain QH1-666 of extraction step one gained, with it for template, adopt bacterial universal primers to carry out pcr amplification, obtain the 16S rDNA fragment of bacterial strain QH1-666, and carry out sequencing, its sequence is sequence 1 in sequence table.Sequence 1 is carried out in GenBank database BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) sequence analysis, determine bacterial classification classification.
According to above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis result, the bacterial strain QH1-666 of step one gained is accredited as Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), and on April 28th, 2015 be preserved in be preserved in April 28 China typical culture collection center (be called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys in the school, Wuhan University's preservation center, postcode 430072), its deposit number is 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 (the Lactococcus lactis subsp.lactis) growth of QH1-666 under differing temps, pH and salt concn
1, the growth of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 under differing temps
Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) the QH1-666CCTCC NO:M 2015255 of activation is inoculated in MRS liquid nutrient medium, constant temperature culture 24h at 5,10,45 and 50 DEG C of temperature respectively, with spectrophotometric determination 600nm place absorbance value (OD600), to observe the growing state of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 under differing temps.
Wherein, the solvent of MRS liquid nutrient medium is water, solute and concentration as follows: peptone 10g/L, extractum carnis 10g/L, yeast extract paste 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, manganous sulfate 0.25g/L; PH6.8.
Result is as shown in table 2, visible Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 grows fine 5 DEG C and 10 DEG C, 45 DEG C of ambient growth are faint, can not grow at 50 DEG C, show that this bacterial strain all has good low temperature resistant energy for growth.
2, the growth of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 under different pH
With the hydrochloric acid of 1mol/L and the sodium hydroxide of 1mol/L, the initial pH of MRS liquid nutrient medium (filling a prescription the same) is adjusted to 3.0 respectively, 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, cultivate 24h, not acid adjustment in culturing process, with spectrophotometric determination 600nm place absorbance value (OD600), to observe the growing state of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 under different pH.
Result is as shown in table 2, visible Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 well-grown in the environment of pH4.0-9.0, shows that this bacterial strain has good acid and alkali-resistance energy for growth.
3, the growth of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 under different salt concn
The MRS liquid nutrient medium being 3% and 6.5% by Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 mass percentage be inoculated in respectively containing NaCl of activation (is filled a prescription the same, pH6.8), in 30 DEG C of constant temperature quiescent culture 24h, with spectrophotometric determination 600nm place absorbance value (OD600), to observe the growing state of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 under different salt concn.
Result is as shown in table 2, visible Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 equal well-grown in the NaCl concentration of 3.00% and 6.50% is faint, shows that this bacterial strain has general salt resistance ability.
4, low temperature (5 DEG C) cultivates results of regular determination pH change
MRS liquid nutrient medium (filling a prescription the same, pH6.8) 1.5ml is put in 2ml centrifuge tube, the mono-bacterium colony of access QH1-666,5 DEG C of cultivations, respectively at 7 days, 10 days, when 14 days, measures the pH value of filtrate with pH acidometer.Experiment repetition 3 times, result represents with the form of mean value.
Result is as shown in table 2, visible cold condition (5 DEG C) liquid culture 7 days, the supernatant liquor pH of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 drops to 4.0, cultivate and drop to 3.5 to pH during 10-14 days, show that its acid producing ability is better.
Table 2 bacterial strain QH1-666 growth characteristics under various circumstances
Note: "+" represents well-grown; " w " represent growth faint (OD600 is greater than 0.3 and is considered as well-grown, be greater than 0.2 be less than or equal to 0.3 for growth faint).
According to above result, the growth adaptation of known Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 is very capable, can the Survival Reproduction of resistance to extreme environment.
The Antibacterial Activity of embodiment 3, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666
For examination bacterium: micrococcus luteus, micrococcus luteus, Salmonellas and intestinal bacteria.
1, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) the QH1-666CCTCC NO:M 2015255 of activation is inoculated in MRS liquid nutrient medium and (fills a prescription the same, pH6.8) in, cultivate 48h for 30 DEG C, the centrifugal 5min of fermentation liquor 10000rpm, gets supernatant liquor for measuring.
2, adopt Oxford cup double-layer agar technique to measure its bacteriostatic activity, be heated to by sterilized nutrient agar and melt completely, be poured in culture dish, every ware 15ml (lower floor), treats that it solidifies.In addition, be cooled to by the PDA substratum of thawing about 50 DEG C to be mixed into for examination bacterium, the substratum 5ml being mixed with bacterium be added to (upper strata) to be solidified on the substratum solidified.Oxford cup (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm) is directly vertically put in media surface with aseptic technique, pressurize gently, make it contact tight with substratum, in cup, add Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 fermented liquid that step 1 obtains.Fill it up with rearmounted 37 DEG C and cultivate 16-18 hour, observations, inhibition zone dipstick metering.Fermented liquid is replaced in contrast with MRS liquid nutrient medium.Experiment repetition 3 times, result represents with the form of mean value.
Result is as shown in table 3, visible: under 30 DEG C of culture condition, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 pairs of micrococcus luteuses and Salmonellas have good restraining effect.
The bacteriostatic activity of table 3 bacterial strain QH1-666
Note: antibacterial circle diameter comprises Oxford cup external diameter (7.8mm); "-" indicates without inhibition zone.
Embodiment 4, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 ensiling wheat stalk
Ensiling raw material: milk stage wheat stalk, moisture content 73.27%, cuts into 1-2cm segment.
One, wheat stalk silage is prepared
1, wheat stalk silage is prepared using Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 as fodder additives
(1) preparation of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 bacteria suspension
Under aseptic condition, extracting lactic acid galactococcus lactic acid subspecies (L.lactis subsp.lactis) QH1-666CCTCC M 2015255 is inoculated in 30 DEG C of overnight incubation in MRS liquid nutrient medium, obtain Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 bacteria suspension, in bacteria suspension, the content of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 is 10
6cfu/ml.
Wherein, the solvent of MRS liquid nutrient medium is water, solute and concentration as follows: peptone 10g/L, extractum carnis 10g/L, yeast extract paste 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, manganous sulfate 0.25g/L; PH6.8.
(2) wheat stalk silage is prepared using Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 as fodder additives
Fringe is spent by after little for milk stage wheat harvesting, chopper is cut into about 1-2cm length (moisture content 73.27%), get 100g and put into bag silo, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 bacteria suspension every bag of 100 μ l step (1) obtained add in bag silo respectively, mixing, utilize Vacuum Packaging Machine sealed after being vacuumized, put into 5 DEG C of refrigerator-freezers.
2, wheat stalk silage is contrasted
Spend fringe by after little for milk stage wheat harvesting, chopper is cut into about 1-2cm length (moisture content 72.37%), gets 100g and puts into bag silo, sterilized water every bag 100 μ l is added in bag silo respectively, mixing, utilizes Vacuum Packaging Machine sealed after being vacuumized, puts into 5 DEG C of refrigerator-freezers.
Two, in low temperature ensilage, the microbiology turbidity of wheat stalk measures
Adopt plate dilution assay method microbiology turbidity.Joined in 90ml sterile distilled water by Feed Sample 10g, utilize vortex oscillator to shake 30s, 10 times of gradient dilutions, then get 10 respectively
-1, 10
-3with 10
-5times each 20 μ l of sample diluting liquid are coated on MRS (for detecting milk-acid bacteria), BLB (for detecting intestinal bacteria), PDA (for detecting yeast and mould, according to colonial morphology, naked eyes are distinguished, and mold colony is fine hair shape, cotton-shaped, spider reticulation, present the color of its spore, and yeast is smaller, shaft-like, spirrillum, spherical, bacterium colony smooth surface or thickness or drying) NA (for detecting aerobic bacteria) substratum, by 10
-1with 10
-2times sample diluting liquid 1ml is after 75 DEG C of water-bath water-bath 15min, get 20 μ l respectively and be coated on NA (for detecting genus bacillus) and CLO (for the detecting clostridium) substratum (formula of CLO substratum: containing peptone 15g in often liter of substratum, soy peptone 7.5g, yeast extract paste 7.5g, extractum carnis 7.5g, ferric ammonium citrate 1g, sodium bisulfite 1g, L-cysteine hydrochloride 0.75g, agar 15g, surplus is water) on, these coated substratum are inverted and are put in 30 DEG C of constant incubators cultivation 48h, wherein MRS and CLO substratum should be positioned over anaerobic culture box, other substratum is positioned over common constant incubator.Cultivating 48 hours postscript list bacterium colony numbers is n, bacterium colony (logCFU/g)=log
10[(n × 10)/20 × 10
-3].Experiment repetition 3 times, result represents with the form of mean+SD.
Result is as shown in table 4, visible: under low temperature (5 DEG C) condition, ensiling the 1st day, add the ensiling wheat stalk of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 compared with control group, high 6 orders of magnitude of lactic acid bacterium number; Ensiling the 3rd and the 7th day, add the ensiling wheat stalk of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 compared with control group, high 3 orders of magnitude of lactic acid bacterium number; Ensiling the 3rd day and the 7th day, the intestinal bacteria quantity of adding in the ensiling wheat stalk of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 is extremely remarkable in control group (P≤0.01), ensiling the 30th day, the silage adding Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 did not detect intestinal bacteria; Ensiling the 7th day, the silage adding Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 does not detect genus bacillus.In a word, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 is added in ensiling wheat stalk, Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 ramp is dominant strain, effectively can suppress intestinal bacteria, the growth of harmful miscellaneous bacteria such as genus bacillus.
The microbiology turbidity (logCFU/g) of wheat stalk in table 4 low temperature ensiling (5 DEG C) process
a
Note:
amean+SD (n=3).ND represents and does not detect.Same file contains different capitalization and represents otherness extremely significantly (P≤0.01); Same file contains different lowercase alphabet and shows significant difference (P≤0.05).
Three, the mensuration of the pH of wheat stalk, free-water and dry weight content change in low temperature ensilage
1, pH measuring method
Feed Sample 10g is joined in 90ml sterile distilled water, utilizes vortex oscillator to shake 30s, get liquid by filter paper filtering, utilize pH acidometer to measure the pH value of filtrate.Experiment repetition 3 times, result represents with the form of mean+SD.
2, the measuring method of free water content and dry weight content
65 DEG C, 48h desiccating method, first orders into a paper bag, analytical balance is weighed to paper bag by a blank sheet of paper, be designated as W1, Feed Sample is put into paper bag, claim gross weight, be designated as W2, constant temperature ventilates after 65 DEG C of dry 48h, is positioned in moisture eliminator and weighs after 30min, be designated as W3.Free water content (%)=(W2-W3)/(W2-W1) × 100.Dry weight (%)=100%-free water content (%).Experiment repetition 3 times, result represents with the form of mean+SD.
Result is as shown in table 5, visible: in whole low temperature (5 DEG C) ensilage, add all extremely remarkable control group (P≤0.01) lower than not adding microbial inoculum of wheat stalk silage pH of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255, add the silage of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255, when ensiling the 7th day, pH dropped to the level being extremely beneficial to ensiling of 3.79, show under 5 DEG C of low temperature environments, add Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 and effective being reduced to by ensiling wheat stalk pH can be beneficial to ensiling level rapidly.Compared with control group, interpolation Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 pairs of free water contents and dry weight content have no significant effect, and show 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) in table 5 low temperature ensiling (5 DEG C) process
a
Note:
amean+SD (n=3); " * " represents significant difference (P≤0.05); " NS " represents difference not significantly (p > 0.05).
Four, the mensuration of the crude fat of wheat stalk, crude protein and neutral detergent fiber change in low temperature ensilage
1, the mensuration of crude fat
Adopt aether extraction.Feed Sample is 3h extractive distillation crude fat in ether, and except neutral fat, phosphoric acid lipid, free fatty acids, fat-soluble pigment etc. are also included.
(1) fatty bottle is positioned in 100 DEG C of baking ovens after dry 2h, and taking-up is placed in moisture eliminator and cools 1h, weighs, is designated as W1.
(2) analytical balance takes 1g (W2) left and right Feed Sample and is placed on filter paper, is filled in fatty bucket by filter paper, block up with absorbent cotton after wrapping.
(3) the fatty bucket that Feed Sample is housed is put into fatty withdrawing device, connect fatty bottle, add about 50ml ether in fatty bottle bottle, open cooling water system, start more than 3h extracting.
(4) complete after extracting, take out fatty bucket, after fatty bottle being placed in 100 DEG C of baking oven 3h drying, taking-up is placed in moisture eliminator and cools 30m, weighs, is designated as W3.
Crude fat (%)=(W3-W1)/W2 × 100.Experiment repetition 3 times, result represents with the form of mean+SD.
2, the mensuration of crude protein
Employing disappears and boils quantitative method mensuration crude protein.
(1) take about 1g (W1) Feed Sample by analytical balance, pan paper is put into disappear after wrapping and is boiled pipe, adds 0.2g copper sulfate and 6.0g potassium sulfate.
(2) adding the 20ml vitriol oil gently boils in pipe in disappearing, and shakes gently, and sample is fully distributed in concentrated sulfuric acid.
(3) will disappear and boil pipe connection and be positioned over to disappear and boil on pipe, stop the volatilization of sulfuric acid.
(4) open circulating water device, will disappear and boil pipe and be placed on to disappear and boil on device, and start to be heated to 420 DEG C.
(5) wait for disappearing and boil after liquid in pipe becomes green transparent, continue 420 DEG C and disappear and boil 2h.
(6) disappear and boil after decomposition completes, turning off disappears boils device, and taking off disappears boils pipe and be put on thermal baffle and cool.
(7) after cooling, alimentary canal is put into kjeldahl apparatus, start titration (analyzing according to the method that AOAC (Official Methods of Analysis [M] .15th ed.Association of official analytical chemists.Arlington, VA.1999) describes).
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: the concentration of titrating solution HCl; W1: example weight.
Experiment repetition 3 times, result represents with the form of mean+SD.
3, the mensuration of neutral detergent fiber (neutral detergent fiber, NDF)
Plant feed boils after process through neutral detergent, and undissolved residue is neutral detergent fiber, is mainly cell wall constituent, and neutral detergent fiber comprises Mierocrystalline cellulose, this quality and hemicellulose, can be used as the food consumption of quantitative Livestock roughage.
(1) 20min opens crude fiber test instrument device and cooling circulating water in advance.
(2) analytical balance takes about 0.5g (W1) left and right Feed Sample and is positioned in glass pot.
(3) upper machine, adds 100ml neutral detergent 3% (3g/100ml) sodium lauryl sulphate and appropriate octyl alconyl makes defoamer, and 100 DEG C are heated to boiling, keeps about 50 DEG C to heat 70min.
After suction filtration (4), take off glass pot, utilize acetone rinsing three times.
(5) glass pot is put in dried overnight in stink cupboard.
(6) glass pot later for drying is positioned in baking oven, 135 DEG C of constant temperature more than air seasoning 2h, puts into after moisture eliminator cools 1h, weigh, be designated as W2.
(7) glass pot is put on electric furnace and carries out ashing, until cigarette disperses, after use crucible tongs taking-up crucible is positioned over and heats ashing 2h in 530 DEG C of retort furnaces, taking-up is positioned over 1h in moisture eliminator and cools, and weighs, is designated as W3.
NDF(％)＝(W2-W3)/W1×100。
Experiment repetition 3 times, result represents with the form of mean+SD.
Result is as shown in table 6, visible: in whole low temperature (5 DEG C) ensilage, crude protein and the neutral detergent fiber content of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 pairs of wheat stalk silages have no significant effect; Ensiling the 1st day and ensiling the 3rd day, the crude fat content change big rise and fall of ensiling wheat stalk, the experimental group that all the other ensiling time adds Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 does not have noticeable change compared with control group.In a word in 5 DEG C of low temperature ensilages, crude protein, neutral detergent fiber and crude fat composition all retain better.
The change (%/fresh weight) of the crude fat of wheat stalk, crude protein and neutral detergent fiber in table 6 low temperature ensiling (5 DEG C) process
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 sum, visible Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666CCTCC NO:M 2015255 breeds and produces acid rapidly and falls pH in low temperature ensiling (5 DEG C) process, effective suppression is harmful to growth or the generation of miscellaneous bacteria, the nutritive ingredients such as crude protein, crude fat, robust fibre effectively retain, and reach long-term effect of preserving silage.
Claims (10)
1. Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666, it is CCTCC NO:M 2015255 at China typical culture collection center deposit number.
2. a microbial inoculum, is characterized in that: the activeconstituents of described microbial inoculum is Lactococcus lactis subsp.lactis according to claim 1 (Lactococcus lactis subsp.lactis) QH1-666.
3. a silage additive, is characterized in that: the activeconstituents of described silage additive is Lactococcus lactis subsp.lactis according to claim 1 (Lactococcus lactis subsp.lactis) QH1-666.
4. a silage, is characterized in that: containing silage additive according to claim 3 in described silage.
5. silage according to claim 4, is characterized in that: described silage prepares according to the method described in claim 9 or 10.
6. Lactococcus lactis subsp.lactis according to claim 1 (Lactococcus lactis subsp.lactis) QH1-666 or microbial inoculum according to claim 2 application in arbitrary as follows:
(a1) anti-bacteria;
(a2) bacterial inhibitor is prepared;
(a3) silage additive according to claim 3 is prepared;
(a4) silage according to claim 4 is prepared;
In described (a1) and described (a2), described bacterium is specially micrococcus luteus and/or Salmonellas.
7. the application according to claim 5 or 6, is characterized in that: described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 or described microbial inoculum are the suppression under 30 DEG C of conditions to the suppression of described bacterium.
8. the application of silage additive according to claim 3 in the silage described in preparation claim 4 or 5.
9. prepare the method for silage described in claim 4, comprise: ensiling raw material is mixed with Lactococcus lactis subsp.lactis according to claim 1 (Lactococcus lactis subsp.lactis) QH1-666, carry out solid anaerobic fermentation, collect all tunnings, obtain described silage;
Described ensiling raw material is specially wheat stalk.
10. method according to claim 9, is characterized in that: in described method, and the proportioning of described wheat stalk and described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) QH1-666 is 100g:10
5cfu; And/or
Described fermentation is low temperature fermentation; Described low temperature is 5 DEG C; And/or
The time of described fermentation is 30 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510340152.0A CN104894030B (en) | 2015-06-18 | 2015-06-18 | One plant of Lactococcus lactis subsp. lactis and its application in low temperature ensiling |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510340152.0A CN104894030B (en) | 2015-06-18 | 2015-06-18 | One plant of Lactococcus lactis subsp. lactis and its application in low temperature ensiling |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104894030A true CN104894030A (en) | 2015-09-09 |
| CN104894030B CN104894030B (en) | 2017-09-26 |
Family
ID=54026991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510340152.0A Active CN104894030B (en) | 2015-06-18 | 2015-06-18 | One plant of Lactococcus lactis subsp. lactis and its application in low temperature ensiling |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104894030B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047773A (en) * | 2016-08-10 | 2016-10-26 | 江南大学 | Lactococcal lactis and application thereof |
| CN106282055A (en) * | 2016-08-10 | 2017-01-04 | 江南大学 | One strain lactococcus lactis |
| CN106282053A (en) * | 2016-08-10 | 2017-01-04 | 江南大学 | The one acidproof lactococcus lactis of strain and application thereof |
| CN112515045A (en) * | 2020-11-30 | 2021-03-19 | 成都理工大学 | Silage additive for pasture silage |
| CN113832072A (en) * | 2021-10-20 | 2021-12-24 | 江南大学 | Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0906952A2 (en) * | 1997-09-26 | 1999-04-07 | Medipharm AB | Bacterial strain for use in ensiling of straw fodder |
| CN102851233A (en) * | 2012-06-12 | 2013-01-02 | 华南农业大学 | Low-temperature-resistant lactobacillus strain lactobacillus plantarum CCZZ1 and application thereof |
| CN103781898A (en) * | 2011-06-29 | 2014-05-07 | 雪印种苗株式会社 | Novel lactic acid bacterium and method for preparing silage or fermented feed by using the same |
-
2015
- 2015-06-18 CN CN201510340152.0A patent/CN104894030B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0906952A2 (en) * | 1997-09-26 | 1999-04-07 | Medipharm AB | Bacterial strain for use in ensiling of straw fodder |
| CN103781898A (en) * | 2011-06-29 | 2014-05-07 | 雪印种苗株式会社 | Novel lactic acid bacterium and method for preparing silage or fermented feed by using the same |
| CN102851233A (en) * | 2012-06-12 | 2013-01-02 | 华南农业大学 | Low-temperature-resistant lactobacillus strain lactobacillus plantarum CCZZ1 and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| HUILI PANG等: "Phenotypic and Phylogenetic Analysis of Lactic Acid Bacteria Isolated from Forage Crops and Grasses in the Tibetan Plateau", 《THE JOURNAL OF MICROBIOLOGY》 * |
| KUIKUI NI等: "Effect of Cellulase and Lactic Acid Bacteria on Fermentation Quality and Chemical Composition of Wheat Straw Silage", 《AMERICAN JOURNAL OF PLANT SCIENCES》 * |
| 储徐建等: "耐低温乳酸菌的分离与优化培养", 《草业科学》 * |
| 李东霞等: "高抗菌活性乳酸菌的筛选及菌种鉴定", 《饲料工业》 * |
| 杨文琦等: "玉米青贮中细菌多样性分析", 《新疆农业科学》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047773A (en) * | 2016-08-10 | 2016-10-26 | 江南大学 | Lactococcal lactis and application thereof |
| CN106282055A (en) * | 2016-08-10 | 2017-01-04 | 江南大学 | One strain lactococcus lactis |
| CN106282053A (en) * | 2016-08-10 | 2017-01-04 | 江南大学 | The one acidproof lactococcus lactis of strain and application thereof |
| CN106282055B (en) * | 2016-08-10 | 2019-06-21 | 江南大学 | One plant of Lactococcus lactis |
| CN106047773B (en) * | 2016-08-10 | 2019-06-21 | 江南大学 | One plant of Lactococcus lactis and its application |
| 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 |
| CN113832072A (en) * | 2021-10-20 | 2021-12-24 | 江南大学 | Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof |
| CN113832072B (en) * | 2021-10-20 | 2023-10-27 | 江南大学 | Lactococcus lactis subspecies lactate with inulin utilization capacity and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104894030B (en) | 2017-09-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104946564A (en) | Lactobacillus plantarum and application thereof in low temperature ensiling | |
| CN103911323B (en) | Bacillus licheniformis, bacillus subtilis and lactobacillus plantarum preparation and preparation | |
| CN104894030A (en) | Lactococcus lactis subsp.lactis and application of lactococcus lactis subsp.lactis in low-temperature silage | |
| CN101338283B (en) | Lactobacillus casei and applications thereof in solid-state fermentation | |
| CN103205374B (en) | Lactic acid bacterium capable of reducing aflatoxin B1 in fermented feed and application of lactic acid bacterium | |
| CN107446841A (en) | A kind of direct putting type Lactobacillus plantarum high-density cultivation method and obtained freeze-dried powder and application | |
| CN104694409A (en) | Lactobacillus plantarum and uses thereof | |
| CN103898017B (en) | The application of the Lactobacillus rhamnosus that comes from Bama of Guangxi Changshou village aspect auxiliary antilipemic | |
| CN104894029A (en) | Leuconostoc mesenteroides and application of leuconostoc mesenteroides in low-temperature silage | |
| CN107217020B (en) | Culture medium suitable for lactobacillus acidophilus and preparation method thereof | |
| CN109161498A (en) | Bacillus subtilis M406 and its preparing the application in bacteriocin and cellulase | |
| CN103952336B (en) | Bacillus licheniformis, bacillus subtilis and lactobacillus casei preparation and preparation | |
| CN104894031A (en) | Leuconostoc mesenteroides and application of leuconostoc mesenteroides in low-temperature silage | |
| CN106497842B (en) | A kind of preparation method and application of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation | |
| CN103315058B (en) | Application of leuconostoc mesenteroides strain | |
| CN110583852A (en) | Bacterium-enzyme synergistic fermentation method for camellia seed meal | |
| CN104694431A (en) | Lactobacillus plantarum and application thereof | |
| CN103992969A (en) | Lactobacillus plantarum containing bacteriocin with antibacterial activity and application thereof | |
| CN104911131A (en) | Enterococcus mundtii and application thereof in low-temperature ensiling | |
| CN106085888A (en) | Saccharomycopsis fibuligera new strains and cultural method thereof and purposes | |
| CN106689391A (en) | Sour soybean milk with high viable count of lactic acid bacteria and high acidity, and preparation method of sour soybean milk | |
| CN104830730B (en) | One strain has Lactobacillus plantarum AB-4 and the application thereof of anti-Fructus Melo Fusarium oxysporum and anti-Fructus Melo phytophthora activity | |
| CN110934290A (en) | Kiwi fruit enzyme and application thereof in increasing anti-glycosylation and improving gastrointestinal immune function | |
| CN106754464B (en) | One plant of bacillus cereus and its application | |
| Prado et al. | Isolation and expeditious morphological, biochemical and kinetic characterization of propolis-tolerant ruminal bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |