CN110157636A - A kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis - Google Patents
A kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis Download PDFInfo
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Abstract
It is preserved in China typical culture collection center the invention discloses a kind of bacillus licheniformis, screening technique, using and containing the feed of the bacillus licheniformis, the bacillus licheniformis, deposit number is CCTCC M 2019165.Screening technique includes collecting soil sample, the culture of bacterial strain and divide bacterium, bacillus licheniformis isolates and purifies, Molecular Identification, biochemical identification, identified according to the result of Molecular Identification and biochemical identification and to filter out the bacillus licheniformis, the bacillus licheniformis filtered out is used to prepare feed mulberry ensilage, the protein content of feed is improved, animal is set to have sufficient nitrogen source to supply, its crude fiber content needs to be declined, animal is set to possess more easily decomposed carbohydrate, the ratio of its ammoniacal nitrogen and total nitrogen needs to be declined, the albumen and amino acid degree of decomposition for making ensilage reduce, form better ensilage quality.
Description
Technical field
The present invention relates to microbe to screen and applied technical field, in particular to a kind of bacillus licheniformis, screening side
Method, the feed using and containing the bacillus licheniformis.
Background technique
Mulberry tree has more than 5000 years cultivation histories in China, and possesses maximum cultivated area in China, and mulberry leaf are rich
Containing nutriments such as protein, vitamin, polysaccharide, mineral elements, protein of folium mori matter content is higher, and about 15~30%, it is fresh
Leaf moisture content is high, it is difficult to which long-term preservation, ensiling at present is its most appropriate storage method, but is stored with such mode
Mulberry leaf feed palatability it is poor, protein utilization is low, the ratio of ammoniacal nitrogen and total nitrogen is high, crude fibre cannot effectively drop
Solution is at sugar, so that mulberry leaf cannot function as conventional feed and be fed with animal extensively.Therefore, if can using microorganism solid fermentation come
Improve this situation, then by with great economic significance, in order to reach this target, firstly, need to find securely and reliably again
It can be by the bacterial strain of animal use.Secondly, the ensilage mulberry of bacterium to be added need using microorganism solid fermentation come
Realize the quality that can preferably improve feed.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of bacillus licheniformis, screening technique, answer
With and containing the bacillus licheniformis feed, the feeding strain in mulberry leaf soil can be filtered out and identified, micro- life is utilized
Bacillus licheniformis is applied to by object solid-state fermentation technology to be prepared in feed mulberry ensilage, and the protein for improving feed contains
Amount, reduces the ratio of ammoniacal nitrogen and total nitrogen, and so that animal is preferably obtained nitrogen at sugar more lignocellulose degradations
Source absorbs digestible carbohydrate.The bacillus licheniformis is preserved in China typical culture collection center, protects
Hiding number is CCTCC M 2019165.
The purpose of the present invention is achieved through the following technical solutions:
A kind of screening technique of bacillus licheniformis, comprising the following steps:
S1, it collects soil sample: 90~110g of mulberry leaf planting site soil is collected using line-of-sight course；
S2, bacterial strain culture and divide bacterium: the soil sample being collected into is placed in the sterile taper of the PBS equipped with 800~1000ml
Bottle in, shake up 18~22 minutes and soil sample be diluted to sample suspension, after according to 10 times of coubling dilutions to the sample suspension into
The dilution of one step；Choosing 100ml dilution is 10-4~10-5Sample suspension be coated on TSA plate, the TSA plate is set
22~26 hours are cultivated in 37 DEG C of insulating boxs, obtain bacterium colony；
S3, bacillus licheniformis isolate and purify: there is the Huang of protuberance fold in the centre that 3~5mm is screened on the TSA plate
White flat colony, and by after TSB Liquid Culture, taking fresh bacterium solution in optical microscopy 100 × times oily microscopic observation bacterium colony
Form, the bacterium colony that the length for filtering out elongated rod shape is 2.0~6.5 μm, width is 0.3~1.0 μm, dyeing is in bluish violet, then
It is isolated purifying and obtains the fresh strain of plating medium bacillus licheniformis；
S4, Molecular Identification: the Molecular Identification is tested by following two and is realized:
S401, bacterium progress 16S rDNA Testing and appraisal is expanded by primer 2 7F/1492R, by sequencing result and NCBI
It compares, whether preliminary judgement is bacillus；
S402, pass through bacillus licheniformis specific primer BL1/BL2 specific amplification gene gyrase B, examined
Identification is surveyed, whether it is bacillus licheniformis according to detection structure preliminary judgement that amplification size is 600~700bp；
S5, biochemical identification: the biochemical identification is five kinds below carrying out the strain that preliminary judgement is bacillus licheniformis
Test is to identify:
S501, Gram stain test: the fresh strain of the bacillus licheniformis is subjected to Gram's staining, optics is aobvious
The microscopic examination result of micro mirror shows that the strain is the gram-positive bacteria that form is in direct rod shape；
S502, can adaptability elongate member test: by the fresh strain of the bacillus licheniformis be placed in temperature be 15~55 DEG C,
PH value carries out growth test under conditions of being 4.0~8.0；
S503, sugar fermentating test: using bacterium micro biochemical assessor to the fresh strain of the bacillus licheniformis in 37
DEG C culture, respectively carry out lactose, maltose, mannose, sucrose and glucose fermentation test, cultivate 16 in corresponding fermentation tube
~18 hours；
S504, Xi Mengshi citrate measurement test: using bacterium micro biochemical assessor to purified lichens gemma
Bacillus is cultivated in 37 DEG C, carries out citrate test, and 22~24 hours are cultivated in citrate fermentation tube；
S505, H2S measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 37 DEG C
Culture carries out H2S measurement test, in H223~25 hours are cultivated in S fermentation tube；
S506, gelatin measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 4 DEG C
Culture carries out gelatin measurement test, 23~24 hours is cultivated in gelatin fermentation tube.
S6, identify the bacillus licheniformis described in filtering out according to the result of Molecular Identification and biochemical identification.
Further, the line-of-sight course collects the specific steps of mulberry leaf planting site soil are as follows:
S101, the soil work to grow densely respectively in the north of mulberry leaf planting base, southwestern face and the southeast selection mulberry leaf
For sampled point；
S102, the north in the sampled point, southwestern face and the southeast acquisition soil sample are simultaneously uniformly mixed.
Further, the specific steps of the Gram stain test are as follows: first by the fresh strain of the bacillus licheniformis
Smear is fixed through flame, washes away dye liquor with clear water after adding crystal violet solution to contaminate 1~2 minute；Again plus iodine solution contaminates 0.5~1 minute,
Clear water washes away dye liquor, and it is slide 10~30 seconds uniformly to be shaken after 95% ethyl alcohol up to no purple falls off, then use clear water that concentration, which is added,
Wash away dye liquor；Then gaza's Huang redyes liquid again, contaminates 1~1.5 minute, continues to wash away dye liquor with clear water, carries out oil after smear is dry
Mirror microscopy.
Further, the sugar fermentating test specific steps are as follows: be first turned on corresponding ampere bottle, draw 50 μ l test examination
Agent is packed into 2ml sterile EP tube, adds fresh 10 μ l of Bacillus licheniformis liquid in 37 DEG C of 18 hours of culture.
Further, the specific steps of the Xi Mengshi citrate measurement test are as follows: pure with the white pipette tips picking of 10 μ l
In 37 DEG C of 24 hours of culture in the bacillus licheniformis inoculation ampere bottle of change, test result shows the bacillus licheniformis
It cannot be grown in citrate.
Further, the specific steps of institute H2S measurement test are as follows: with the purified lichens gemma of the white pipette tips picking of 10 μ l
Bacillus was inoculated in ampere bottle in 37 DEG C of 24 hours of culture, and test result shows that the bacillus licheniformis cannot generate H2S.
Further, the specific steps of the gelatin measurement test are as follows: with the purified lichens bud of the white pipette tips picking of 10 μ l
Spore bacillus was inoculated in ampere bottle in 4 DEG C of 24 hours of culture, and it is bright that test result shows that the bacillus licheniformis cannot decompose
Glue.
Application of the bacillus licheniformis in terms of preparing ensilage, prepare ensilage when to ensiling
Bacillus licheniformis is added in feedstuff to ferment.
A kind of feed, including the bacillus licheniformis.
Further, the feed is that 5 × 10 are added in the new fresh mulberry leaf and petiole of every 1000~1200g weight6~5.5
×106The bacillus licheniformis of cfu/g fastens sack after manual compaction, is placed in fermentation in horizental silo with rubber belt sealing and obtains.
The beneficial effects of the present invention are: the BL-DY that the present invention filters out can effectively improve feed mulberry Silage Quality,
During mulberry leaf silage fermentation, although the dry matter and crude ash content and control group difference of the experimental group of addition BL-DY bacterial strain
Less, crude fat content significantly reduces, and illustrates that it is utilized by bacterium and produces energy；Add the experimental group of BL-DY bacterial strain
Crude protein content significantly improves, and can fully ensure that the nitrogen source supply of animal；The experimental group for adding BL-DY bacterial strain, which significantly reduces, raises
Expect coarse-fibred content, be the cellulose degraded secreted by bacterium, the carbon absorbed can be easier by animal body by being formed
Hydrate improves animal to coarse-fibred digestion, absorption and utilization rate.Meanwhile the ammonia of the fermentation mulberry leaf of BL-DY is added
State nitrogen/total nitrogen ratio decline, reflecting protein and amino acid degree of decomposition in ensilage reduces, and illustrates ensilage
Quality to improve.
Detailed description of the invention
Fig. 1 is the screening technique of bacillus licheniformis of the invention and the flow diagram of application；
Fig. 2 is the schematic diagram that line-of-sight course of the present invention collects soil sample；
Fig. 3 isolates and purifies figure for bacillus licheniformis of the present invention；
Fig. 4 is bacillus licheniformis primer amplified PCR of the present invention figure；
Fig. 5 is the aspect graph of bacillus licheniformis of the invention under an optical microscope；
Specific embodiment
Below in conjunction with embodiment, technical solution of the present invention is clearly and completely described, it is clear that described
Embodiment is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ability
Field technique personnel every other embodiment obtained under the premise of not making the creative labor, belongs to guarantor of the present invention
The range of shield.
Refering to fig. 1-5, the present invention provides a kind of technical solution:
Bacillus licheniformis of the invention is preserved in China typical culture collection center on March 18th, 2019, institute
The address for stating collection is the Wuhan Wuhan University, China, and deposit number is CCTCC NO:M 2019165.
Embodiment 1
As shown in Figure 1, a kind of screening technique of bacillus licheniformis, comprising the following steps:
S1, it collects soil sample: mulberry leaf planting site soil 90g is collected using line-of-sight course；
As shown in Fig. 2, the line-of-sight course collects the specific steps of mulberry leaf planting site soil are as follows:
S101, the soil work to grow densely respectively in the north of mulberry leaf planting base, southwestern face and the southeast selection mulberry leaf
Stain for sampled point, in the sampled point such as Fig. 2 ● shown；
S102, in the sampled point ● north, southwestern face and the southeast acquire soil sample, such as open circles О institute in Fig. 2
Show, and is uniformly mixed.
S2, bacterial strain culture and divide bacterium: the soil sample being collected into is placed in the sterile conical flask of the PBS equipped with 800ml,
Shake up 18 minutes and soil sample be diluted to sample suspension, after the sample suspension is further diluted according to 10 times of coubling dilutions；
Choosing 100ml dilution is 10-4Sample suspension be coated on TSA plate, the TSA plate is placed in 37 DEG C of insulating boxs
22 hours are cultivated, bacterium colony is obtained；
S3, bacillus licheniformis isolate and purify: there is the yellow-white of protuberance fold in the centre that 3mm is screened on the TSA plate
Flat colony, and by after TSB Liquid Culture, taking fresh bacterium solution in optical microscopy 100 × times oily microscopic observation bacterium colony shape
State, the length for filtering out elongated rod shape is 2.0 μm, width is 0.3 μm, dyeing is in the bacterium colony of bluish violet, then is isolated and purifies
To the fresh strain of plating medium bacillus licheniformis；
S4, Molecular Identification: the Molecular Identification is tested by following two and is realized:
S401, bacterium progress 16S rDNA Testing and appraisal is expanded by primer 2 7F/1492R, by sequencing result and NCBI
It compares, whether preliminary judgement is bacillus；
S402, pass through bacillus licheniformis specific primer BL1/BL2 specific amplification gene gyrase B, examined
Identification is surveyed, whether it is bacillus licheniformis according to detection structure preliminary judgement that amplification size is 600bp；
S5, biochemical identification: the biochemical identification is five kinds below carrying out the strain that preliminary judgement is bacillus licheniformis
Test is to identify:
S501, Gram stain test: the fresh strain of the bacillus licheniformis is subjected to Gram's staining, optics is aobvious
The microscopic examination result of micro mirror shows that the strain is the gram-positive bacteria that form is in direct rod shape；
The specific steps of the Gram stain test are as follows: first by the fresh strain smear of the bacillus licheniformis through fire
Flame is fixed, and washes away dye liquor with clear water after adding crystal violet solution to contaminate 1 minute；Again plus iodine solution contaminates 0.5 minute, and clear water washes away dye liquor, adds
Entering concentration is slide 10 seconds uniformly to be shaken after 95% ethyl alcohol up to no purple falls off, then wash away dye liquor with clear water；Then gaza again
Huang redyes liquid, contaminates 1 minute, continues to wash away dye liquor with clear water, carries out oil mirror microscopy after smear is dry.
S502, can adaptability elongate member test: by the fresh strain of the bacillus licheniformis be placed in temperature be 15 DEG C, pH value
Growth test is carried out under conditions of being 4.0；
S503, sugar fermentating test: using bacterium micro biochemical assessor to the fresh strain of the bacillus licheniformis in 37
DEG C culture, respectively carry out lactose, maltose, mannose, sucrose and glucose fermentation test, respectively in lactose fermentation tube, malt
16 hours are cultivated in sugared fermentation tube, mannose fermentation tube, sucrose fermentation tube and glucose fermentation tube；
The sugar fermentating test specific steps are as follows: be first turned on corresponding ampere bottle, draw 50 μ l test reagents and be packed into 2ml
Sterile EP tube adds fresh 10 μ l of Bacillus licheniformis liquid and cultivates in 37 DEG C.
S504, Xi Mengshi citrate measurement test: using bacterium micro biochemical assessor to purified lichens gemma
Bacillus is cultivated in 37 DEG C, carries out citrate test, and 22 hours are cultivated in citrate fermentation tube；
The specific steps of the Xi Mengshi citrate measurement test are as follows: with the white purified lichens of pipette tips picking of 10 μ l
Bacillus is inoculated in ampere bottle and cultivates in 37 DEG C, and test result shows that the bacillus licheniformis cannot be in citrate
Growth.
S505、H2S measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 37 DEG C
Culture carries out H2S measurement test, in H223 hours are cultivated in S fermentation tube；
Institute H2The specific steps of S measurement test are as follows: with the purified bacillus licheniformis inoculation peace of the white pipette tips picking of 10 μ l
It trains in bottle in 37 DEG C of training samples, test result shows that the bacillus licheniformis cannot generate H2S.
S506, gelatin measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 4 DEG C
Culture carries out gelatin measurement test, 23 hours is cultivated in gelatin fermentation tube；
The specific steps of the gelatin measurement test are as follows: connect with the white purified bacillus licheniformis of pipette tips picking of 10 μ l
In 4 DEG C of 23 hours of culture in kind ampere bottle, test result shows that the bacillus licheniformis cannot decompose gelatin.
S6, identify the bacillus licheniformis described in filtering out according to the result of Molecular Identification and biochemical identification.
As shown in Figure 1, application of the bacillus licheniformis in terms of preparing ensilage, is preparing ensilage
When into ensilage raw material add bacillus licheniformis to ferment.
A kind of feed, including bacillus licheniformis of the present invention.
Preferably, the bacillus licheniformis can also be used to prepare feed mulberry ensilage, and preparation method includes following
Step:
(1) processing and grouping of new fresh mulberry leaf: being cut into 3cm for new fresh mulberry leaf and petiole, and ensiling is packed into after being mixed evenly and is raised
Expect fermentation bag, every bag of weight 1000g, into every bag of mulberry leaf according to 5 × 106The standard of cfu/g adds bacillus licheniformis, mixes 3
Minute, sack is fastened after manual compaction, is placed in horizental silo under the conditions of 36~38 DEG C and is fermented 7~56 days with rubber belt sealing
To fermented feed sample；
(2) fermented feed sample treatment: the 7th, 14,28,56 day after ensiling, 20g is taken to send out from fermentation bag at random
Ferment Feed Sample is put into 250ml conical flask, and 100ml deionized water is added in conical flask, extracts 20 under the conditions of temperature is 4 DEG C
A hour is successively filtered with double gauze incoming call filter paper, filtrate is distributed into several test tubes, cold storage is ready for use in -20 DEG C of refrigerators
Remaining ensilage is dried in the measurement of subsequent ammoniacal nitrogen, and weighing is ready for use on subsequent measurements dry matter, crude protein, thick rouge
Fat, coarse ash and crude fibre；
(3) testing index and method: the ammoniacal nitrogen uses phenol-sodium hypochlorite colorimetric method for determining, and the albumen contains
It measures and uses Kjeldahl's method surely, referring to GB/T 6432-1994 " crude protein determining method in feed "；The crude fat content
Measurement uses soxhlet extraction, referring to GB/T 6433-2006 " measuring method of crude fat in feed "；The crude ash content
Measurement uses calcination method, referring to GB/T 6438-2007 " measuring method of coarse ash in feed "；The crude fiber content measurement
Using the cooking method that disappears, referring to GB/T 6434-2006 " coarse-fibred measuring method in feed ".
Embodiment 2
As shown in Figure 1, a kind of screening technique of bacillus licheniformis, comprising the following steps:
S1, it collects soil sample: mulberry leaf planting site soil 110g is collected using line-of-sight course；
As shown in Fig. 2, the line-of-sight course collects the specific steps of mulberry leaf planting site soil are as follows:
S101, the soil work to grow densely respectively in the north of mulberry leaf planting base, southwestern face and the southeast selection mulberry leaf
Stain for sampled point, in the sampled point such as Fig. 2 ● shown；
S102, in the sampled point ● north, southwestern face and the southeast acquire soil sample, such as open circles О institute in Fig. 2
Show, and is uniformly mixed.
S2, bacterial strain culture and divide bacterium: the soil sample being collected into is placed in the sterile conical flask of the PBS equipped with 1000ml,
Shake up 22 minutes and soil sample be diluted to sample suspension, after the sample suspension is further diluted according to 10 times of coubling dilutions；
Choosing 100ml dilution is 10-5Sample suspension be coated on TSA plate, the TSA plate is placed in 37 DEG C of insulating boxs
26 hours are cultivated, bacterium colony is obtained；
S3, bacillus licheniformis isolate and purify: there is the yellow-white of protuberance fold in the centre that 5mm is screened on the TSA plate
Flat colony, and by after TSB Liquid Culture, taking fresh bacterium solution in optical microscopy 100 × times oily microscopic observation bacterium colony shape
State, the length for filtering out elongated rod shape is 6.5 μm, width is 1.0 μm, dyeing is in the bacterium colony of bluish violet, then is isolated and purifies
To the fresh strain of plating medium bacillus licheniformis；
S4, Molecular Identification: the Molecular Identification is tested by following two and is realized:
S401, bacterium progress 16S rDNA Testing and appraisal is expanded by primer 2 7F/1492R, by sequencing result and NCBI
It compares, whether preliminary judgement is bacillus；
S402, pass through bacillus licheniformis specific primer BL1/BL2 specific amplification gene gyrase B, examined
Identification is surveyed, whether it is bacillus licheniformis according to detection structure preliminary judgement that amplification size is 700bp；
S5, biochemical identification: the biochemical identification is five kinds below carrying out the strain that preliminary judgement is bacillus licheniformis
Test is to identify:
S501, Gram stain test: the fresh strain of the bacillus licheniformis is subjected to Gram's staining, optics is aobvious
The microscopic examination result of micro mirror shows that the strain is the gram-positive bacteria that form is in direct rod shape；
The specific steps of the Gram stain test are as follows: first by the fresh strain smear of the bacillus licheniformis through fire
Flame is fixed, and washes away dye liquor with clear water after adding crystal violet solution to contaminate 2 minutes；Again plus iodine solution contaminates 1 minute, and clear water washes away dye liquor, is added
Concentration is slide 30 seconds uniformly to be shaken after 95% ethyl alcohol up to no purple falls off, then wash away dye liquor with clear water；Then gaza is yellow again
Liquid is redyed, contaminates 1.5 minutes, continues to wash away dye liquor with clear water, carries out oil mirror microscopy after smear is dry.
S502, can adaptability elongate member test: by the fresh strain of the bacillus licheniformis be placed in temperature be 55 DEG C, pH value
Growth test is carried out under conditions of being 8.0；
S503, sugar fermentating test: using bacterium micro biochemical assessor to the fresh strain of the bacillus licheniformis in 37
DEG C culture, respectively carry out lactose, maltose, mannose, sucrose and glucose fermentation test, respectively in lactose fermentation tube, malt
18 hours are cultivated in sugared fermentation tube, mannose fermentation tube, sucrose fermentation tube and glucose fermentation tube；
The sugar fermentating test specific steps are as follows: be first turned on corresponding ampere bottle, draw 50 μ l test reagents and be packed into 2ml
Sterile EP tube adds fresh 10 μ l of Bacillus licheniformis liquid and cultivates in 37 DEG C.
S504, Xi Mengshi citrate measurement test: using bacterium micro biochemical assessor to purified lichens gemma
Bacillus is cultivated in 37 DEG C, carries out citrate test, and 24 hours are cultivated in citrate fermentation tube；
The specific steps of the Xi Mengshi citrate measurement test are as follows: with the white purified lichens of pipette tips picking of 10 μ l
Bacillus is inoculated in ampere bottle and cultivates in 37 DEG C, and test result shows that the bacillus licheniformis cannot be in citrate
Growth.
S505, H2S measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 37 DEG C
Culture carries out H2S measurement test, in H225 hours are cultivated in S fermentation tube；
The specific steps of institute H2S measurement test are as follows: with the purified bacillus licheniformis inoculation peace of the white pipette tips picking of 10 μ l
It trains in bottle and is cultivated in 37 DEG C, test result shows that the bacillus licheniformis cannot generate H2S.
S506, gelatin measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 4 DEG C
Culture carries out gelatin measurement test, 24 hours is cultivated in gelatin fermentation tube；
The specific steps of the gelatin measurement test are as follows: connect with the white purified bacillus licheniformis of pipette tips picking of 10 μ l
It is cultivated 24 hours in kind ampere bottle in 4 DEG C, test result shows that the bacillus licheniformis cannot decompose gelatin.
S6, identify the bacillus licheniformis described in filtering out according to the result of Molecular Identification and biochemical identification.
Application of the bacillus licheniformis in terms of preparing ensilage, prepare ensilage when to ensiling
Bacillus licheniformis is added in feedstuff to ferment.
A kind of feed, including bacillus licheniformis of the present invention.
Preferably, the bacillus licheniformis can also be used to prepare feed mulberry ensilage, and preparation method includes following
Step:
(1) processing and grouping of new fresh mulberry leaf: being cut into 5cm for new fresh mulberry leaf and petiole, and ensiling is packed into after being mixed evenly and is raised
Expect fermentation bag, every bag of weight 1200g, into every bag of mulberry leaf according to 5.5 × 106The standard of cfu/g adds bacillus licheniformis, mixes
Even 6 minutes, sack is fastened after manual compaction, is placed in horizental silo to ferment 7~56 days under the conditions of 38 DEG C with rubber belt sealing and is obtained
Fermented feed sample；
(2) fermented feed sample treatment: the 7th, 14,28,56 day after ensiling, 20g is taken to send out from fermentation bag at random
Ferment Feed Sample is put into 250ml conical flask, and 100ml deionized water is added in conical flask, extracts 24 under the conditions of temperature is 5 DEG C
A hour is successively filtered with double gauze incoming call filter paper, filtrate is distributed into several test tubes, cold storage is ready for use in -20 DEG C of refrigerators
Remaining ensilage is dried in the measurement of subsequent ammoniacal nitrogen, and weighing is ready for use on subsequent measurements dry matter, crude protein, thick rouge
Fat, coarse ash and crude fibre；
(3) testing index and method: with embodiment 1.
Embodiment 3
As shown in Figure 1, a kind of screening technique of bacillus licheniformis, comprising the following steps:
S1, it collects soil sample: mulberry leaf planting site soil 100g is collected using line-of-sight course；
As shown in Fig. 2, the line-of-sight course collects the specific steps of mulberry leaf planting site soil are as follows:
S101, the soil work to grow densely respectively in the north of mulberry leaf planting base, southwestern face and the southeast selection mulberry leaf
Stain for sampled point, in the sampled point such as Fig. 2 ● shown；
S102, in the sampled point ● north, southwestern face and the southeast acquire soil sample, such as open circles О institute in Fig. 2
Show, and is uniformly mixed.
S2, bacterial strain culture and divide bacterium: the soil sample being collected into is placed in the sterile conical flask of the PBS equipped with 900ml,
Shake up 20 minutes and soil sample be diluted to sample suspension, after the sample suspension is further diluted according to 10 times of coubling dilutions；
Choosing 100ml dilution is 10-4~10-5Sample suspension be coated on TSA plate, the TSA plate is placed in 37 DEG C of perseverances
24 hours are cultivated in incubator, obtain bacterium colony；
S3, bacillus licheniformis isolate and purify: there is the yellow-white of protuberance fold in the centre that 4mm is screened on the TSA plate
Flat colony, and by after TSB Liquid Culture, taking fresh bacterium solution in optical microscopy 100 × times oily microscopic observation bacterium colony shape
State, the length for filtering out elongated rod shape is 4.0 μm, width is 0.65 μm, dyeing is in the bacterium colony of bluish violet, then is isolated purifying
Obtain the fresh strain of plating medium bacillus licheniformis；
S4, Molecular Identification: the Molecular Identification is tested by following two and is realized:
S401, bacterium progress 16S rDNA Testing and appraisal is expanded by primer 2 7F/1492R, by sequencing result and NCBI
It compares, whether preliminary judgement is bacillus；
S402, pass through bacillus licheniformis specific primer BL1/BL2 specific amplification gene gyrase B, examined
Identification is surveyed, whether it is bacillus licheniformis according to detection structure preliminary judgement that amplification size is 650bp；
S5, biochemical identification: the biochemical identification is five kinds below carrying out the strain that preliminary judgement is bacillus licheniformis
Test is to identify:
S501, Gram stain test: the fresh strain of the bacillus licheniformis is subjected to Gram's staining, optics is aobvious
The microscopic examination result of micro mirror shows that the strain is the gram-positive bacteria that form is in direct rod shape；
The specific steps of the Gram stain test are as follows: first by the fresh strain smear of the bacillus licheniformis through fire
Flame is fixed, and washes away dye liquor with clear water after adding crystal violet solution to contaminate 1.5 minutes；Again plus iodine solution contaminates 0.8 minute, and clear water washes away dye liquor,
It is slide 20 seconds uniformly to be shaken after 95% ethyl alcohol up to no purple falls off, then wash away dye liquor with clear water that concentration, which is added,；Then add again
Husky of common dye redyes liquid, contaminates 1.25 minutes, continues to wash away dye liquor with clear water, carries out oil mirror microscopy after smear is dry.
S502, can adaptability elongate member test: by the fresh strain of the bacillus licheniformis be placed in temperature be 35 DEG C, pH value
Growth test is carried out under conditions of being 6.0；
S503, sugar fermentating test: using bacterium micro biochemical assessor to the fresh strain of the bacillus licheniformis in 37
DEG C culture, respectively carry out lactose, maltose, mannose, sucrose and glucose fermentation test, respectively in lactose fermentation tube, malt
17 hours are cultivated in sugared fermentation tube, mannose fermentation tube, sucrose fermentation tube and glucose fermentation tube；
The sugar fermentating test specific steps are as follows: be first turned on corresponding ampere bottle, draw 50 μ l test reagents and be packed into 2ml
Sterile EP tube adds fresh 10 μ l of Bacillus licheniformis liquid and cultivates in 37 DEG C.
S504, Xi Mengshi citrate measurement test: using bacterium micro biochemical assessor to purified lichens gemma
Bacillus is cultivated in 37 DEG C, carries out citrate test, and 23 hours are cultivated in citrate fermentation tube；
The specific steps of the Xi Mengshi citrate measurement test are as follows: with the white purified lichens of pipette tips picking of 10 μ l
Bacillus is inoculated in ampere bottle and cultivates in 37 DEG C, and test result shows that the bacillus licheniformis cannot be in citrate
Growth.
S505, H2S measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 37 DEG C
Culture carries out H2S measurement test, in H224 hours are cultivated in S fermentation tube；
The specific steps of institute H2S measurement test are as follows: with the purified bacillus licheniformis inoculation peace of the white pipette tips picking of 10 μ l
It is trained in bottle in 37 DEG C of 24 hours of culture, test result shows that the bacillus licheniformis cannot generate H2S.
S506, gelatin measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis in 4 DEG C
Culture carries out gelatin measurement test, 23.5 hours is cultivated in gelatin fermentation tube；
The specific steps of the gelatin measurement test are as follows: connect with the white purified bacillus licheniformis of pipette tips picking of 10 μ l
In 4 DEG C of 23.5 hours of culture in kind ampere bottle, test result shows that the bacillus licheniformis cannot decompose gelatin.
S6, identify the bacillus licheniformis described in filtering out according to the result of Molecular Identification and biochemical identification.
Application of the bacillus licheniformis in terms of preparing ensilage, prepare ensilage when to ensiling
Bacillus licheniformis is added in feedstuff to ferment.
A kind of feed, including bacillus licheniformis of the present invention.
Further, the bacillus licheniformis can also be used to preparing feed mulberry ensilage, preparation method include with
Lower step:
(1) processing and grouping of new fresh mulberry leaf: being cut into 4cm for new fresh mulberry leaf and petiole, and ensiling is packed into after being mixed evenly and is raised
Expect fermentation bag, every bag of weight 1100g, into every bag of mulberry leaf according to 5.25 × 106The standard of cfu/g adds bacillus licheniformis, mixes
Even 5 minutes, sack is fastened after manual compaction, is placed in horizental silo to ferment 7~56 days under the conditions of 37 DEG C with rubber belt sealing and is obtained
Fermented feed sample；
(2) fermented feed sample treatment: the 7th, 14,28,56 day after ensiling, 20g is taken to send out from fermentation bag at random
Ferment Feed Sample is put into 250ml conical flask, and 100ml deionized water is added in conical flask, extracts under the conditions of temperature is 4.5 DEG C
23 hours are successively filtered with double gauze incoming call filter paper, filtrate are distributed into several test tubes, cold storage is spare in -20 DEG C of refrigerators
In the measurement of subsequent ammoniacal nitrogen, remaining ensilage is dried, weighing is ready for use on subsequent measurements dry matter, crude protein, thick rouge
Fat, coarse ash and crude fibre；
(3) testing index and method: with embodiment 1.
In above-described embodiment 1-3, doubtful bacillus licheniformis is isolated and purified as shown in Figure 3；In the Molecular Identification:
The first: expanding bacterial 16 S rDNA by primer 2 7F/1492R:
27F:5 '-AGAGTTTGATCCTGGCTCAG-3 '
1492R:5 '-GGTTACCTTGTTACGACTT-3 '
PCR reaction system:
PCR reaction condition:
Sequencing result: the sequencing of amplified production Song Shenggong bioengineering limited liability company, 16S rDNA sequencing result is such as
Under:
CTTCGGCGGCTGGCTCAAAGAGGTTACCCTACCGACTTCGGGTGTTACAAACTCTCGTGGTGTG ACG
GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATT CCAGCTTCAC
GCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAAC CTCGCGGTTTCGCTGCC
CTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATG ATGATTTGACGTCATCCCCACCTT
CCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAA TGCTGGCAACTAAGATCAAGGGTTGCGCTCG
TTGCGGGACTTAACCCAACATCTCACGACACGAGCTG ACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAG
GGGACGTCCTATCTCTAGGATTGTCAGAGG ATGTCAAGACCTGGTAAGGGTTCTTCGCGTTGCTTCAAATAAAAC
ACACATGCTCCACCGCTTGTGCG GGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCG
GAGTGCTTAATGCGTT AGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTG
GACTACCAG GGTATCTAATCCTGTTCGCTCCCCACGCTCTCGCTCTCTCAGCGTCAGTACACAGACACAGAGAGT
CG CCTTCGCACACTGTGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCC TCTT
CTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCCGGGGGCTCTCATATCAC ACTAAAAGGAC
CCCGCCGCGCGAGCCCTTACACGCTCAAAACATCCCGGACAACGCTTGCCCACCTAC GTATTACCGCCGGCTGCT
GGGCACGTAGTTAGCCCGTGTGCCTTCTCTGGTTTAAGTATACGTCTCAA GG
It is compared by NCBI, tentatively predicates bacillus.
Second: passing through bacillus licheniformis specific primer BL1/BL2 specific amplification gene gyrase B, amplification
Size is 600-700bp.
BL1:5’-AKACGGAAGTGACGGGAAC-3’
BL2:5’-AGAAACTTTTCRAGCGCTT-3’
Primarily determine bacterium kind.
PCR reaction system:
PCR reaction condition:
PCR runs cementing fruit as shown in figure 4, it can be seen that NO.4 bacterium solution amplifies the segment of 600-700bp size, into one
Step is tentatively judged as bacillus licheniformis, we are named as BL-DY.
For Gram's staining result of the invention as shown in figure 5, being shown by microscopic examination result, the BL-DY is Gram-positive
Bacterium is in direct rod shape.
In all embodiments of the invention, growth temperature can be fitted and grow the statistical data of pH value as shown in table 1.1:
1.1 bacillus licheniformis of table fits growth temperature and pH value
Note :+, normal growth；, do not grow.
The result shows that BL-DY growth conditions threshold value is wide (15~55 DEG C of temperature, pH4.0~8.0 can normal growth).
Sugar fermentating test of the invention is carried out using the bacterium micro biochemical assessor of Hai Bo Bioisystech Co., Ltd
Analysis, is tested as a control group using Quality-control strains bacillus subtilis (BS), and the experimental condition of control group is the same as implementation
Example 1-3, the result of test is as shown in table 1.2:
The sugar fermentating test of 1.2 bacillus licheniformis of table
Note: reagent indicates that result is positive in yellow, is "+"；Reagent indicates that result is negative in green or blue, is "-".
The results show that BL-DY fermentating maltose, mannose, sucrose and glucose, azymic lactose.
Xi Mengshi citrate of the invention measures test, uses the micro life of the bacterium of Hai Bo Bioisystech Co., Ltd
Change assessor to be analyzed, be tested as a control group using Quality-control strains bacillus subtilis (BS), the examination of control group
Condition is tested with embodiment 1-3, the result of test is as shown in table 1.3:
The Xi Mengshi citrate of 1.3 bacillus licheniformis of table measures test
Note: reagent expression result blue is positive, is "+"；Reagent indicates that result is negative in green, is "-".
The results show that BL-DY cannot be grown in citrate.
H2S of the invention measures test, is carried out using the bacterium micro biochemical assessor of Hai Bo Bioisystech Co., Ltd
Analysis, is tested as a control group using Quality-control strains bacillus subtilis (BS), and the experimental condition of control group is the same as implementation
Example 1-3, the result of test is as shown in table 1.4:
The H of 1.4 bacillus licheniformis of table2S measurement test
Note: site of puncture, in umbrella growth and the blackening expression result positive, is "+" along puncture line；Site of puncture is without blackening
It indicates result ginkgo, is "-".
The results show that BL-DY cannot generate H2S.
The gelatin of invention measures test, is carried out using the bacterium micro biochemical assessor of Hai Bo Bioisystech Co., Ltd
Analysis, is tested as a control group using Quality-control strains bacillus subtilis (BS), and the experimental condition of control group is the same as implementation
Example 1-3, the result of test is as shown in table 1.5:
The gelatin of 1.5 bacillus licheniformis of table measures test
Note: 0-4 DEG C of reagent, which is in a liquid state, indicates the result positive, is "+"；0-4 DEG C of reagent indicates that result is negative in solid-state, is
“-”。
The results show that BL-DY cannot decompose gelatin.
In the present invention as bacillus subtilis (BS) deposit number of standard Quality-control strains be ATCC6633, be the U.S.
Culture Collection Center ATCC strain.
Therefore, binding molecule identification and biochemical results determine that bacterium is a kind of bacillus licheniformis Bacillus
Licheniformes, the related biomaterial feature of the bacillus licheniformis are as follows: its bacteria colony white, are in flattened round, intermediate
There is fold and swell, edge is irregular, amphimicrobian；That 4~6 are cultivated in agarose media is small by 1:100 for the fresh bacterium solution
When enter stationary phase；Bacterium energy metabolism glucose, sucrose, maltose and the mannose, are unable to metabolising lactose；The bacterium is suitble to grow
Temperature range can be 30~37 DEG C, and highest tolerable temperature can reach 80 DEG C；The bacterium can be in the range growth of pH4.5~8.0, most
The long pH range of adaptability is 5.5~7.5；Colonial morphology, long 2.0~6.5 μ of bacterium are observed by optical microscopy 100 × times oil mirror
M, it is 0.3~1.0 μm wide, it is in elongated rod shape；Gram's staining is in bluish violet, is gram-positive bacteria.
Bacillus licheniformis of the invention is applied when preparing feed mulberry ensilage, we have also done comparative experiments,
The processing of new fresh mulberry leaf and grouping stage, by treated, new fresh mulberry leaf is packed into ensilage fermentation bag, takes out 24 bags at random,
Control group and each 12 bags of experimental group, control group do not add the lichen bacillus ferments, and experimental group adds bacillus licheniformis hair
Ferment, the method for the process of preparation with embodiment 13.
The 7th after ensiling, 14,28,56 days, random each 3 bags of ensilage fermentation bag for taking out control group and experimental group,
By fermented feed sample by extraction and filtering, ammoniacal nitrogen, dry matter, crude protein, crude fat, coarse ash and crude fibre are carried out
Measurement:
Crude protein content measurement uses Kjeldahl's method in the present invention, referring to GB/T 6432-1994 " crude protein in feed
Measuring method "；Crude fat content measurement uses soxhlet extraction, referring to the GB/T 6433-2006 " measurement of crude fat in feed
Method "；Crude ash content measurement uses calcination method, referring to GB/T 6438-2007 " measuring method of coarse ash in feed "；Slightly
Fiber content measurement is using the cooking method that disappears, referring to GB/T 6434-2006 " coarse-fibred measuring method in feed "；Ammoniacal nitrogen uses
Phenol-sodium hypochlorite colorimetric method for determining, these methods are all the common measuring methods of those skilled in the art, no longer superfluous herein
It states.
The testing result of dry matter is as shown in table 1.6:
Influence of 1.6 BL-DY of table to dry matter in mulberry leaf ensilage
Note: p≤0.05 is significant difference.
The result shows that experimental group and the control group dry matter content for adding BL-DY bacterial strain are poor during mulberry leaf silage fermentation
It is different unobvious.
The testing result of crude protein is as shown in table 1.7:
Influence of 1.7 BL-DY of table to crude protein in mulberry leaf ensilage
Note: p≤0.05 is significant difference.
After mulberry leaf silage fermentation 14 days, the crude protein content for adding the experimental group of BL-DY bacterial strain is significantly higher than control group.
The testing result of crude fat is as shown in table 1.8:
Influence of 1.8 BL-DY of table to crude fat in mulberry leaf ensilage
Note: p≤0.05 is significant difference.
After mulberry leaf silage fermentation 14 days, the crude fat content for adding the experimental group of BL-DY bacterial strain is substantially less than control group.
The testing result of coarse ash is as shown in table 1.9:
Influence of 1.9 BL-DY of table to coarse ash in mulberry leaf ensilage
Note: p≤0.05 is significant difference.
During mulberry leaf silage fermentation, experimental group and the control group crude ash content difference for adding BL-DY bacterial strain are unknown
It is aobvious.
Coarse-fibred testing result is as shown in table 1.10:
1.10 BL-DY of table is on coarse-fibred influence in mulberry leaf ensilage
Note: p≤0.05 is significant difference.
During mulberry leaf silage fermentation, the crude fiber content for adding the experimental group of BL-DY bacterial strain is substantially less than control group.
The testing result of ammoniacal nitrogen is as shown in table 1.11:
1.11 BL-DY of table is to ammoniacal nitrogen/total nitrogen ratio in mulberry leaf ensilage
Note: p≤0.05 is significant difference.
During mulberry leaf silage fermentation, add BL-DY bacterial strain experimental group ammoniacal nitrogen/total nitrogen ratio be substantially less than pair
According to group.
Bacillus licheniformis (Bacillus Licheniformis) BL-DY of the invention can be used for preparing ensiling feeding
Material, especially prepares feed mulberry ensilage, the life of bacillus licheniformis (Bacillus Licheniformis) BL-DY
Object feature are as follows: gram-positive bacteria, in the shape of a rod, growth rate is high, and growth conditions threshold value is wide: 15~55 DEG C of temperature, pH4.0
~8.0 can normal growth, also metabolizable various saccharides will not generate poisonous and harmful substance.
The BL-DY that the present invention filters out can effectively improve feed mulberry Silage Quality, during mulberry leaf silage fermentation,
The dry matter and crude ash content and control group difference of the experimental group of addition BL-DY bacterial strain are little, and crude fat content significantly drops
It is low, illustrate that it is utilized by bacterium and produces energy；But the crude protein content for adding the experimental group of BL-DY bacterial strain significantly improves, energy
Fully ensure that the nitrogen source supply of animal；The experimental group for adding BL-DY bacterial strain significantly reduces the coarse-fibred content of feed, is thin
Cellulose degraded secreted by bacterium, the carbohydrate absorbed can be easier by animal body by being formed, and improve animal pair
Coarse-fibred digestion, absorption and utilization rate.Meanwhile ammoniacal nitrogen/total nitrogen ratio decline of the fermentation mulberry leaf of BL-DY, reflection is added
Protein and amino acid degree of decomposition reduce in ensilage, illustrate the quality of ensilage to raising.
The above is only a preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein
Form, should not be regarded as an exclusion of other examples, and can be used for other combinations, modifications, and environments, and can be
In contemplated scope described herein, modifications can be made through the above teachings or related fields of technology or knowledge.And those skilled in the art institute
The modifications and changes of progress do not depart from the spirit and scope of the present invention, then all should be in the protection model of appended claims of the present invention
In enclosing.
SEQUENCE LISTING
<110>Chengdu Academy of Agriculture and Forestry Science
<120>a kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis
<130> 2019.4.1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1018
<212> DNA
<213>unknown
<400> 1
cttcggcggc tggctcaaag aggttaccct accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480
acctggtaag ggttcttcgc gttgcttcaa ataaaacaca catgctccac cgcttgtgcg 540
ggcccccgtc aattcctttg agtttcagtc ttgcgaccgt actccccagg cggagtgctt 600
aatgcgttag ctgcagcact aaggggcgga aaccccctaa cacttagcac tcatcgttta 660
cggcgtggac taccagggta tctaatcctg ttcgctcccc acgctctcgc tctctcagcg 720
tcagtacaca gacacagaga gtcgccttcg cacactgtgt gttcctccac atctctacgc 780
atttcaccgc tacacgtgga attccactct cctcttctgc actcaagttc cccagtttcc 840
aatgaccctc cccggttgag cccgggggct ctcatatcac actaaaagga ccccgccgcg 900
cgagccctta cacgctcaaa acatcccgga caacgcttgc ccacctacgt attaccgccg 960
gctgctgggc acgtagttag cccgtgtgcc ttctctggtt taagtatacg tctcaagg 1018
Claims (10)
1. a kind of bacillus licheniformis, it is characterised in that: the bacillus licheniformis is preserved in China typical culture collection
The heart, deposit number are CCTCC M2019165.
2. the screening technique of bacillus licheniformis as described in claim 1, it is characterised in that: the following steps are included:
S1, it collects soil sample: 90~110g of mulberry leaf planting site soil is collected using line-of-sight course；
S101, it is used as adopts in the soil that the north of mulberry leaf planting base, southwestern face and the southeast selection mulberry leaf grow densely respectively
Sampling point；
S102, the north in the sampled point, southwestern face and the southeast acquisition soil sample are simultaneously uniformly mixed；
S2, bacterial strain culture and divide bacterium: the soil sample being collected into is placed in the sterile conical flask of the PBS equipped with 800~1000ml,
Shake up 18~22 minutes and soil sample be diluted to sample suspension, after it is further dilute to the sample suspension according to 10 times of coubling dilutions
It releases；Choosing 100ml dilution is 10-4~10-5Sample suspension be coated on TSA plate, the TSA plate is placed in 37 DEG C
22~26 hours are cultivated in insulating box, obtain bacterium colony；
S3, bacillus licheniformis isolate and purify: the centre that 3~5mm is screened on the TSA plate has the yellow-white of protuberance fold flat
Flat bacterium colony, and by after TSB Liquid Culture, taking fresh bacterium solution in optical microscopy 100 × times oily microscopic observation bacterium colony form,
The length for filtering out elongated rod shape is 2.0~6.5 μm, width is 0.3~1.0 μm, dyeing is in the bacterium colony of bluish violet, then is isolated
Purifying obtains the fresh strain of plating medium bacillus licheniformis；
S4, Molecular Identification: the Molecular Identification is tested by following two and is realized:
S401, bacterium progress 16S rDNA Testing and appraisal is expanded by primer 2 7F/1492R, sequencing result and NCBI are compared,
Whether preliminary judgement is bacillus；
S402, pass through bacillus licheniformis specific primer BL1/BL2 specific amplification gene gyrase B, carry out detection mirror
Fixed, whether it is bacillus licheniformis according to detection structure preliminary judgement that amplification size is 600~700bp；
S5, biochemical identification: the biochemical identification is that the strain that preliminary judgement is bacillus licheniformis is carried out following five kinds of tests
To identify:
S501, Gram stain test: carrying out Gram's staining for the fresh strain of the bacillus licheniformis, optical microscopy
Microscopic examination result shows that the strain is the gram-positive bacteria that form is in direct rod shape；
S502, can the test of adaptability elongate member: the fresh strain of the bacillus licheniformis is placed in temperature is 15~55 DEG C, pH value is
Growth test is carried out under conditions of 4.0~8.0；
S503, sugar fermentating test: the fresh strain of the bacillus licheniformis is trained in 37 DEG C using bacterium micro biochemical assessor
It supports, carries out lactose, maltose, mannose, sucrose and glucose fermentation test respectively, 16~18 are cultivated in corresponding fermentation tube
Hour；
S504, Xi Mengshi citrate measurement test: using bacterium micro biochemical assessor to purified bacillus licheniformis
It is cultivated in 37 DEG C, carries out citrate test, 22~24 hours are cultivated in citrate fermentation tube；
S505、H2S measurement test: cultivating purified bacillus licheniformis in 37 DEG C using bacterium micro biochemical assessor, into
Row H2S measurement test, in H223~25 hours are cultivated in S fermentation tube；
S506, gelatin measurement test: cultivating purified bacillus licheniformis in 4 DEG C using bacterium micro biochemical assessor,
Gelatin measurement test is carried out, 23~24 hours are cultivated in gelatin fermentation tube；
S6, identify the bacillus licheniformis described in filtering out according to the result of Molecular Identification and biochemical identification.
3. the screening technique of bacillus licheniformis according to claim 1, it is characterised in that: the Gram stain test
Specific steps are as follows: first the fresh strain smear of the bacillus licheniformis is fixed through flame, add crystal violet solution contaminate 1~2 point
Zhong Houyong clear water washes away dye liquor；Again plus iodine solution contaminates 0.5~1 minute, and clear water washes away dye liquor, and it is uniform after 95% ethyl alcohol that concentration, which is added,
Slide 10~30 seconds is shaken up to no purple falls off, then washes away dye liquor with clear water；Then gaza's Huang redyes liquid again, contaminates 1~1.5 point
Clock continues to wash away dye liquor with clear water, carries out oil mirror microscopy after smear is dry.
4. the screening technique of bacillus licheniformis according to claim 1, it is characterised in that: the sugar fermentating test is specific
Step are as follows: be first turned on corresponding ampere bottle, draw 50 μ l test reagents and be packed into 2ml sterile EP tube, add fresh lichens gemma
10 μ l of bacillus bacterium solution is in 37 DEG C of 18 hours of culture.
5. the screening technique of bacillus licheniformis according to claim 1, it is characterised in that: the Xi Mengshi citrate
Measure the specific steps of test are as follows: trained in 37 DEG C in the purified bacillus licheniformis inoculation ampere bottle of the white pipette tips picking of 10 μ l
24 hours are supported, test result shows that the bacillus licheniformis cannot grow in citrate.
6. the screening technique of bacillus licheniformis according to claim 1, it is characterised in that: the H2The tool of S measurement test
Body step are as follows: it is inoculated in ampere bottle with the purified bacillus licheniformis of the white pipette tips picking of 10 μ l in 37 DEG C of 24 hours of culture,
Test result shows that the bacillus licheniformis cannot generate H2S。
7. the screening technique of bacillus licheniformis according to claim 1, it is characterised in that: the gelatin measurement test
Specific steps are as follows: it is inoculated in ampere bottle with the purified bacillus licheniformis of the white pipette tips picking of 10 μ l in 4 DEG C of 24 hours of culture,
Test result shows that the bacillus licheniformis cannot decompose gelatin.
8. application of the bacillus licheniformis according to claim 1 in terms of preparing ensilage, it is characterised in that: be used for
Bacillus licheniformis is added when preparing feed mulberry ensilage into raw material to ferment.
9. a kind of feed, it is characterised in that: including bacillus licheniformis as described in claim 1.
10. feed according to claim 9, it is characterised in that: in the new fresh mulberry leaf and petiole of every 1000~1200g weight
It is added 5 × 106~5.5 × 106The bacillus licheniformis of cfu/g fastens sack after manual compaction, is placed in ensiling with rubber belt sealing
It ferments in cellar.
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| CN112980918A (en) * | 2019-12-17 | 2021-06-18 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
| CN114574386A (en) * | 2022-03-01 | 2022-06-03 | 广东石油化工学院 | Bacillus licheniformis and application thereof |
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| CN114574386A (en) * | 2022-03-01 | 2022-06-03 | 广东石油化工学院 | Bacillus licheniformis and application thereof |
| CN114574386B (en) * | 2022-03-01 | 2023-08-11 | 广东石油化工学院 | Bacillus licheniformis and application thereof |
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