CN107047978A - A kind of Lactobacillus plantarum and its application in ensilage is prepared - Google Patents
A kind of Lactobacillus plantarum and its application in ensilage is prepared Download PDFInfo
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- CN107047978A CN107047978A CN201710278399.3A CN201710278399A CN107047978A CN 107047978 A CN107047978 A CN 107047978A CN 201710278399 A CN201710278399 A CN 201710278399A CN 107047978 A CN107047978 A CN 107047978A
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Abstract
The invention discloses the lactobacillus plantarum for belonging to microbe application and feed preparation manufacture field and its application in ensilage is prepared.Lactobacillus plantarum (Lactobacillus plantarum) CAU a214 of the present invention, preserving number is CGMCC No.13609, available for preparing ensilage, especially alfalfa silage.Lactobacillus plantarum (Lactobacillus plantarum) CAU a214 of the present invention can quickly reduce pH value, and accelerated fermentation processes improve ensiling speed；The breeding of undesirable microorganism can be suppressed, alfalfa silage quality is improved, preferably preserve clover nutrition, add the Vitro Digestibility of ensilage, the problem of clover fermentation is difficult is overcome.With the present invention Lactobacillus plantarum (Lactobacillus plantarum) CAU a214 prepare additive silage effect than existing commercially available lactic bacteria additive more preferably, and cost it is low, safely, be easy to utilize.
Description
Technical field
The invention belongs to microbe application and feed preparation manufacture field, and in particular to a lactobacillus plantarum and its system
Application in standby ensilage.
Background technology
Clover is the big tame forage grass of China first, and the fast development and rural area plant structure with China's animal husbandry are not
Disconnected adjustment, the plantation of clover large area and industrialization development are fast-developing.China's alfalfa industry is using alfalfa hay as processing base
Plinth, but drench with rain or the moist loss for easily causing nutriment in production and storage process, seriously limit alfalfa industry health
Development, therefore seek new approach, solve clover processing storage problem very necessary.Ensiling be solve the above problems it is ideal
Measure.Ensiling refers to make green forage fermentation under air-proof condition within considerable time its quality can be kept relative
They are converted into organic acid by a kind of stable preservation technology, i.e. microorganism by the use of the carbohydrate in plant tissue as substrate
So as to realize the process of crop fresh-retaining preserving.
Protein content of alfalfa is high, resiliency is high, water-soluble carbohydrate content is low, while Herba Medicaginis stem tissue is hollow,
With the presence of large quantity of air, alfalfa ensilage is set to be difficult to modulate.To ensure silage effect, ensiling addition is usually added in alfalfa ensilage
Agent, traditional additives for ensiling such as formic acid, formaldehyde have certain corrosivity, exist domestic animal feeding potential safety hazard, addition it is larger,
Cost is high and operates the defects such as inconvenience.In recent years the research of microbe additive causes the extensive concern of academia.
Numerous studies show, ensiling success, and can be heavily dependent on lactic acid bacteria breed rapidly and in large quantities.
Lactic acid bacteria is a kind of facultative anaerobic bacteria, ensilage can be accelerated to ferment, and quickly produces lactic acid, pH value is reduced rapidly, so as to suppress
Spoilage organisms activity, reduces nutrient component damages, greenfeed nutrient is preferably preserved.Lactic acid bacteria is added, on the one hand can be with
Improve the nutritive value and security of ensilage；On the other hand, substantial amounts of biodiasmin enters cud with ensilage, right
Domestic animal also has prebiotic effect.Improvement of the lactic bacteria additive of ensilage modulation to fermentation quality has been recognized extensively
Can, but commercially available microbial inoculum produces acid less during alfalfa ensilage, growth is slow, therefore, feed preparation manufacture field high-function breast
The exploration and application of the probiotic lactobacillus with care treatment effect are significant in sour bacterium and animal alimentary canal.
The content of the invention
The invention aims to overcome in ensilage preparation process in the prior art, lactic acid bacteria growth is slow, it is low to produce acid
And cause the problem of Silage Quality is low, propose that one kind can quickly reduce pH value, accelerate fermentation process, effectively improve clover blue or green
Store the Lactobacillus plantarum of quality.
Therefore, the concrete technical scheme of the present invention is as follows：
A kind of Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214, preserving number is CGMCC
No.13609。
A kind of active component of silage additive is Lactobacillus plantarum (Lactobacillus plantarum) CAU-
a214。
The preparation method of described silage additive, comprises the following steps：By described Lactobacillus plantarum
(Lactobacillus plantarum) CAU-a214 is cultivated on MRS culture mediums, obtains silage additive.
Described Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 answering in ensilage preparation
With.
In above-mentioned application, the ensilage is alfalfa silage.
A kind of preparation method of ensilage, comprises the following steps：
1) will feed cutting be fermented, be well mixed；
2) to step 1) the middle Lactobacillus plantarum (Lactobacillus plantarum) added described in claim 1
CAU-a214；
3) by step 2) in feed vacuum sealing, storage, tunning is ensilage.
Step 1) in treat fermented feed be clover.
Step 1) in feed cutting length to be fermented be 1~2cm.
Step 2) in every gram treat that fermented feed is inoculated with the Lactobacillus plantarum (Lactobacillus described in 5~8log cfu
plantarum)CAU-a214。
Step 3) in holding conditions be：15~45 DEG C of temperature, 30~60d of time.
Beneficial effects of the present invention：
1st, Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 of the invention can be used for preparing ensiling feeding
Material, especially prepares alfalfa silage.
2nd, Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 of the invention can quickly reduce pH
Value, accelerated fermentation processes improve ensiling speed；The breeding of undesirable microorganism can be suppressed, alfalfa silage quality is improved, preferably protect
Clover nutrition is deposited, the Vitro Digestibility of ensilage is added, the problem of clover fermentation is difficult is overcome.
3rd, the additive green grass or young crops prepared with Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 of the present invention
Store effect than existing commercially available lactic bacteria additive more preferably, and cost it is low, safely, be easy to utilize.
Biomaterial preservation is proved
Classification And Nomenclature：Lactobacillus plantarum；Strain number：CAU-a214
Preservation mechanism：China General Microbiological culture presevation administrative center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On January 13rd, 2017
Collection is registered on the books numbering：CGMCC No.13609
Embodiment
Following embodiment facilitates a better understanding of the present invention, but is not limited to the present invention.Such as without special in following embodiments
Illustrate, be conventional method.
GFG (business microbial inoculum), purchased from Sichuan Gaofuji Biotechnology Co., Ltd., FG1 (business microbial inoculum), by Japanese livestock products
Grassland Research Institute is provided.Culture medium is from extensive and profound in meaning star bio tech ltd (Beijing) purchase in Beijing.
Embodiment 1：Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 separation is identified with bacterial strain
1st, the separation of bacterial strain
Using dilution-plate method separating plant lactobacillus：The 10g alfalfa ensilages sample of 60 days is added to 90mL sterilizing steamings
Distilled water shaken well, 10 times of gradient dilutions, then takes 10 respectively-1、10-3With 10-5Times each 20 μ L of sample diluting liquid are coated on MRS
On culture medium, 30 DEG C of constant incubator culture 48h；According to features such as bacterium colony size, form and colors, picking colonies typical enters
Row catalase activity is determined, Gram's staining and microscopy are tested.Every Gram-positive, negative catalase
Bacterial strain can be primarily determined that as lactobacillus, and it is rule into purifying twice on MRS culture mediums, be then stored in 1ml and added 10%
In the NB culture mediums of dimethyl sulfoxide (DMSO), preserved in -80 DEG C of ultra low temperature freezers.
MRS culture mediums are：Peptone (Proteose peptone NO.3) 10.0g；Beef extract (Beef extract)
10.0g；Yeast extract (Yeast extract) 5.0g；Glucose (Dextrose) 20.0g；Tween (Polysorbate
80)1mL；Ammonium citrate (Ammonium citrate) 2.0g；Sodium acetate (NaAc) 5.0g；Magnesium sulfate (MgSO4·7H2O)
0.1g；Manganese sulfate (MnSO4·4H2O)0.05g；Dipotassium hydrogen phosphate (K2HPO4)2.0g；Distilled water (H2O) 1000mL, solid culture
Base adds 20g/L agar (Agar) again, and 121 DEG C, sterilize 20min.
2nd, screening high acid, the lactobacillus of fast growth
To the lactobacillus separated from the feed of the natural ensiling 60 days of clover, squeezed the juice training with reference to Physiology and biochemistry method and clover
Foster method filters out that acid production speed is fast, acid-fast ability is strong and eugonic Lactobacillus plantarum CAU-a214.The physiological of bacterial strain
Shape, which is determined, to be included：Salt tolerant (NaCl) concentration, pH range growths, temperature range growth test etc.；Biochemical trait, which is determined, to be included：Peroxide
Change hydrogen enzyme test, glucose fermentation aerogenesis experiment (homotype or heterofermentation experiment), carbon source through fermentation experiment etc..
Physiological and biochemical test method is as follows：
(1) Gram's staining is with reference to the elegant pearl chief editor's in east《Common bacteria system identification handbook》.
(2) Lactobacillus plantarum growth pH regulations use 2mol/L NaOH and 2mol/L HCl.
(3) Catalase determination is tested：Experimental strain is seeded on MRS culture mediums and in anaerobic culture box culture
48h, picking single bacterium colony, which is coated on, to be added dropwise on the culture dish of 3% (w/w) hydrogenperoxide steam generator, has seen whether bubble generation,
Produce bubble for catalase positive, bubble is not produced is then feminine gender.
(4) glucose fermentation aerogenesis determination test：Experimental strain is seeded on MRS culture mediums and in anaerobic culture box training
48h is supported, picking single bacterium colony is inoculated in the test tube containing 5mL MRS fluid nutrient mediums (pH6.5) that (Du Shi tubules tip upside down on test tube
It is interior), common 30 DEG C of culture 48h of constant incubator are positioned over, observes and records result.
(5) carbon source through fermentation determination test：For Gram-positive, the experimental strain of negative catalase, using containing
49 kinds of carbon sources, Analytical Profile Index (API 50CH, bioMerieux, France) test bar of a control
Tested under part, experimental method is operated according to specification, be incubated at 30 DEG C of common constant incubators.48h record experiments
As a result, positive findings is the acid production in culture medium shown in contained Bromocresol purple flavescence, and aesculin determines (No. 25
Pipe) it is black for the positive by purple stain.
(6) clover is squeezed the juice cultural method：By the fresh alfalfa of chopping and distilled water according to 1：3 ratio mixing is squeezed the juice and mistake
Filter, by 121 DEG C of sterilization treatment 15min of filtrate, can adapt to clover habitat in this, as Screening of Media and quickly produces the bacterium of acid
Strain.
As a result show, Lactobacillus plantarum CAU-a214 is inoculated in well-grown in clover juice culture medium, the quick (table of production acid
1) clover habitat can, be well adapted to.Lactobacillus plantarum CAU-a214 bacterial strains be Gram-positive, homo-fermentative bacillus,
It can all be grown under the conditions of 10~50 DEG C and pH3.5~9.0, with stronger acid resistance (table 2)；Can be fermented most of carbohydrate (tables
3)。
The Lactobacillus plantarum CAU-a214 of table 1 ferments pH and organic acid content (mg/mL) after 24h in clover juice
The Lactobacillus plantarum CAU-a214 of table 2 colonial morphology and physio-biochemical characteristics
Note：-, do not grow；W, growing way is faint；+, normal growth.
The Lactobacillus plantarum CAU-a214 carbon source through fermentation experimental results of table 3
Note：-, do not grow；W, growing way is faint；+, normal growth.
3rd, the identification of bacterial strain
The Lactobacillus plantarum CAU-a214 of screening is seeded in 0.5mL MRS fluid nutrient mediums, 30 DEG C, 180rmp cultures
Overnight, strain idenfication is carried out using bacterium solution PCR.
Universal primer of the primer from the 16S rDNA gene magnifications of bacterium：
27F：5’-AGAGTTTGATCCTGGCTCAG-3’
1492R：5’-GGTTACCTTGTTACGACTT-3’
PCR reaction systems (50 μ L)：
Reaction condition：
Amplified production send Huada gene company to be sequenced, and 16S rDNA sequences are as shown in SEQ ID No.1, on GeneBank
Sequence alignment is carried out, with reference to bio-chemical characteristics data, the final bacterial strain for determining separation belongs to Lactobacillus plantarum
Lactobacillus plantarum, are named as CAU-a214.
Embodiment 2：The preparation of alfalfa silage
The preparation of alfalfa silage comprises the following steps：
(1) clover is dried to moisture 60% or so, and is cut to 1cm~2cm, be well mixed；
(2) into step (1) clover according to 6log cfu g-1Inoculated plant lactobacillus (Lactobacillus
Plantarum) CAU-a214, and be control with no added (Control), GFG (business microbial inoculum), FG1 (business microbial inoculum), each
Handle 3 repetitions；
(3) step (2) clover loads to 30cm × 20cm polyethylene bag silo, each three bags of processing, per it is packed enter
100g, is evacuated with vacuum sealing machine, sealed, and is placed in room temperature storage 60d.
Nutritional ingredient, fermentation quality, microbiological analysis and External digestion experiment are carried out after alfalfa ensilage 60d.
1st, the identification of ensilage nutritional ingredient and fermentation quality
Ensiling is opened after bag, weighs representative sample 10g, adds 90mL distilled water, is well mixed, and is soaked after immersion 12h
Go out liquid, the pH value of leachate is determined with pH meter.With the filtering with microporous membrane that aperture is 0.22 μm, 10mL centrifuge tubes are loaded after filtering
In, be put into -20 DEG C it is standby, to determine organic acid (lactic acid, acetic acid, propionic acid, butyric acid) and ammoniacal nitrogen (NH3- N) content.It is organic
Acidity test uses Shimadzu GC-14 type high performance liquid chromatograph (chromatographic columns：KC-811column, Shimadzu, Japan；Detector：
SPD-M10AVP, mobile phase：3mmol L-1Perchloric acid, is accurately weighed in 0.8493g perchloric acid, the volumetric flask of constant volume to 2L,
Flow velocity 1mL min-1；50 DEG C of column temperature；Detection wavelength 210nm, the μ L of sample size 5), remaining ensiling sample is placed in 65 DEG C of baking ovens and dried
48h, determines dry (DM) content.The sample comminution of drying is crossed to be put into valve bag after 1mm sieves and preserved, for determining routine
Chemical composition and External digestion experiment.The measure of crude protein (CP) content uses Kjeldahl's method；Neutral detergent fiber (NDF),
The measure of acid detergent fiber (ADF) uses Fan Shi fibre analysis methods；The measure of soluble sugar (WSC) uses Anthrone Sulphuric acid ratio
Color method.
Nutritional ingredient and fermentation quality after alfalfa ensilage change as shown in table 4, table 5, add CAU-a214 treatment group
PH value is minimum, and ammonia nitrogen content is minimum, and lactic acid content highest, dry matter content highest, crude protein and soluble sugar content are higher than
Control, GFG and FG1；Clostridium is the harmful microorganism in ensiling, and it produces butyric acid and ammonia, reduces ensilage quality,
The butyric acid composition of various concentrations is detected in control group and commercial strain addition group, and CAU-a214 treatment groups are not detected by fourth
Sour composition, illustrates that bacterial strain CAU-a214 can effectively suppress the growth and breeding and proteolysis of the undesirable microorganisms such as clostridium.Therefore,
Addition Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 can improve alfalfa silage nutritional ingredient and
Fermentation quality.
Table 4 alfalfa ensilage, 60 days after fermentation qualities
Note：DM, dry weight.
5 alfalfa ensilage of table nutritional ingredient after 60 days
2nd, microbiological analysis
Content of microorganisms, which is determined, uses spread plate counting method, weighs the 90mL distilled water that 10g ensilings sample adds sterilizing
In, after mixing, then 10 times of gradient dilutions take 10 respectively-1、10-3With 10-5Times each 20 μ L of sample diluting liquid be coated on MRS, she
Detection lactic acid bacteria, Escherichia coli, mould and yeast are respectively used on red methylene blue, rose bengal medium；By 10-1With 10-2Times sample
Product dilution 1mL is after 75 DEG C of water-bath water-bath 15min, and taking 20 μ L to be coated on nutrient agar respectively is used to detect bud
Spore bacillus, by these coated 30 DEG C of culture 48h of culture medium, wherein MRS culture mediums should be positioned over anaerobic culture box, Qi Tapei
Foster base is positioned over common constant incubator.
Yihong methylene blue medium component (g/L)：Peptone 10g；Beef extract powder 3g；Lactose 10g；Sodium chloride 5g；Eosine
0.4g；Methylenum careuleum 0.065g；Agar 14g.
Rose bengal medium composition (g/L)：Peptone 5g；Dipotassium hydrogen phosphate 1g；Magnesium sulfate 0.5g；Glucose 10g；Chlorine
Mycin 0.1g；Rose-bengal 0.033g；Agar 18.5g.
Nutrient agar composition：Peptone 10g；Powdered beef 3g；Sodium chloride 5g, distilled water 1L, pH value 7.2 ± 0.2.
121 DEG C of high pressure steam sterilization 15min..
Content of microorganisms change after alfalfa ensilage is as shown in table 6, addition Lactobacillus plantarum (Lactobacillus
Plantarum) CAU-a214 can significantly reduce the content of the undesirable microorganisms such as Escherichia coli and bacillus.
The content of microorganisms change (log CFU/g of FM) after 60 days of 6 alfalfa ensilage of table
Note：In table significant difference (p is represented with column data shoulder mark lowercase difference<0.05)；FM, fresh weight.
3rd, External digestion experiment
Rumen fluid is derived from 3 milk cows for being provided with permanent fistula, 1h collections rumen fluid before being raised in morning, and equivalent is mixed after collection
Merge through four layers of filtered through gauze, 39 DEG C of water-bath insulations, be constantly passed through CO2Keep anaerobic environment.0.220g samples are loaded and sent into
In 100mL glass syringes.The buffer solution of rumen fluid and preheating is added, is well mixed.It is inoculated in the type microbial fermentations of AGRS- III
Micro aerogenesis automatic recording instrument, 39 DEG C of continuous culture 72h, records gas production.Each processing 3 is parallel, and 3 blank are set in addition.
After fermentation ends, then Nylon Bag tap water rinse is cleaned 2 times to limpid to water with distilled water.65 DEG C of drying are counted to constant weight
Calculate dry digestibility.
External digestion experiment result as shown in table 7, adds Lactobacillus plantarum (Lactobacillus after alfalfa ensilage
Plantarum) CAU-a214 can dramatically increase the dry digestibility of alfalfa silage, can significantly improve gas production,
Illustrate that addition Lactobacillus plantarum (Lactobacillus plantarum) digestion of CAU-a214 to ensilage is beneficial.
The alfalfa silage 72h external digestion aerogenesis dynamics of table 7 and vitro Dry Matter Digestibility
Note：IVDMD, vitro Dry Matter Digestibility；GP, gas production；A, theoretical maximum gas production；C, gas production rate；Lag,
Aerogenesis lag time；AGPR, average gas production rate.
SEQUENCE LISTING
<110>China Agricultural University
<120>A kind of Lactobacillus plantarum and its application in ensilage is prepared
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1426
<212> DNA
<213>Lactobacillus plantarum（Lactobacillus plantarum）
<400> 1
ctggttccta aaaggttacc ccaccgactt tgggtgttac aaactctcat ggtgtgacgg 60
gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc 120
gattccgact tcatgtaggc gagttgcagc ctacaatccg aactgagaat ggctttaaga 180
gattagctta ctctcgcgag ttcgcaactc gttgtaccat ccattgtagc acgtgtgtag 240
cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc 300
ggcagtctca ccagagtgcc caacttaatg ctggcaactg ataataaggg ttgcgctcgt 360
tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtat 420
ccatgtcccc gaagggaacg tctaatctct tagatttgca tagtatgtca agacctggta 480
aggttcttcg cgtagcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcctttg agtttcagcc ttgcggccgt actccccagg cggaatgctt aatgcgttag 600
ctgcagcact gaagggcgga aaccctccaa cacttagcat tcatcgttta cggtatggac 660
taccagggta tctaatcctg tttgctaccc atactttcga gcctcagcgt cagttacaga 720
ccagacagcc gccttcgcca ctggtgttct tccatatatc tacgcatttc accgctacac 780
atggagttcc actgtcctct tctgcactca agtttcccag tttccgatgc acttcttcgg 840
ttgagccgaa ggctttcaca tcagacttaa aaaaccgcct gcgctcgctt tacgcccaat 900
aaatccggac aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt 960
ggctttctgg ttaaataccg tcaatacctg aacagttact ctcagatatg ttcttcttta 1020
acaacagagt tttacgagcc gaaacccttc ttcactcacg cggcgttgct ccatcagact 1080
ttcgtccatt gtggaagatt ccctactgct gcctcccgta ggagtttggg ccgtgtctca 1140
gtcccaatgt ggccgattac cctctcaggt cggctacgta tcattgccat ggtgagccgt 1200
taccccacca tctagctaat acgccgcggg accatccaaa agtgatagcc gaagccatct 1260
ttcaaactcg gaccatgcgg tccaagttgt tatgcggtat tagcatctgt ttccaggtgt 1320
tatcccccgc ttctgggcag gtttcccacg tgttactcac cagttcgcca ctcactcaaa 1380
tgtaaatcat gatgcaagca ccaatcaata ccagagttcg ttcgac 1426
Claims (10)
1. a kind of Lactobacillus plantarum (Lactobacillus plantarum), bacterial strain is CAU-a214, and preserving number is CGMCC
No.13609。
2. a kind of silage additive, it is characterised in that the active component of the additive is the plant described in claim 1
Lactobacillus (Lactobacillus plantarum) CAU-a214.
3. the preparation method of the silage additive described in claim 2, it is characterised in that comprise the following steps：By right
It is required that Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 described in 1 is cultivated on MRS culture mediums, obtain
Silage additive.
4. Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 described in claim 1 is in ensilage system
Application in standby.
5. application according to claim 4, it is characterised in that the ensilage is alfalfa silage.
6. a kind of preparation method of ensilage, it is characterised in that comprise the following steps：
1) will feed cutting be fermented, be well mixed；
2) to step 1) middle Lactobacillus plantarum (Lactobacillus plantarum) CAU- added described in claim 1
a214；
3) by step 2) in feed vacuum sealing, storage, tunning is ensilage.
7. preparation method according to claim 6, it is characterised in that step 1) in treat that fermented feed is clover.
8. preparation method according to claim 6, it is characterised in that step 1) in feed cutting length to be fermented be 1~
2cm。
9. preparation method according to claim 6, it is characterised in that step 2) in every gram treat fermented feed inoculation 5~
Lactobacillus plantarum (Lactobacillus plantarum) CAU-a214 described in 8log cfu claims 1.
10. preparation method according to claim 6, it is characterised in that step 3) in holding conditions be：Temperature 15~45
DEG C, 30~60d of time.
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