CN107603923A - 3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling - Google Patents
3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling Download PDFInfo
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Abstract
The invention discloses 3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling, their optimum growth temperature is 15 DEG C, has the features such as the speed of growth is fast, production acid is more, advantage is provided for ensiling modulation.Fermentation (≤20d) can be rapidly completed under cryogenic, increase lactic acid and total acid content, reduce pH value, ammoniacal nitrogen/total nitrogen ratio, propionic acid and butyric acid content, improve ensilage fermentation quality, ensilage crude protein content and Vitro Dry Matter Digestibility are improved, can be used in ensiling modulated process as additive.
Description
Technical field
The invention belongs to animal husbandry field, and in particular to 3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling.
Background technology
With the development of animal husbandry, lactobacillus preparation is in ensilage using more and more extensive.Numerous studies show, newborn
Sour bacterium additive effect difference because of the difference of metabolic type, property of raw material and climatic environment.Cool regional forage grass attachment breast
Sour bacterium number amount and composition are different from Temperate Region in China and torrid areas.The commercial lactic acid bacterium used at present is mainly derived from temperate zone ring
Border, while there is part research to be intended to screen the advantage lactic acid bacteria that torrid areas adapts to ensiling modulation.
However, Northwest Sichuan alpine pastoral regions are directed to, so the forage grass of Qinghai-Tibet Alpine-arctic Pastoral extreme environment adheres in itself
The research of lactic acid bacteria physio-biochemical characteristics and application foundation etc. is few.Low temperature lactic acid bacteria is divided into two types, i.e., thermophilic cold breast
Sour bacterium
(Obligate LAB) and cold-resistant lactic acid bacteria (Faculative LAB).Thermophilic cold lactic acid bacteria can be in 0 DEG C of growth, advantage
Growth temperature≤15 DEG C, 20 DEG C of maximum growth temperature；Cold-resistant lactic acid bacteria can also grow at 0 DEG C, but optimum growth temperature >=20 DEG C.
Probiotics is screened, suppresses the aerobic microbiologicals such as mould, saccharomycete and produces mycotic spore and mycotoxin, be that ensiling is ground in recent years
The focus studied carefully.It is well known that the harmful microorganism that causes a disease can reduce ensilage fermentation quality, reduce ensilage palatability and
Utilization rate, the parasite harm of increase ruminant.Butyric reduces Forage quality in ensilage, and this can be suppressed by screening
The probiotics of class harmful microorganism growth is still less.Correlative study report points out that some lactic acid bacterias have antibacterial activity, can
Harmful microorganism quantity and composition are influenceed, and reduces mycotoxin levels in ensilage.At the same time, it is high and cold in North-West Sichuan
Pastoral area, be particularly in remote, economics of underdevelopment, region backward in technique, because being lagged by equipment, financial strain, technology shortcoming, production
Can the objective condition such as limited restrict, plant equipment promoted the use of in ensiling modulated process improve ensiling improving storage temperature and raise
The measure of item matter is simultaneously unrealistic.Therefore, for Northwest Sichuan alpine pastoral regions microthermal climate feature, low temperature lactic acid bacteria is screened, is extended
Production sour (lactic acid) time, it is favorably improved ensilage quality.
The content of the invention
The technical problems to be solved by the invention are：How a kind of lactic acid bacteria for being suitable for Alpine-arctic Pastoral ensiling is provided.
The technical scheme is that：3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling, they are that deposit number is respectively
CGMCC No.14117 Lactobacillus plantarum (lactobacillus plantarum) 148, deposit number CGMCC
No.14116 Lactobacillus paracasei (Lactobacillus paracasei) 171, deposit number are CGMCC No.14183's
The piece of resistance to ethanol coccus (Pediococcus ethanolidurans) P-14.
Lactobacillus plantarum (lactobacillus plantarum) 148 is referred to as bacterial strain 148, Lactobacillus paracasei
(Lactobacillus paracasei) 171 is referred to as bacterial strain 171, the piece of resistance to ethanol coccus (Pediococcus
Ethanolidurans) P-14 is referred to as bacterial strain P-14.
Application of above-mentioned 3 strains of lactic acid bacteria in ensiling.
On May 11st, 2017, bacterial strain P-14 was deposited in the micro- life of China on May 24th, 2017 for bacterial strain 148 and bacterial strain 171
Thing bacterial strain preservation administration committee common micro-organisms center, preservation address are：In Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of microbiology of the academy of sciences of state.
Compared with prior art, the invention has the advantages that：
The bacterial strain P-14 of the present invention, the optimum growth temperature of bacterial strain 148 and bacterial strain 171 are 15 DEG C, have the speed of growth it is fast,
The features such as production acid is more, modulate for ensiling and provide advantage.Fermentation (≤20d) can be rapidly completed under cryogenic, increased
Lactic acid and total acid content, pH value, ammoniacal nitrogen/total nitrogen ratio, propionic acid and butyric acid content are reduced, improve ensilage fermentation quality,
Ensilage crude protein content and Vitro Dry Matter Digestibility are improved, can be used in ensiling modulated process as additive.
Brief description of the drawings
PH value and thalline absorbance (OD after the green juice fermentation 48h of Fig. 1 inoculating strains600)；
Fermented green juice OD after Fig. 2 inoculating lactic acid bacterial strains600Curve；
Fermented green juice pH curves after Fig. 3 inoculating lactic acid bacterial strains；
Lactic acid, acetic acid, propionic acid and ethanol content after the green juice fermentation 48h of Fig. 4 inoculating strains；
Fermented green juice lactic acid curve after Fig. 5 inoculating lactic acid bacterial strains；
Influence (48h) of Fig. 6 temperature to inoculating lactic acid bacterium fermented green juice pH value；
Fig. 7 refers to lactic acid bacteria pcr amplified fragment electrophoresis pattern；
Fig. 8 institutes screening lactobacillus pcr amplified fragment electrophoresis pattern；
Influence of Fig. 9 lactic acid bacterias to ensilage pH value；
Influence of Figure 10 lactic acid bacterias to ensilage lactic acid content；
Influence of Figure 11 low temperature lactic acid bacteria to ensilage ammoniacal nitrogen/total nitrogen ratio.
Embodiment
1 materials and methods
The separation of 1.1 lactic acid bacterias
The collection natural ensilage in Northwest Sichuan alpine pastoral regions (siberian wildrye, oat) sample amounts to 86 parts, and aseptic nipper sandwiches
In sterile valve bag, sealing, car refrigerator transports laboratory back, and -80 DEG C of low-temperature preservations are standby.
Frozen sample is placed in superclean bench thaw at RT, takes 5g samples in the sterile triangular flasks of 250mL, adds 45mL
0.9% sterile saline, preservative film sealing, shakes 2~3h, is filtered with sterile gauze, takes filtrate 1m to carry out gradient dilution, point
Do not take 10-3、10-4、10-5Dilution factor sample liquid 1mL is coated in aseptic culture medium.Each dilution factor is coated with 3~5 flat board (Φ
10cm).Culture medium is respectively MRS culture mediums, CaCO3+ MRS complex mediums, M17 culture mediums and (all cultures of GYP culture mediums
Base is provided by Chengdu Li Tian Centrix Technology Ltd., similarly hereinafter).Using anaerobic culture box culture, cultivation temperature is 15 DEG C, the time
48-96h.Selection milky, yellow are repeatedly purified with the bacterium colony of molten decorative cover, by Gram's staining (positive),
Catalase reaction (feminine gender) is initially identified as lactic acid bacteria (127 plants).According to count's bodyguard handbook, pass through colonial morphology, nitric acid
Salt reduction test, gelatin liquefaction test, H2The experiment of S aerogenesis, indole test, glucose produce sour aerogenesis experiment, carbohydrate hair
(all biochemical tubes and assessor are provided by Chengdu Li Tian centuries bio tech ltd) such as ferment production acid experiments, is classified as 44 plants
Lactic acid bacteria.The bacterial strain gone out according to preliminary screening purifies standby after MRS broth/M17broth+ -20 DEG C of Storage in refrigerator of glycerine again
With.
Fermented green juice characteristic after the screening and inoculation of 1.2 lactic acid bacterias
Jointing stage silage corn (or florescence clover) is squeezed the juice, filtered with sterile gauze, filtrate is collected and is fermented as green juice
Lactic acid bacteria pH and the tunning composition (lactic acid, acetic acid, propionic acid and butyric acid) that liquid Evaluating and screening goes out.Fermented green juice 4500r/
Supernatant is taken after min centrifugations, 2% sucrose is added, stirs.It is inoculated in after the bacterial strain of preservation is taken out from ultra low temperature freezer
Activated in MRS broth or M17broth, 5% (v/v) inoculum concentration progressively expands culture, is adjusted by spectrophotometer
The same absorbance of bacteria suspension, it is inoculated in afterwards according still further to 5% (v/v) inoculum concentration in fermented green juice, in 15 DEG C of anaerobic culture boxes
Culture 3,6,12,24,48h respectively.Meanwhile respectively be inoculated with different strains in fermented green juice, different temperatures (5 DEG C, 10
DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C) under cultivate 48h.Using plum Teller acidometer, (A20, Shanghai is wealthy to think the limited public affairs of electronics
Department) measure fermented green juice pH value；Using spectrophotometer (Shimadzu model UV-2501；Shimadzu Corp.,
Tokyo, Japan) measure fermented green juice absorbance (OD600)；The fermented green juice zymotic fluid 10000r/min for cultivating 48h is low
Using the method measure lactic acid and volatile fatty acid of chapter 1 1.4 after (4 DEG C) centrifugations of temperature, and pass through high performance liquid chromatography
(SCR-101H, 10 μm of 8.0mm × 30cm, Shim-pack, Shimadzu, Japan；30 DEG C of column temperature；Flow velocity 0.6mL/min is (high
Chloric acid)；210nm, SPD detector) measure ethanol content.This process filter out 31 plants can quickly reduce fermented green juice pH and
Increase the lactic acid bacteria strains of organic acid content.
To evaluate the growth characteristics of lactic acid bacteria, the culture medium of different cultivation temperatures, pH and NaCl concentration is set, observes its life
Long situation.Inoculating lactic acid bacterium is in MRS broth or M17broth, 15 DEG C of Anaerobic culturel 48h, adjustment bacteria suspension concentration (OD560),
It is stand-by.0.1mL lactic acid bacteria bacteria suspensions are taken, are inoculated in MRS broth or M17broth, respectively at 10 DEG C, 15 DEG C, 30 DEG C, 45 DEG C
48~72h is cultivated in constant-temperatureanaerobic anaerobic incubator, observes lactobacter growth situation；Be inoculated in respectively simultaneously different pH value (2.0,
3.0th, 4.0) fluid nutrient medium, different NaCl concentrations (2.5,4.5,6.5, g/L%) fluid nutrient medium and lactic acid concn (1.0,
3.0th, 5.0, v/v%) fluid nutrient medium, using pH7.0 fluid nutrient mediums as control, (15 DEG C) culture 48h of anaerobism constant temperature, measure is not
With the absorbance (OD of zymotic fluid560), if the absorbance (OD of inoculating lactic acid bacterium culture medium560 inoculations) with the extinction of control medium
Spend (OD560 controls) ratio be more than 1.0, illustrate that lactic acid bacteria can grow under this kind of condition of culture.Each processing sets 3~5 to put down
OK.
The safety evaluatio of 1.3 lactic acid bacterias
Using the drug resistance of disk diffusion method measure screening lactobacillus bacterial strain.Different strains be inoculated in MRS broth or
M17broth, (15 DEG C) culture 48h of anaerobism constant temperature, it is same absorbance (OD to adjust bacteria suspension with physiological saline560).Ultra-clean
1mL bacteria suspensions are drawn in workbench in sterile petri dish, aseptic culture medium MRS or M17 is added, shakes up, it is fixed, stick certainly
The round resistance scraps of paper (Φ 10mm), (15 DEG C) culture 48h of anaerobism constant temperature, observe inhibition zone.The antibiotic used is big mould for celebrating
Plain (10 μ g/mL), amikacin (30 μ g/mL), Ciprofloxacin (5 μ g/mL), vancomycin (30 μ g/mL), chloramphenicol (30 μ g/
ML), penicillin (10 μ g/mL) and tetracycline (30 μ g/ pieces), every kind of antibiotic setting 3~5 are parallel.
Lactic acid bacteria strains Activities of Fermentation Broth is determined using agar hole diffusion method.Indicator bacteria (Escherichia coli,
Escherichiacoli DH5a；Pseudomonas aeruginosa, Pseudomonas aeruginosa；Bacillus subtilis, Bacillus
subtilis；Salmonella, Salmonella derby；C.perfringens, Clostridium perfringens；Black song
It is mould, Aspergillus niger；Pichia pastoris phaff, Pichia pastoris) by Sichuan Province Grassland research institute from
Separation screening is obtained in ensilage, and bacterium is preserved using LB culture mediums, and fungi is preserved using YPD culture mediums.Taking-up has been sieved
The bacterial strain selected, MRS broth or M17broth are inoculated in, 5% inoculum concentration expands culture step by step, takes its supernatant, adds
Proper amount of hydrogen peroxide enzyme, the warm bath 1h in 37 DEG C of thermostat water baths, its pH 6.0 is adjusted with 0.1mol/L NaOH solutions, excluded
The influence of some peroxide and acidic materials to fungistatic effect is produced in lactobacter growth metabolic process.By indicator bacteria bacteria suspension
Adjust to same concentration, take 50mL bacteria suspensions to be down flat plate after the mixing of indicator bacteria solid medium, beaten after its solidification with sterile
Hole device punches (Φ 10mm), and 0.1mL lactic acid bacteria strains fermented supernatant fluids are added in hole, and front is placed, 37 DEG C of 18~24h of culture,
Fungistatic effect is observed, and using pH identicals buffer solution as negative control, Amp (30ug/mL) is used as positive control.Every plant of lactic acid
Bacterium setting 3~5 is parallel.
The identification of 1.4 lactic acid bacterias
Using 16S rRNA bacterial universal primers (7F 1540R5 '-
CAGAGTTTGATCCTGGCTAGGAGGTGATCCAGCCGCA-3 ' and 27F 1492R, 5 '-
AGTTTGATCMTGGCTCAGGGTTACCTTGTTACGACTT-3 ') carry out lactic acid bacteria identification.The lactic acid bacteria strains of screening exist
Bacteria suspension is trained at suitable temperature.Bacteria suspension 2mL is taken in sterile EP pipes, 8000r/min centrifugation 5min, collects thalline,
The 580mL TE buffer solutions of lysozyme containing 2.0mg/mL are added, 37 DEG C of processing 1h, lactobacillus cell wall is destroyed, uses bacterial genomes
DNA extraction kit (SK8255, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) obtains lactic acid bacteria genomic DNA.Will
The template that chromosomal DNA expands as PCR, PCR amplification system (50 μ L)：4.0 μ L 2.5mmol/L dNTP, 25 μ L 10 ×
buffer Mg2+, each 0.4 μ L rTaqDNA polymerases of two kinds of primers (50 μm of ol/L) and 2.0 μ L template DNAs, mend ddH2O to 50 μ
L.PCR response procedures：94 DEG C of 4min, 94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 1.0min, 30 circulations；Last 72 DEG C of extensions 10min.
Take the μ L of amplified production 3.0, carry out electrophoresis with 1.0% Ago-Gel, voltage is 150V 100mA 20min, electrophoresis liquid be 50 ×
TAE.After electrophoresis, dyed with ethidium bromide (EB) and observed under 10min, uviol lamp.Successful PCR primer will be expanded and deliver to Shanghai life
Work biotechnology Services Co., Ltd is sequenced, and the preferable fragment adjustment of peak type is intercepted after the 16S rDNA sequences of sequencing
After the sequence of reverse complemental, analysed and compared with the known array in GenBank, relevant bacterium is obtained from LPSN databases
The recognised standard sequence data of kind.
The laboratory ensiling evaluation of 1.5 lactic acid bacterias
The 10 plants of low temperature lactic acid bacterias filtered out, expand culture, adjustment bacteria suspension cell concentration 1 × 10 step by step9cfu/mL。
With milk stage corn and branching stage~florescence alfalfa (wilting to water content 70% or so) for test material, shred to 1~
2cm, it is fitted into special ensiling bag (30cm × 50cm × 0.14mm, every bag of 1kg), adds bacteria suspension (105Cfu/g FM), and
With additive-free processing for negative control (CK, 3mL/kg FM), saccharose treatment (S, 2%FM), potassium citrate (PC, 6.7g/kg
FM), formic acid (FA, 5mL/kg FM) and blue or green precious No. II (FS, 3g/t FM) are positive control, are mixed, using vacuum packing machine
(DZQ-600, Shanghai Shen Yue package packing machinies Co., Ltd) vacuum sealing, as (15 DEG C) of constant incubator storage 5d,
10d, 15d, 20d, 30d, 60d, 90d, 120d, open, determine fermentation quality, nutritional ingredient, the micro organism quantity of ensilage,
Specific method is with reference to national standard.Each handle 3 repetitions of each time point setting.
1.6 data analysis
Data are arranged using excel 2007；Variance analysis is carried out to data using SPSS19.0 softwares
And Multiple range test (ANOVA)；With Graph prime 5 to fermentation parameter mapping analysis；The significance level of difference is P<0.05.
2 results and analysis
The separation of 2.1 lactic acid bacterias
1990 plants of bacteriums are separated to from siberian wildrye and the natural ensilage of oat.Through Gram's staining and catalase
Reaction and physiological and biochemical property are initially identified as 127 plants of lactic acid bacteria.According to count's bodyguard handbook, pass through colonial morphology, Physiology and biochemistry
Feature, glycometabolism reaction etc. are classified as 44 plants of advantage lactic acid bacterias.
The screening of 2.2 lactic acid bacterias
In 44 plants of advantage lactobacillus inoculum fermented green juices, the absorbances of 48h corn fermented green juices for 0.102~
2.644, pH be 2.64~4.88；48h clover fermented green juices absorbance is that 0.791~2.797, pH is 2.64~4.78 (figures
1).Wherein 31 plants of bacterium quickly can utilize the soluble carbon hydrate in fermented green juice quickly to rise in value, and enter in the short period
Logarithmic phase (Fig. 2).
In screening process, 68,101,108,139,146,151, WK5, WK6, WK88, WK121, WK130, CC3, CC31
Grown Deng 13 plants of bacterium in fermented green juice poor, it is impossible to effectively reduce pH, be eliminated in the process.The production of part bacterial strain
Sour ability is relatively strong, can quickly reduce the pH value (Fig. 3) of fermented green juice.
The metabolite that different lactic bacteria strains are analyzed in fermented green juice latter stage (48h) is understood, is inoculated with different lactic acid bacteria bacterium
Lactic acid, acetic acid, propionic acid and butyric acid content difference are notable in fermented green juice after strain.Lactic acid content in corn fermented green juice is
2.15~3.40g/L, acetic acid content are 0.51~0.73g/L, and propionic acid content is 0.01~0.18g/L, total acid content 1.73
~4.11g/L, ethanol content are 1.73~4.54mg/L (Fig. 4).Lactic acid content in clover fermented green juice for 1.18~
2.85g/L, acetic acid content are 0.40~0.58g/L, and propionic acid content is 0.01~0.04g/L, total acid content is 1.83~
3.57g/L, ethanol content are 2.78~5.38mg/L (Fig. 4).By comparing, find CC1, CC7, SC2, SC4, SC7, SC29,
The strains of lactic acid bacteria of SC1, WK3, CC17 etc. 9 has a relatively low lactic acid production, higher ethanol production, is eliminated in screening process.Hair
Ferment product belongs to homofermentation based on lactic acid, with bacterial strain P-8, P-11, P-14,65,148,157,159,171,192,220
Lactic acid generation Deng 10 strains of lactic acid bacteria is fastest, and fermented green juice lactic acid content is higher (such as Fig. 5).
Strain growth situation is as shown in table 1 under different temperatures, acidity, NaCl, lactic acid condition of culture.All bacterial strains are in high temperature
Under the conditions of (45 DEG C) can not grow；Bacterial strain MU4Y-5, MU4Y-7 and MU4Y-25 can not grow in lower temperature (10 DEG C)；
Bacterial strain MU4Y-5, MU4Y-25 and M17-171 can not grow higher than 30 DEG C.Also, bacterial strain MU4Y-5, MU4Y-7, MU4Y-
25 and M17-171 can not the high lactic acid growing environment of enduring high-concentration.Part lactic acid bacteria can be quick at a lower temperature
The pH value of fermented green juice is reduced, the most suitable growth temperature is 20 DEG C (Fig. 6).
Table 1：Strain growth situation under different temperatures, acidity, NaCl, lactic acid condition of culture
Note：Strain growth inoculating strain absorbance divided by control absorbance (△ OD560=OD560 inoculations/OD560 controls) represent
(+, △ OD560>1；-, △ OD560<1)。
The drug resistance and biocidal property of bacterial strain are as shown in table 2 and table 3.Bacterial strain MQ0-2 is to amikacin, chloramphenicol, penicillin
There is stronger drug resistance with tetracycline, bacterial strain 177 has stronger drug resistance, bacterial strain to gentamicin, A Ka meter stars and tetracycline
MU4Y-8 and MU4Y-15 has stronger drug resistance to vancomycin and penicillin, and U4Y-14 has to vancomycin and chloramphenicol
Stronger drug resistance.The zymotic fluid of bacterial strain 9, P-5 and NZ-2 is without fungistatic effect.Therefore, in screening process, due to bacterial strain 9,177,
MQ0-2, P-5, MUY4-8, MUY4-14, MU4Y-15, NZ-2 etc. have stronger drug resistance or weaker biocidal property, are eliminated.
Table 2：The drug resistance of different lactic bacteria strains
Note：Antibiotic resistance represented with antibacterial circle diameter (-, 10mm (antibiotic scraps of paper diameter)；W, 10-15mm；+,
>15mm)。
The fungistatic effect of table 1-3 different strains
Note：Bacteriostatic activity represented with antibacterial circle diameter (-, 10mm (card punch mouth diameter)；W, 10-15mm；+,>
15mm)。
The identification of 2.3 lactic acid bacterias
The type strain DNA of all extractions is subjected to PCR primer amplification, being put into gel imager after agargel electrophoresis enters
Row is taken pictures and observed, and clear band (Fig. 7) occurs in 1500bp right positions, then shows to expand successfully.It is low by what is filtered out
Warm lactic acid bacteria DNA carries out PCR primer amplification, and amplified production carries out agargel electrophoresis, is put into gel imager and takes pictures and observe
(Fig. 8), by being accredited as 7 lactobacillus plantarums (L.plantarum), 1 plant of excrement chain after its 16S rRNA fragment amplification is sequenced
Coccus (E.faecium), the 1 plant of piece of resistance to ethanol coccus (P.ethanolidurans) and 1 plant of Lactobacillus paracasei
(L.paracasei) (table 4).
Table 4：The molecular biology identification of lactic acid bacteria
The laboratory ensiling evaluation of 2.4 lactic acid bacterias
2.4.1 the nutritional ingredient of ensiling raw material
Corn silage raw material dry, soluble-carbohydrate, crude protein, neutral detergent fiber, acid detergent fiber
Content and dry digestibility be respectively 281.6g/kg, 100.7g/kg DM, 113.8g/kg DM, 487.0g/kg DM,
275.8g/kg DM、608.9g/kg DM.Alfalfa ensilage raw material dry, soluble-carbohydrate, crude protein, neutral detergent
Fiber, acid detergent fiber content and dry digestibility be respectively 308.7g/kg, 70.1g/kg DM, 224.3g/kg DM,
426.3g/kg DM、381.4g/kg DM、632.6g/kg DM。
2.4.2 the microorganism group of ensiling raw material and ensilage into
Ensiling raw material is into as shown in table 5 with ensilage microorganism group.By 120d storage at normal temperature, ensilage yeast
Bacterium, filamentous fungi and Escherichia coli quantity are substantially less than ensiling raw material (P<0.05).Ensiling after bacterial strain P-14,148 and 171 are handled
Feed lactic acid bacterium number is significantly higher than CK (P<0.05).All bacterial strain processing ensilage Escherichia coli quantity are less than CK.It is degerming
Outside strain P-14,148 and 171, other bacterial strains processing ensilage filamentous fungi quantity and the not notable (P of contrast difference>0.05).Remove
Outside bacterial strain 220, other bacterial strains processing ensilage Escherichia coli quantity is substantially less than CK (P<0.05).Bacterial strain handles ensilage
Lactic acid bacterium number is higher than CK, and bacterial strain P-14,148 and 171 handle ensilage lactic acid bacterium number and are significantly higher than the processing of other bacterial strains
(P<0.05)。
Table 5：Lactic acid bacteria to ensilage microorganism group into influence (log cfu/g FM)
Influence of the 2.3 advantage lactic acid bacterias to ensilage fermentation quality
The fermentation quality of corn Silage is as shown in table 6.In addition to bacterial strain 65 and 157, other bacterial strains can significantly drop
Low corn Silage pH value (P<0.05) the ensilage pH value that, bacterial strain 148 is handled is minimum (4.16).Low temperature lactic acid bacteria is handled
Corn Silage lactic acid content be significantly higher than CK (P<0.05), and ammoniacal nitrogen/total nitrogen ratio, acetic acid and propionic acid content shows
Work is higher than CK (P<0.05).In corn Silage, bacterial strain 148 handles lactic acid content highest (41.0g/kg DM), at bacterial strain 65
It is minimum (5.8g/kg DM) to manage acetic acid content, the processing propionic acid content of bacterial strain 171 is minimum (0.4g/kg DM), and bacterial strain 148 is handled always
Acid content highest (48.5g/kg DM), it is minimum (68.6g/kgTN) that bacterial strain 171 handles ammoniacal nitrogen/total nitrogen ratio.All maize stalks
Store in feed and be not detected by butyric acid.Overall merit finds that 10 strains of lactic acid bacteria filtered out are added in ensilage, bacterial strain P-
11st, 65,157,159 and 220 acid producing abilities are relatively weak, and ammoniacal nitrogen/total nitrogen ratio is of a relatively high, is washed in a pan in screening process
Eliminate.
Table 6：Influence of the lactic acid bacteria to corn Silage fermentation quality
Note：SEM, mean standard error；Different lowercase letter mean differences significantly (P in same row<0.05) under,
Together.
The fermentation quality of alfalfa silage is as shown in table 7.Bacterial strain processing can significantly reduce alfalfa ensilage and raise pH value
With ammoniacal nitrogen/total nitrogen ratio (P<0.05) the alfalfa silage pH value that, bacterial strain 171 is handled is minimum (4.31), at bacterial strain P-14
It is minimum (82.8g/kg TN) to manage alfalfa silage ammoniacal nitrogen/total nitrogen ratio.All bacterial strain processing alfalfa silage lactic acid contain
Amount is significantly higher than control (P<0.05), bacterial strain 148 handles alfalfa silage lactic acid content highest (37.2g/kg DM).It is degerming
Outside strain P-11,148,171 and 220, other bacterial strains can significantly reduce alfalfa silage acetic acid content (P<0.05).Bacterial strain
157 processing alfalfa silage acetic acid contents are minimum (5.1g/kg DM).In addition to bacterial strain P-14 and 148, other bacterial strains processing lucerne
Mu ensilage propionic acid content is significantly higher than CK (P<0.05).Bacterial strain processing alfalfa silage butyric acid content is substantially less than CK (P
<0.05), the processing of bacterial strain P-14 and 171 alfalfa silage butyric acid content is substantially less than other bacterial strains processing (P<0.05).Bacterial strain
The alfalfa silage butyric acid content of the processing of P-8, P-11,65,157,159,192 and 220 is higher than 1.0g/kg DM, quality compared with
Difference, it is eliminated in screening process.The fermentation quality of bacterial strain P-14,148 and 171 is preferable, beneficial to the preservation of ensilage.
Table 7：Influence of the lactic acid bacteria to alfalfa silage fermentation quality
Table 2-3.The effects of LAB isolates on the fermentation quality of
alfalfa silage
Influence of the lactic acid bacteria to ensilage pH value is as shown in Figure 9.The ensilage storage 20d pH of inoculating lactic acid bacterium
Value maintains relatively stable (P>0.05) it is blue or green precious No. II, to be not added with or add sucrose, potassium citrate and commercial lactic acid bacterium preparation
Ensilage storage 30d pH value just maintain relatively stablize (P>0.05).
Influence of the lactic acid bacteria to ensilage lactic acid content is as shown in Figure 10.Relative to CK and PC, all low temperature lactic acid bacterias
It can speed up the generation (P of corn Silage lactic acid<0.05)；Relative to S and FA, bacterial strain P-14, bacterial strain 148 and bacterial strain 171
The corn Silage storage 20d lactic acid contents of processing maintain relatively stable (P>0.05).Relative to CK, S, PC, FA and
FS, the lactic acid that lactic acid bacteria can speed up alfalfa silage produce (P<0.05)；The clover of the processing of bacterial strain P-14,148 and 171 is blue or green
Store feed storage 20d lactic acid contents and maintain relatively stable (P>0.05).
Influence of the lactic acid bacteria to ensilage ammoniacal nitrogen/total nitrogen ratio is as shown in figure 11.With the increase of period of storage, S,
Corn Silage ammoniacal nitrogen/total nitrogen ratio of PC, FA and FS processing continues to increase (P<0.05), the maize stalks of lactic acid bacteria processing
20d dramatically increases (P before storage feed ammoniacal nitrogen/total nitrogen ratio storage<0.05), maintain to stablize (P relatively afterwards>0.05).At PC
Alfalfa silage ammoniacal nitrogen/total nitrogen ratio of reason continues to increase (P<0.05), other processing alfalfa silage ammoniacal nitrogens/
30d dramatically increases (P before the storage of total nitrogen ratio<0.05).
Influence of the 2.4 advantage lactic acid bacterias to ensilage nutritional ingredient
Influence of the lactic acid bacteria to corn Silage nutritional ingredient is as shown in table 8.The corn Silage of bacterial strain processing can
Soluble carbohydrate and acid detergent fiber content and the not notable (P of CK differences>0.05) corn silage that, bacterial strain 159 is handled
Feed water-soluble carbohydrate content is minimum (25.0g/kg DM).The corn Silage of the processing of bacterial strain P-14,149 and 171
Crude protein content is significantly higher than CK (P<0.05) the corn Silage crude protein content that, bacterial strain P-8 and bacterial strain 157 are handled is notable
Less than CK (P<0.05), the corn Silage crude protein content of other bacterial strains processing and the not notable (P of CK differences>0.05).Respectively
The corn Silage digestibility of bacterial strain processing is significantly higher than CK (P<0.05), the jade of the processing of bacterial strain P-14,148,171 and 192
Rice ensilage Vitro Dry Matter Digestibility is higher than other bacterial strains (P<0.05).
Table 8：Influence of the lactic acid bacteria to corn Silage nutritional ingredient
Influence of the lactic acid bacteria to alfalfa silage nutritional ingredient is as shown in table 9.The alfalfa silage of the processing of bacterial strain 148
Soluble-carbohydrate matter content is 24.8g/kg DM, is significantly higher than CK (P<0.05).In addition to bacterial strain 65 and 157, other bacterium
The alfalfa silage crude protein content of strain processing is significantly higher than CK (P<0.05).The alfalfa silage of all bacterial strain processing is done
Substance digestion rate is substantially less than CK, Vitro Dry Matter Digestibility highest (the 608.3g/kg DM, P of the processing of bacterial strain 148<0.05).
Table 9：Influence of the lactic acid bacteria to alfalfa silage nutritional ingredient
3 conclusions
127 strains of lactic acid bacteria are isolated from 86 parts of natural ensiling samples, are made up of Physiology and biochemistry, tunning, resistance
Property, biocidal property etc. filter out 10 plants of low temperature lactic acid bacterias, respectively 7 lactobacillus plantarums, 1 plant of streptococcus fecalis, the 1 plant of piece of resistance to ethanol ball
Bacterium and 1 plant of plan Lactobacillus casei.The optimum growth temperature of these bacterial strains is 15 DEG C, has the features such as the speed of growth is fast, production acid is more,
Advantage is provided for ensiling modulation.Wherein, bacterial strain P-14, bacterial strain 148 and bacterial strain 171 can be rapidly completed under cryogenic
Ferment (≤20d), increase lactic acid and total acid content, reduce pH value, ammoniacal nitrogen/total nitrogen ratio, propionic acid and butyric acid content, improve blue or green
Feed fermentation quality is store, ensilage crude protein content and Vitro Dry Matter Digestibility is improved, can make in ensiling modulated process
Used for additive.
Bacterial strain P-14 is the piece of resistance to ethanol coccus (Pediococcus ethanolidurans), deposit number CGMCC
No.14183；Bacterial strain 148 is Lactobacillus plantarum (lactobacillus plantarum), deposit number CGMCC
No.14117；Bacterial strain 171 is Lactobacillus paracasei (Lactobacillus paracasei), deposit number CGMCC
No.14116。
Embodiment described above only expresses the embodiment of the application, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the application protection domain can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, on the premise of technical scheme design is not departed from, various modifications and improvements can be made, these belong to this
The protection domain of application.
Claims (2)
1. 3 strains of lactic acid bacteria of Alpine-arctic Pastoral ensiling, they are the plant breast bar that deposit number is CGMCC No.14117 respectively
Bacterium (lactobacillus plantarum) 148, deposit number are CGMCC No.14116 Lactobacillus paracasei
(Lactobacillus paracasei) 171, deposit number are the CGMCC No.14183 piece of resistance to ethanol coccus
(Pediococcus ethanolidurans)P-14。
2. application of 3 strains of lactic acid bacteria of the Alpine-arctic Pastoral ensiling according to belonging to claim 1 in ensiling.
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| CN114276949A (en) * | 2021-11-10 | 2022-04-05 | 丰唐生态农业科技研发(山东)有限公司 | Lactobacillus paracasei and application thereof in preparation of broussonetia papyrifera silage |
| CN114621900A (en) * | 2022-04-21 | 2022-06-14 | 四川农业大学 | Lactobacillus paracasei and application thereof |
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