CN111363701B - Heat-resistant bacillus licheniformis and application thereof - Google Patents
Heat-resistant bacillus licheniformis and application thereof Download PDFInfo
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Abstract
The invention discloses heat-resistant bacillus licheniformis and application thereof, and belongs to the technical field of microbial fermentation and transformation. The ginkgo leaf fermentation microbial agent prepared by fermenting ginkgo leaves with the strain LZTT-001 is used as a feed additive, so that the stress capability and disease resistance of livestock and poultry can be improved, and the growth is promoted; the ginkgo leaf fermentation microbial agent prepared by the invention is used as a feed additive, has the characteristics of environmental protection, excellent performance and the like, and has better economic benefit and social benefit.
Description
Technical Field
The invention relates to the technical field of microbial fermentation and transformation, in particular to heat-resistant bacillus licheniformis and application thereof.
Background
The chemical components of the ginkgo leaf extract comprise flavonoids, terpenoids, lactones, phenolic acids, alkaloids, polyisoprene and other compounds. Flavonoids occur in two forms in ginkgo leaves: one is fat-soluble flavone, such as flavonoid aglycone; second is water soluble flavone, such as flavonoid glycoside. More than 95% of the flavone in the stem and leaf of natural plant exists in the form of glucoside. A large number of literature data reports that the biological activity of the flavonoid aglycone for removing the oxygen free radicals of the human body is obviously superior to that of the glucoside of the flavonoid aglycone, and only the aglycone type flavonoid can directly enter the blood of animals to be absorbed and utilized. The microbial hydrolase treatment of the plant tissue is beneficial to the release and conversion of natural flavonoids, and the enzymes such as alpha-rhamnosidase, beta-glucosidase, xylanase, cellulase, pectinase, alpha-amylase and the like can effectively biologically convert glucoside type flavone into aglycon type flavone. The folium Ginkgo extract has effects of reducing blood sugar and blood lipid, resisting oxidation, relieving inflammation, killing bacteria, and enhancing immunity. Clinical resistance is becoming increasingly severe due to the abuse of antibiotics in the field of animal farming. The development of novel botanical antibacterial drugs to replace traditional antibacterial drugs is one of the hotspots in the field of feed additives in recent years. The application of the ginkgo biloba extract as an additive in the field of breeding is vigorously developed by combining the uniqueness of ginkgo biloba resources, the biological function of the ginkgo biloba extract is fully utilized, and partial problems existing in the breeding industry at present can be improved.
Therefore, the problem to be solved by the technical personnel in the field is to provide a heat-resistant bacillus licheniformis and application thereof.
Disclosure of Invention
In view of the above, the invention provides heat-resistant bacillus licheniformis and an application thereof, the heat-resistant bacillus licheniformis is prepared into a ginkgo leaf fermentation microbial agent, and the ginkgo leaf fermentation microbial agent is added into feed as a feed additive, so that the daily gain of livestock and poultry can be increased, and the diarrhea rate can be reduced.
In order to achieve the purpose, the invention adopts the following technical scheme:
a heat-resistant Bacillus licheniformis (Bacillus licheniformis) strain LZZT-001 with the preservation number of CGMCC No. 18672; preserved in China general microbiological culture Collection center (CGMCC) for 10, 12 and 2019; address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the classification is named as Bacillus licheniformis.
The heat-resistant Bacillus licheniformis LZZT-001 can produce beta-glucosidase and galactosidase.
Further, the heat-resistant bacillus licheniformis LZZT-001 is applied to preparation of a ginkgo leaf fermentation microbial agent.
Further, the preparation method of the ginkgo leaf fermentation microbial agent comprises the following specific steps:
(1) activating the heat-resistant Bacillus licheniformis LZTT-001, transferring the activated strain into seed culture solution, performing shake culture at 30-50 deg.C for 1-3d at a vibration frequency of 150-9CFU/ml; the concentration of the preferred bacterial liquid of the seed liquid is 109-1010CFU/ml。
(2) Adding the ginkgo flavone extract into the seed liquid obtained in the step (1), fermenting and culturing for 3-10h to ensure that the activity of the glucosidase is more than 300 units/ml and the activity of the galactosidase is more than 50 units/ml; obtaining a fermented seed solution; adding 20-50mg of ginkgetin extract into each liter of seed liquid, wherein the content of total flavone in the ginkgetin extract is more than or equal to 24%; the preferred total flavone content in the ginkgo flavone extract is 24%.
(3) Inoculating the fermented seed liquid obtained in the step (2) into a solid substrate for solid fermentation culture, and specifically comprising the following steps:
inoculating fermented seed liquid into a solid substrate to obtain a mixture; the inoculation amount of the fermentation seed liquid is 1-10% of the solid matrix, and the bacterial liquid concentration of the fermentation seed liquid is more than or equal to 10%9CFU/ml, the concentration of the preferred bacterium liquid of the fermentation seed liquid is 109-1010CFU/ml; the solid matrix contains 10-30% of crushed ginkgo leaves, and the balance of agricultural organic waste; the carbon-nitrogen ratio of the mixture is 25-35: 1, the water content is 45-65%, and the oxygen content is 5-14%; stirring for 1 time at intervals of 2-5 d;
secondly, when the temperature of the reactor is reduced to 45-50 ℃, inoculating the fermented seed liquid again, wherein the inoculation amount is 2-6% of the solid matrix, and the temperature of the reactor is controlled to be 65-75 ℃;
thirdly, after stacking and fermenting for 10-20 days, air-drying the stacked materials to obtain the ginkgo leaf fermentation microbial agent; the content of ginkgetin in each kg of the ginkgo leaf fermentation microbial agent is more than or equal to 2g, and the preferable content of ginkgetin in each kg of the ginkgo leaf fermentation microbial agent is 2-3 g; the content of heat-resistant bacillus licheniformis LZZT-001 in per g of folium Ginkgo fermentation microbial agent is not less than 109CFU, the content of heat-resistant Bacillus licheniformis LZZT-001 in per g of folium Ginkgo fermentation microbial agent is preferably 109-1010CFU。
The temperature of 30-50 ℃ is the proper culture temperature of the heat-resistant bacillus licheniformis LZTT-001, and the strain LZTT-001 can survive under the high temperature condition of 60-80 ℃; when the heap temperature is between 65 and 75 ℃, the activity of the strain LZZT-001 is not influenced.
Further, the strain activation operation step in the step (1) is as follows: inoculating the preserved heat-resistant bacillus licheniformis LZZTT-001 on a solid culture medium, wherein the culture temperature is 30-50 ℃.
Further, the formula of the seed culture solution in the step (1) is as follows: 3-5g of soluble starch, 8-11g of tryptone and 3-5g of sodium chloride, and adding water to 1000 ml; the pH is 7.2-7.4.
Further, the agricultural organic waste in the step (3) is at least one of wheat bran, wood chips, peanut shell powder, furfural, bean pulp and sugar residues.
The ginkgo leaf fermentation microbial agent is applied as a feed additive, and the addition amount of the ginkgo leaf fermentation microbial agent is 0.30-0.60% of the basic ration.
According to the technical scheme, compared with the prior art, the heat-resistant bacillus licheniformis and the application thereof are disclosed and provided, the ginkgo leaf fermentation microbial agent prepared by fermenting ginkgo leaves with the strain LZZT-001 is used as a feed additive, is a high-efficiency microbial agent integrating probiotics and traditional Chinese medicine effective components, and has the functions of diminishing inflammation, sterilizing and promoting growth; the ginkgo leaf fermentation microbial agent developed by using an active strain LZZT-001 with various hydrolases and using ginkgo leaves as a fermentation substrate through a solid state fermentation technology contains bioactive components, is rich in probiotics or feed complex enzyme, can be widely used for various livestock and poultry and aquatic animals such as pigs, chickens, cows, fishes, shrimps and the like as a substitute of a growth promoter and antibiotics, and reduces the diarrhea rate; the ginkgo leaf fermentation microbial agent prepared by the invention is used as a feed additive, has the characteristics of environmental protection, excellent performance and the like, and has better economic benefit and social benefit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing of a phylogenetic tree of the strain LZT-001 of the present invention;
FIG. 2 is a drawing showing morphological characteristics of LZZT-001 strain of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 isolation and characterization of Bacillus licheniformis LZTT-001
Collecting ginkgo leaf, cleaning soil and dust on the surface of the leaf by using a test tube brush, shearing necrotic tissues, cutting into materials with proper sizes, quickly wiping the surface of the tissues by using a 75% alcohol cotton ball, and quickly baking residual ethanol on the surface of the tissues by using alcohol lamp flame; naturally air drying for about 1 day, and separating to obtain folium Ginkgo endophyte. Before separation, surface disinfection is needed, before disinfection, a large amount of clear water is used for cleaning dust on the surface of the explant and the dust is absorbed by filter paper, and the surface disinfection program is as follows: 1) soaking in 99% (V/V) ethanol for sterilization for 1 min; 2) 3.125% (effective chlorine/V) NaClO soaking and sterilizing for 6 min; 3) soaking in 99% (V/V) ethanol for 30s for disinfection; 4) repeatedly rinsing with sterile water for 4-6 times. Then under the aseptic condition, cutting the ginkgo leaf slices with the surfaces sterilized into small sections or slices of 1-2cm multiplied by 1-2cm by using a scalpel, clamping and inoculating the ginkgo leaf slices on a separation culture medium by using aseptic tweezers, and uniformly inoculating 8-10 plant samples on each culture dish. The separation culture medium adopts humic acid vitamin agar (HVA) culture medium (NaH)2PO40.5 g，KCl 1.7g，MgSO4·7H2O 0.5g，CaCO30.02g，FeSO4·7H20.01g of O, 1.0g of humic acid, 0.5mg of riboflavin, 0.5mg of thiamine, 0.5mg of nicotinic acid, 0.25mg of biotin, 0.5mg of inositol and VB60.5mg, p-aminobenzoic acid 0.5mg, pantothenic acid 0.5mg, agar 15.0g, distilled water 1000mL, pH 7.2). After culturing in an incubator at 37 ℃ for about 3 days, newly grown bacterial colonies were picked up and streaked on a TSB solid medium (tryptone 15.0g, soytone 5.0g, NaCl 5.0g, agar 18.0g, distilled water 1000mL, pH 7.2). Separating and picking microorganism, primarily removing weight by colony morphology and gram staining method, inoculating to screening culture medium (folium Ginkgo extract 5g/L, agar 20g/L, magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 10g/L, geniposide 0.1g/L, sodium glutamate 10g/L, ammonium sulfate 2g/L), hydrolyzing geniposide in the culture medium by microorganism-produced beta-glucosidase to generate genipin, combining with sodium glutamate to form blue geniposide blue,obtaining a bacterial strain which grows vigorously and is gram-positive. Using the obtained strain DNA as a template, selecting a primer (27 f): 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1; and primer (1492 r): 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 2; its 16S rDNA sequence was PCR amplified. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 35 sec, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 60 sec, 35 cycles. The obtained 16S rDNA gene sequence is compared with an NCBI Genebank database, the similarity with the strain Bacillus licheniformis is highest, and the homology is 99.4%; the results are shown in FIG. 1. The strain is identified as Bacillus licheniformis by combining the growth characteristics of the strain, named as LZZT-001, and the morphological characteristics are shown in figure 2; and preserved in the China general microbiological culture Collection center (address: No.3 of West Lu No.1 of the sunward area, Beijing, Wenyu institute of microbiology, China academy of sciences, postal code 100101) in 2019, 10 months and 12 days, and is classified and named as Bacillus licheniformis, with the preservation number of CGMCC No. 18672.
EXAMPLE 2 preparation of bacterial Strain LZZT-001 microbial inoculum
The production strain LZTT-001 slant stored at 4 ℃ is transferred to a TSB culture medium plate, cultured at 30-50 ℃, then transferred to a 5L triangular flask filled with 2L TSB liquid culture medium, shake-cultured for 1 day at 30-50 ℃ on a shaker and at the rotating speed of 150-.
Inoculating the shake flask seed solution into a fermentation seed tank containing TSB liquid culture medium according to 0.1-1% of the liquid culture medium volume ratio for fermentation culture. The fermentation conditions are 35-38 deg.C, aeration and stirring, and the aeration amount is 0.45m3At stirring speed of 150-.
Inoculating seed liquid in the seed tank to a fermentation tank containing TSB liquid culture medium according to 0.1-1% of the liquid culture medium volume ratio for fermentation culture. The fermentation conditions are 35-38 deg.C, aeration and stirring, and the aeration amount is 0.45m3At stirring speed of 150-Stopping culturing, and obtaining the zymogen liquid.
Subpackaging the fermentation liquor into liquid microbial inoculum, dehydrating the fermentation liquor in a plate-frame or centrifugal mode, and spray drying to obtain the microbial powder.
Example 3 detection of Activity of Strain LZTT-001 high temperature resistant glucosidase and Gene sequence thereof
The activity of LZTT-001 glucosidase was measured by DNS method, 800. mu.L of 0.05mol/L glycine-sodium hydroxide buffer (pH 9.0) and 100. mu.L of 45mmol/L pNPG were preheated at 45 ℃ for 5 minutes, 100. mu.L of the fermented broth prepared in example 2 was added, reaction was carried out for 10 minutes, and 2mol/L Na was added2CO3Stopping enzyme reaction by using 2mL of solution, adding 10mL of distilled water, uniformly mixing, adding a proper amount of reaction solution into a cuvette, measuring an OD value at a wavelength of 405nm, and calculating the activity of the glucosidase according to a standard curve. The alpha-glucosidase enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1. mu. mol of substrate pNPG to product pNP per minute under optimal reaction conditions. The method adopts an alpha-glucosidase characteristic primer agf: 5'-ATGAAATTTTCAGACGGCTA-3', SEQ ID NO. 3; and agr: 5'-TTAGAAAAAAATGAAGACATCCC-3', SEQ ID NO.4, amplifying, and carrying out PCR amplification reaction by taking the strain LZZT-001DNA as a template, wherein the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 minutes, denaturation at 95 ℃ for 30 seconds, annealing at 54 ℃ for 30 seconds, extension at 72 ℃ for 90 seconds, 35 cycles, extension at 72 ℃ for 10 minutes, detecting by electrophoresis to obtain a nucleotide fragment with the size of about 2.3kb, sequencing, and comparing the sequence in NCBI GeneBank database by using Blast program to obtain the sequence fragment coding alpha-glucosidase gene, wherein the gene sequence is shown in SEQ ID NO. 5.
Example 4 detection of the galactosidase Activity of the Strain LZT 001 and the Gene sequences related thereto
After preheating at 45 ℃ for 2min, 100. mu.L of the fermented broth prepared in example 2 and 100. mu.L of pNPG solution (10mmol/L) were mixed, reacted at the corresponding temperature for 10min, and 800. mu.L of 0.5mol/L Na was added2CO3The solution is stopped to react, 200 mu L of the solution is added into a 96-well plate after being mixed evenly, the absorbance value at the position with the wavelength of 405nm is measured by a microplate reader, and the absorbance value is substituted into a standard curve regression equation which is measured by p-nitrobenzene (pNP) in advance to obtain the activity value of the alpha-galactosidase.Alpha-galactosidase enzyme activity definition: the amount of enzyme required to hydrolyze pNPG per minute under certain conditions to produce 1. mu. mol of pNP is defined as 1. alpha. -galactosidase enzyme activity unit (U). Using galactosidase specific primer galctosidase-F: 5'-ATGGTCAAACCGTATCCCCC-3', SEQ ID NO. 6; and galactosidase-R: 5'-CTAGGCTTTGCGGCTTCTC-3', SEQ ID NO. 7; and carrying out PCR amplification reaction by using the strain LZZT-001DNA as a template, wherein the PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 minutes, denaturation at 95 ℃ for 30 seconds, annealing at 55 ℃ for 45 seconds, extension at 72 ℃ for 90 seconds, 35 cycles, extension at 72 ℃ for 10 minutes, detecting by electrophoresis to obtain a nucleotide fragment with the size of about 2.0kb, sequencing, and comparing the sequence in NCBI GeneBank database by using Blast program to obtain a sequence fragment encoding galactosidase gene, wherein the gene sequence is shown in SEQ ID NO. 8.
Example 5 preparation of high efficiency feed additive by solid fermentation of Ginkgo biloba leaf with bacterial strain LZTT-001
Mixing 30% of pulverized folium Ginkgo with 60% of testa Tritici and 10% of soybean meal; adjust moisture and increase porosity. The effective volume of the reactor is 500 liters, the weight of the stockpile is 200kg, the carbon-nitrogen ratio is approximately equal to 30, the water content is about 45-65%, and the organic matter content is about 60%. Inoculating LZTT-001 microbial inoculum at 3%, performing forced ventilation aerobic fermentation for 10-20 days, and turning over for 1 time every 1-2 days. After stacking and fermenting, turning the stacks, airing at room temperature or drying at 60 ℃, crushing, screening by a screening machine, and packaging to obtain the ginkgo leaf fermentation microbial agent which is used as a feed additive product.
Example 6 detection of Ginkgo flavone content in feed additives
Sampling from a ginkgo leaf fermentation microbial agent, weighing 1g each time, crushing, extracting with 10ml of absolute ethanol (the material-liquid ratio is 1: 10) for 2 hours, then filtering with filter paper, concentrating a sample by adopting a reduced pressure distillation mode, adsorbing the obtained concentrated solution by using D101 macroporous resin, eluting flavone substances from the resin by using ethanol, and dissolving the obtained concentrated sample by using butanediol after the reduced pressure distillation. Adopting an HPLC method to detect the ginkgetin, wherein the detection parameters are as follows: the chromatographic column is Intersil ODS-34.6X 150 mm; the column temperature was 25 ℃; the flow rate is 1 mL/min; the mobile phase is methanol: 0.04mol/L phosphoric acid (50:50, V/V); the detection wavelength was 360 nM. Quercetin, kaempferide, and isorhamnetin as reference substances. The total flavone content (quercetin content + kaempferol content + isorhamnetin content) x2.51, and the measured total flavone content of semen Ginkgo is 5.3-6.5 mg/g.
Example 7 detection of Ginkgo flavone aglycone content in feed additive
Sampling from a ginkgo leaf fermentation microbial agent, weighing 1g each time, crushing, extracting with 10ml of absolute ethanol (the material-liquid ratio is 1: 10) for 2 hours, then filtering with filter paper, concentrating a sample by adopting a reduced pressure distillation mode, adsorbing the obtained concentrated solution by using D101 macroporous resin, eluting flavone substances from the resin by using ethanol, and dissolving the obtained concentrated sample by using butanediol after the reduced pressure distillation. The detection conditions of the ginkgo flavonoid glycoside high performance liquid chromatography are as follows: gradient elution was carried out using 1% formic acid (A) and methanol (B) as mobile phases in the order shown in Table 1, with a flow rate of 0.5mL/min, a sample volume of 5. mu.L, a column temperature of 30 ℃ and a detection wavelength of 360nm, using a C18 reverse phase chromatography column (4 μm, 3.9 mm. times.150 mm).
TABLE 1 Ginkgolin aglycone liquid phase detection gradient elution procedure
| Time | 1% formic acid | Methanol |
| 0 minute | 70％ | 30％ |
| 3 minutes | 65％ | 35％ |
| 9 minutes | 65％ | 35％ |
| 14 minutes | 60％ | 40％ |
| 16 minutes | 50％ | 50％ |
| 18 minutes | 35％ | 65％ |
| 20 minutes | 25％ | 75％ |
| 23 minutes | 0％ | 100％ |
Taking hydrolysate quercetin of rutin which is the main component of ginkgo flavone as a main control standard, and considering that the ginkgo flavone is partially hydrolyzed by solid fermentation when elution peak of the quercetin appears when liquid phase retention time is 21.12 minutes.
Example 8 application of fermented Ginkgo biloba feed additive to growth promotion of fattening pig
The ginkgo leaf fermentation microbial agent prepared according to the method of example 5 is used as a feed additive and is respectively added into basic daily ration in an amount of 0.30-0.60%, two experimental groups and a control group are adopted to feed growing pigs, and the growth conditions of the growing pigs are observed and recorded. The feeding subjects select healthy growing pigs with the weight of about 25kg, the healthy growing pigs are divided into 3 groups at random, 10 pigs in each group are respectively fed with 2 groups of growing pigs to prepare the feed containing 0.30-0.60% of the ginkgo leaf fermentation microbial agent feed additive, the other group of growing pigs is used as a control group, the feed without the ginkgo leaf fermentation microbial agent feed additive is fed, and the feeding time is 21 days. During the test period, the feeding environment of each group of pigs is kept consistent, the feeding, drinking and the like are kept consistent, the pigs are weighed on an empty stomach every day, and the average daily gain and diarrhea rate of the pigs are calculated. The test results are shown in table 2.
| Group of | Initial weight (kg/head) | Terminal weight (kg/head) | Daily gain (g/head) | Rate of diarrhea/%) |
| Experimental group 1 | 25.34 | 44.01 | 889.05 | 1.01 |
| Experimental group 2 | 25.23 | 43.89 | 888.57 | 2.32 |
| Control group | 25.16 | 42.10 | 806.67 | 10.53 |
The initial and final weights in Table 2 are the average weights of the pigs on the first and last days of the test, and the daily gains and daily feed intake are the average weight gain and average feed intake per day and per head for the pigs in each group. The experimental results show that the growing pig feed prepared by the embodiments of the invention obviously reduces the diarrhea rate (P < 0.05) of the growing pig, the daily gain of the growing pig of the experimental group is obviously higher than that of the growing pig of the control group (P < 0.05), and the growing pig feed added with the ginkgo leaf fermentation microbial agent as the feed additive can obviously reduce the diarrhea of the pig, is beneficial to improving the disease resistance and the digestibility of the pig and promotes the growth of the pig.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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gtctggatca acccttacat cgcccaaaaa tcgaagcttt ttcaggaagg aaaagaacac 1080
ggctactttt taaaaaacag ccggggggat gtgtggcagt gggaccggtg gcaggccggc 1140
atggcaatcg tcgacttcac caatcgaaaa gcgcgtgact ggtattgttc aaaacttgaa 1200
agcctgctcg acatgggcgt cgactgtttc aagaccgatt tcggagaacg gattccgaga 1260
gacgcggttt actttgacgg atcagacccc gacagaatgc acaattatta ttccttttta 1320
tacaacaaaa ccgtatttga cctgttgaag caaaagcgcg gcgaacggga agcggtcgtg 1380
tttgcaaggt cggcgacggc cggaggccag caatttcccg tacactgggg cggggattgt 1440
tttgccagct atgattcaat ggctgaaagc cttcgcggcg gcctgtcgct ttccctttca 1500
ggcttcggct tctggagcca cgatatcggc ggatttgaaa gcacggcaac agccgatttg 1560
tataagcgct ggaccgcatt cggcctcttg tcgacgcaca gccgcctgca cgggagcgaa 1620
tcatatcgcg ttccgtggct gtttgacgaa gaagcggccg atgtgatgcg gtattttgtc 1680
aaactgaagc acaggctgat gccttatttg tacgccgccg cgcatgaagc gcacgctgaa 1740
ggcattccga tgatgcgggc gatgctcctc gaatttcccg gggacaacac ttgccactgg 1800
cttgacagac agtacatgct cggcggaaga ctcctcgtcg ctcccgtttt tcaggaagac 1860
ggcgttgtcc gttattattt gccaaaaggc acctggacac accttttgac gggaaaagag 1920
acggaaggcg gagaatggaa agaagaacag tacgggtata tggggcttcc cctgttcgtc 1980
agggaaaata cgctgctgcc gctcgggcag gaatccgggc gacctgatta tgattatctc 2040
gacggtgtga cgttctgcct ttaccaatta aaagacggct gcacagccga acagacgatt 2100
tacaatgaaa agtgcgaagc aatgacagtc agagcttcac ggcatgaaaa tacgattact 2160
gtgaaaacaa caggcgtcca aaagaaatgg aagctgctca tccgcgatct gtctgtcact 2220
tctgtcataa acggcgcagc ggaagcgctt caggagggaa cagcaattca gccaaaagaa 2280
aaggaccggg atgtcttcat ttttttctaa 2310
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
atggtcaaac cgtatccccc 20
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 7
ctaggctttg cggcttctc 19
<210> 8
<211> 2073
<212> DNA
<213> Artificial Sequence
<400> 8
atggtcaaac cgtatccccc gatcagccgg aaagttcccc gactattgca tggagccgat 60
tacaaccctg atcaatggct taaatatccg gacgttttaa aagaagatat ccgcctgatg 120
aagctgtccc gctgcaatgt gatgtctgtc ggcattttct catgggtttc gctcgagcct 180
gaagaaggaa ggtttacatt tgattggctc gatcaggttc ttgatacttt caaggaaaac 240
ggaatttatg cgttcttggc tacaccgagc ggtgccagac cggcttggat gtccaaaaag 300
tatcccgagg tgctgagaac ggagcgcaac cggatcagaa accttcacgg aaagcggcac 360
aatcattgct atacgtcgcc tgtctaccgc aggaaaacgg cgattataaa cggaaggctt 420
gcggagcgct atgcgcatca cccggctgtc atcggctggc atatctccaa tgaatacggc 480
ggagagtgtc attgtgaact ttgccaagat aagttcagag agtggctgct cgcgaaatat 540
aaaacgctgg accgccttaa ccatgcatgg tggacatcgt tttggagcca cacctataca 600
gattgggacg agatcgagtc gccggctccg cacggcgagg atgccgttca cggtctgaat 660
ttggactgga agcggtttgt cacagatcag acggttgatt tttgccggca tgaaatcgcc 720
catgtccgtt cttttaaccc tgaactgccc gtgacgacga attttatggg aacgtatgcc 780
ggattgaatt attggaagtt tgtcgatttg cttgatgtca tctcgtggga ttcttatccg 840
gcatggcaca acagccggca ggaagacagc cggcttggag ccgatattgc ttttgttcat 900
gatatcaatc ggtctcttaa aggcggccgg ccgttcatgc tgatggaaag tacgccgagc 960
atgacgaact ggcaggacgt cagcaagctg aagcgccccg gtatgcacga attgtcatca 1020
ttacaggcag tcgctcacgg atcggacact gttcagtatt ttcaatggcg gaaaagccgc 1080
ggctcaagcg aaaagtttca tggggcagtc gttgatcatg cgggatcaga gcatacgcgc 1140
gttttccagg aagtaaaaaa acttggggaa aagctcgaaa aacttgagga tgtcgcagga 1200
acgacagtcg aacccgaggc cgcgattata tttgactggg agaatcgctg ggccttggaa 1260
gacgcccaag gtccgcgcaa tatcggcatg cattatgaac ggacagccaa atcgttttat 1320
gaaccgtttt ggcgtgaagg tgtaccggtt gatgtgattc atatggaggc cgatttctcc 1380
cgctataagc ttttggtcgc cccgatgctc tacatgctga aagaaggcac cgccgaaagg 1440
attgaacagt tcgttcaaaa agggggtaca tttgtcgcca cttattggtc gggcattgtc 1500
aatgagaatg atcttgtgtt cttgggaggg tttcctggag gcttaagaaa aacgctcggc 1560
atttggtctg aggagatcga tgcgcttcat gacggacaga aaaatacgat tgtgctggat 1620
gcagataacc ggctcgggtt aagcgggagc tatgaaacca gcgagctttg cgacctgatt 1680
catttagaag gagccgaagc gctcgctctg tacggggaag atttttacgc gggtcggccc 1740
gcgctgacgg tcaatttgtt cggagacggg aaggcctact atatcgcttc aaggaatgag 1800
gcggcgttcc aagaggcgtt tatatcgagg ctcattgaag agtgcgggat tgccaaagtg 1860
ctcgatacag agcttccgaa aggcgtcacc gcccagctcc gcacagacgg ccgccacgat 1920
tatattttcg tgatgaattt tacagcggaa aaccgcacgg tctcgcttcc ggaagaaaca 1980
tatacggacg tcttaaaagg aacccggaaa aaaggcgctc tcaaactggt tccatacgga 2040
acgagcattt tgcggagaag ccgcaaagcc tag 2073
Claims (4)
1. The application of the ginkgo leaf fermentation microbial agent prepared by heat-resistant bacillus licheniformis LZZTT-001 as a fattening pig feed additive is characterized in that the addition amount of the ginkgo leaf fermentation microbial agent is 0.30-0.60% of the basic ration;
the preservation number of the heat-resistant bacillus licheniformis LZZTT-001 is CGMCC No. 18672;
the preparation method of the ginkgo leaf fermentation microbial agent comprises the following specific steps of:
(1) activating the heat-resistant Bacillus licheniformis LZTT-001, transferring the activated strain into seed culture solution, performing shake culture at 30-50 deg.C for 1-3d at a vibration frequency of 150-9CFU/ml；
(2) Adding the ginkgo flavone extract into the seed liquid obtained in the step (1), and fermenting and culturing for 3-10h to obtain fermented seed liquid; adding 20-50mg of ginkgetin extract into each liter of seed liquid, wherein the content of total flavone in the ginkgetin extract is more than or equal to 24%;
(3) inoculating the fermented seed liquid obtained in the step (2) into a solid substrate for solid fermentation culture, and specifically comprising the following steps:
inoculating fermented seed liquid into a solid substrate to obtain a mixture; the inoculation amount of the fermentation seed liquid is 1-10% of the solid matrix, and the bacterial liquid concentration of the fermentation seed liquid is more than or equal to 10%9CFU/ml; the solid matrix contains 10-30% of crushed ginkgo leaves, and the balance of agricultural organic waste; the carbon-nitrogen ratio of the mixture is 25-35: 1, the water content is 45-65%, and the oxygen content is 5-14%; stirring for 1 time at intervals of 2-5 d;
secondly, when the temperature of the reactor is reduced to 45-50 ℃, inoculating the fermented seed liquid again, wherein the inoculation amount is 2-6% of the solid matrix, and the temperature of the reactor is controlled to be 65-75 ℃;
thirdly, after stacking and fermenting for 10-20 days, air-drying the stacked materials to obtain the ginkgo leaf fermentation microbial agent; the content of ginkgetin in each kg of the ginkgo leaf fermentation microbial agent is more than or equal to 2 g; the content of heat-resistant bacillus licheniformis LZZT-001 in per g of folium Ginkgo fermentation microbial agent is not less than 109CFU。
2. The application of the ginkgo leaf fermentation microbial agent prepared from the heat-resistant bacillus licheniformis LZZTT-001 as a fattening pig feed additive according to claim 1, wherein the strain activation operation in the step (1) is as follows: inoculating the preserved heat-resistant bacillus licheniformis LZZTT-001 on a solid culture medium, wherein the culture temperature is 30-50 ℃.
3. The application of the folium ginkgo fermentation microbial agent prepared by the heat-resistant bacillus licheniformis LZZTT-001 as a fattening pig feed additive according to claim 1 is characterized in that the formula of the seed culture solution in the step (1) is as follows: 3-5g of soluble starch, 8-11g of tryptone and 3-5g of sodium chloride, and adding water to 1000 ml; the pH is 7.2-7.4.
4. The application of the folium ginkgo fermentation microbial agent prepared by the heat-resistant bacillus licheniformis LZZTT-001 as a fattening pig feed additive according to claim 1, wherein the agricultural organic waste in the step (3) is at least one of wheat bran, wood chips, peanut shell powder, furfural, bean pulp and sugar residues.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103005159A (en) * | 2012-12-18 | 2013-04-03 | 南京林业大学 | Preparation method of ginkgo leaf biological feed additive |
| CN104771419A (en) * | 2015-04-20 | 2015-07-15 | 龙岩学院 | Preparation method of ginkgo leaf fermentation preparation |
| CN108713558A (en) * | 2018-05-12 | 2018-10-30 | 湖南科技学院 | The growth-promoting preparation of general flavone content in a kind of promotion ginkgo leaf |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103005159A (en) * | 2012-12-18 | 2013-04-03 | 南京林业大学 | Preparation method of ginkgo leaf biological feed additive |
| CN104771419A (en) * | 2015-04-20 | 2015-07-15 | 龙岩学院 | Preparation method of ginkgo leaf fermentation preparation |
| CN108713558A (en) * | 2018-05-12 | 2018-10-30 | 湖南科技学院 | The growth-promoting preparation of general flavone content in a kind of promotion ginkgo leaf |
Non-Patent Citations (2)
| Title |
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| 产黄酮银杏内共生真菌的分离及其发酵条件的优化;张洋等;《工业微生物》;20150430;第45卷(第2期);第1.1.1-2.3.2部分 * |
| 发酵银杏叶制备新型生物饲料添加剂的研究;高树峰;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150415(第04期);D050-7页，具体参见摘要，第19页第2.1部分至2.2.3部分，第37页第2.5部分至第38页第2.8部分 * |
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