CN109517761A - The bacillus licheniformis of cellulase-producing, its microbial fermentation preparation and its application - Google Patents
The bacillus licheniformis of cellulase-producing, its microbial fermentation preparation and its application Download PDFInfo
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- CN109517761A CN109517761A CN201811560739.2A CN201811560739A CN109517761A CN 109517761 A CN109517761 A CN 109517761A CN 201811560739 A CN201811560739 A CN 201811560739A CN 109517761 A CN109517761 A CN 109517761A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 51
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- 229940106157 cellulase Drugs 0.000 title claims abstract description 20
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
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- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
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Abstract
The present invention relates to a kind of bacillus licheniformis 50 of cellulase-producing and its microbial fermentation preparation and applications, the bacterium is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is CGMCC No.15012.Bacillus licheniformis 50 of the invention being capable of High Cellulase Production, improve the degradation capability to cellulose in corn processing by-product, it can be adapted for using corn processing by-product as in the application of the feed fermentation of raw material, fiber content in degradable fermented feed is conducive to efficient utilization of the fowl poultry kind to feed to improve the quality of feed.
Description
Technical field
The present invention relates to field of microbial fermentation, specifically, the present invention relates to the lichens gemma of one plant of cellulase-producing
Bacillus, and the microbial fermentation preparation comprising the bacterial strain and its answering in the fermentation of the feed comprising corn processing by-product
With.
Background technique
Corn is the main crops product in China, and annual output is about at 200,000,000 tons.Wherein, per directly 1 ton of corn of processing,
It can produce 0.09 ton of corn pulp, 0.07 ton of plumule dregs of rice, 0.056 ton of corn protein powder and 0.13 ton of corn bran；Simultaneously with corn
It ferments for raw material, can produce many fermented products, such as alcohol fuel, citric acid, amino acid；Also it can produce a large amount of pair
Product, such as DDGS, citric acid grain.Corn processing product such as corn bran, DDGS, citric acid grain etc. is usually added directly to
It is used for livestock and poultry cultivation in feed, but it to contain the non-starch polysaccharide such as higher cellulose, hemicellulose, chitin, directly adds
It is difficult to be digested by livestock and poultry such as pig, chickens into feed, while influencing the absorption of the mineral ions such as phosphorus, reducing the utilization of nutriment,
The problems such as easily causing livestock and poultry diarrhea, slow growth.Various unfavorable factors limit corn processing by-product answering in feed
With.
Fermentative feedstuff of microbe, by the metabolic activity of microorganism itself, selectively will under manual control condition
Anti-nutritional factors in vegetalitas, animality and mineral substance is decomposed or conversion, and generating can more be searched for food by livestock and poultry, digest and assimilate
And the feed of nonhazardous effect.Wherein, solid microbe fermentation technology is applied relatively broad in China's feedstuff industry, mainly
Anti-nutritional factors (such as non-starch polysaccharide) content in raw material is reduced by the Enzymatic characteristic and its metabolite of microorganism,
Improve the product quality and digestibility of feed.
Bacillus licheniformis (Bacillus licheniformis) is strong with accommodative ability of environment, has enzyme abundant
It is, there is wider antimicrobial spectrum, adjusts the excellent performances such as intestinal flora, the speed of growth be fast, is used frequently as feed addition bacterial strain.It
Right bacillus licheniformis can produce a variety of enzymes, such as phytase, amylase, protease, cellulase, but its Enzymatic characteristic
With environment (such as soil constituent), incubation time, condition of culture (such as carbon source) and change.For example, being using microcrystalline cellulose
When sole carbon source, bacillus licheniformis does not generate cellulase (referring to " bacillus licheniformis and other microorganism enzymatic productivities
Compare ", Bai Jianling, feed research, 2003 (7): 4-6).And in the art it has not been found that in corn processing product
Cellulose have higher fiber element enzyme activity bacillus licheniformis.
In order to solve the problems, such as above-mentioned corn processing by-product encountered in feed applications, the present inventor it is expected to obtain high
The bacillus licheniformis of cellulase-producing is prepared into microorganism formulation using the bacillus licheniformis and carries out microbe in solid state hair
Ferment, the fiber content being effectively reduced in corn processing by-product improve the quality of fermented feed, are conducive to fowl poultry kind to feed
Efficient utilization.
Summary of the invention
In order to solve the above-mentioned technical problem, in a first aspect, the present invention provides the lichens gemma of one plant of cellulase-producing
Bacillus (Bacillus licheniformis) 50 (also referred to as " BL50 "), deposit number are CGMCC No.15012.The bacterial strain
The cellulase (enzyme activity of fermentation liquid is up to 114U/mL) of high yield can be generated, and using the bacterial strain to including richness
The fiber content when feed of cellulose-containing corn processing by-product is fermented, in the feed that can effectively degrade.The bacterial strain is not
Containing virulence gene, belong to safe bacterial strain.
In second aspect, the present invention provides a kind of microbial fermentation preparations, wherein the microbial fermentation preparation includes
Bacillus licheniformis 50 described in first aspect, the microbial fermentation preparation can be used for adding to comprising the corn containing cellulose
The feed of work by-product ferments.
In the third aspect, the present invention provides described in first aspect bacillus licheniformis 50 or second aspect described in it is micro-
Purposes of the biofermentation preparation in fermenting to the feed comprising the corn processing by-product containing cellulose.
Advantageous effects of the invention are as follows: bacillus licheniformis 50 of the invention is free of virulence gene, belongs to safety
Bacterial strain is colonized in the enteron aisle of fowl poultry kind after being eaten with feed, is played a role as healthy intestinal microorganism；The present invention
The cellulase activity that generates of bacillus licheniformis 50 it is high (enzyme activity of fermentation liquid is up to 114U/mL), matched using the bacterial strain
The feed that other strain fermentations include the corn processing by-product containing cellulose is closed, it can fiber effectively in degradation of fibers feed
(fermented feed has lower acid washing fiber, neutral washing fiber and crude fiber content), fermentation time is short, fermentation
Number of viable is high in feed afterwards, obtains the feed for being conducive to the high-quality of fowl poultry kind digestion and absorption.
Detailed description of the invention
Fig. 1 is the 16S rDNA sequence of bacillus licheniformis 50.
Fig. 2 is the photo of state of the bacillus licheniformis 50 on CMC screening and culturing medium plate.
Specific embodiment
In the present invention, the bacillus licheniformis isolation and purification method of the cellulase-producing includes: using lichens gemma bar
Bacterium culture medium separates bacillus licheniformis from Shanxi Fenjiu yeast sample, using gradient dilution method, is coated on ground for dilution
On clothing bacillus solid medium, 12-24h is cultivated at 30-37 DEG C, bacillus licheniformis is purified by plate streak,
Obtain form single colonie identical with the morphological feature of bacillus licheniformis；The bacillus licheniformis dibbling that separation obtains is existed
On CMC screening and culturing medium, then handles, filter out with larger diameter (about through Congo red solution and sodium chloride solution respectively
4cm) the single colonie of transparent circle extracts single colonie genome, its 16S rDNA sequence (SEQ ID NO.:1) is sequenced, with
Ncbi database is compared, and carries out molecular biology identification.Meanwhile according to the qualification result of physiological and biochemical property (referring to " primary
Outstanding Bacteria Identification handbook ", Science Press, 1984 and " common bacteria system identification handbook ", Science Press, 2001
In record identified), finally the bacterial strain is identified and is named as 50 (Bacillus of bacillus licheniformis
licheniformis 50).The bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number are CGMCC No.15012, and the deposit date is 2017 12
The moon 4, specific name are bacillus licheniformis (Bacillus licheniformis).
In one embodiment, the present invention provides the bacillus licheniformis (Bacillus of one plant of cellulase-producing
Licheniformis) 50, deposit number is CGMCC No.15012.
In a preferred embodiment, the cellulose enzyme vigor of the fermentation liquid of the bacillus licheniformis 50 may be up to
114U/mL.In the present invention, unless otherwise indicated, enzyme activity is also referred to as enzymatic activity, refers to the energy that enzymatic centainly chemically reacts
Power.The size of enzyme activity can be under certain condition, and the conversion rate of a certain chemical reaction that it is catalyzed indicates, i.e. enzyme
The conversion rate of catalysis is faster, and the vigor of enzyme is higher.
In one embodiment, the present invention provides a kind of microbial fermentation preparations, wherein the microbial fermentation system
Agent includes above-mentioned bacillus licheniformis 50.
In some preferred embodiments, the microbial fermentation preparation further comprises in feed science or veterinarily
Acceptable additive, such as various antibiotic, antioxidant, mould inhibitor, adhesive, colorant and/or flavoring agent etc..Ability
Field technique personnel can select the amount of additive according to actual needs, and this point can't limit the invention.
In some preferred embodiments, the microbial fermentation preparation also includes in feed science or veterinarily beneficial
Other microorganisms, such as, but not limited to yeast, bacillus subtilis, bifidobacterium bifidum and/or lactobacillus.
In further preferred embodiment, the microbial fermentation preparation further include lactobacillus plantarum and/or
Candida utili.
In some preferred embodiments, in the microbial fermentation preparation, the bacillus licheniformis 50 contains
Amount is at least 1 × 108CFU/mL, preferably 1 × 108CFU/mL-10×108CFU/mL。
In a further preferred embodiment, in the microbial fermentation preparation, the lactobacillus plantarum
Content is 1 × 108CFU/mL-10×108CFU/mL。
In another further preferred embodiment, in the microbial fermentation preparation, the Candida utilis ferment
Female content is 1 × 108CFU/mL-10×108CFU/mL。
Wherein, the lactobacillus plantarum and candida utili can be commercially available from from the market, it is possible to use this field
It is known separation and/or screening lactobacillus plantarum and candida utili method from containing them source (such as fermentation ox
Cream, fermented feed) in separation and/or screening get.The selection of the source of lactobacillus plantarum and candida utili is not limiting upon
The scope of the present invention.
In one embodiment, the present invention also provides bacillus licheniformis 50 of the present invention or of the present invention
Purposes of the microbial fermentation preparation in fermenting to the feed comprising the corn processing by-product containing cellulose.
In a preferred embodiment, the corn processing by-product containing cellulose refers to that corn processing process generates
The by-product that can be used to that feed is made, such as corn bran, whitewashing maize peel, DDGS (doing full vinasse), DDS (solvable dry wine
Smart grain), corn flour or corn starch residue or their any combination.
In a preferred embodiment, the temperature of the fermentation is 28 DEG C -37 DEG C, preferably 30 DEG C.In preferred embodiment party
In formula, the pH value of real-time monitoring fermentation system during the fermentation stops fermentation when pH value reaches 4.5 or less.It is logical
Often, fermentation time can be 3-6 days.
In a preferred embodiment, after carrying out the fermentation, the viable bacteria of the bacillus licheniformis 50 in the feed
Sum is 1 × 108CFU/mL-10×108CFU/mL。
For purposes of illustration, the purpose of the present invention can be for example achieved by content described in following paragraph:
1. the bacillus licheniformis (Bacillus licheniformis) 50 of one plant of cellulase-producing, deposit number are
CGMCC No.15012。
2. the bacillus licheniformis 50 as described in paragraph 1, wherein the cellulose of the fermentation liquid of the bacillus licheniformis 50
Enzyme enzyme activity is up to 114U/mL.
3. a kind of microbial fermentation preparation, wherein the microbial fermentation preparation includes lichens bud described in paragraph 1 or 2
Spore bacillus 50.
4. the microbial fermentation preparation as described in paragraph 3, wherein the microbial fermentation preparation further comprises feed science
Upper or veterinarily acceptable additive.
5. the microbial fermentation preparation as described in paragraph 4, wherein the additive is selected from antibiotic, antioxidant, mould proof
Agent, adhesive, colorant and/or flavoring agent.
6. the microbial fermentation preparation as described in any one of paragraph 3-5, wherein the microbial fermentation preparation also includes
In feed science or veterinarily beneficial other microorganisms.
7. the microbial fermentation preparation as described in paragraph 6, wherein beneficial other microorganisms are selected from yeast, withered grass
Bacillus, bifidobacterium bifidum and/or lactobacillus.
8. the microbial fermentation preparation as described in paragraph 7, wherein the yeast is candida utili.
9. the microbial fermentation preparation as described in paragraph 7 or 8, wherein the lactobacillus is lactobacillus plantarum.
10. the microbial fermentation preparation as described in paragraph 7, wherein the microbial fermentation preparation further includes plant
Lactobacillus and/or candida utili.
11. the microbial fermentation preparation as described in either segment in paragraph 3-10, wherein the bacillus licheniformis 50 contains
Amount is at least 1 × 108CFU/mL。
12. the microbial fermentation preparation as described in paragraph 11, wherein the content of the bacillus licheniformis 50 be 1 ×
108CFU/mL-10×108CFU/mL。
13. the microbial fermentation preparation as described in paragraph 8 or 10, wherein the content of the candida utili be 1 ×
108CFU/mL-10×108CFU/mL。
14. the microbial fermentation preparation as described in paragraph 9 or 10, wherein the content of the lactobacillus plantarum be 1 ×
108CFU/mL-10×108CFU/mL。
15. microbial fermentation system described in either segment in bacillus licheniformis 50 or paragraph 3-14 described in paragraph 1 or 2
Purposes of the agent in fermenting to the feed comprising the corn processing by-product containing cellulose.
16. the purposes as described in paragraph 15, wherein the corn processing by-product containing cellulose is selected from corn bran
Skin, whitewashing maize peel, DDGS, DDS, corn flour or corn starch residue or their any combination.
17. the purposes as described in paragraph 15 or 16, wherein the temperature of the fermentation is 28-37 DEG C.
18. the purposes as described in paragraph 17, wherein the temperature of the fermentation is 30 DEG C.
19. the purposes as described in either segment in paragraph 15-18, wherein real-time monitoring is fermented during the fermentation
The pH value of system stops fermentation when pH value reaches 4.5 or less.
20. the purposes as described in either segment in paragraph 15-19, wherein the time of the fermentation is 3-6 days.
21. the purposes as described in either segment in paragraph 15-20, wherein the ground after carrying out the fermentation, in the feed
The total viable count of clothing bacillus 50 is 1 × 108CFU/mL-10×108CFU/mL。
Embodiment
In order to keep the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with the drawings and the specific embodiments,
The present invention will be described in further detail.It should be appreciated that the specific embodiments described herein are only used to explain this hair
It is bright, it is not intended to limit the present invention.Experimental method described in following embodiments is unless otherwise specified conventional method.It is following
Experimental material used in embodiment, unless otherwise instructed, whole reagents are public purchased from the extensive and profound in meaning star biotechnology Limited Liability in Beijing
Department.
Reagent used in embodiment:
1. bacillus licheniformis culture medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, glucose
7.0,121 DEG C of sterilizing 15min of 20.0g, distilled water 1000mL, pH.
2. bacillus licheniformis solid medium: tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, grape
Sugared 20.0g, agar 20.0g, 7.0,121 DEG C of sterilizing 15min of distilled water 1000mL, pH.
3.CMC screening and culturing medium: sodium carboxymethylcellulose (CMC) 20.0g, tryptone 10.0g, yeast extract 5.0g,
7.0,121 DEG C of sterilizing 15min of agar 20.0g, distilled water 1000mL, pH.
4. cellulase-producing test media: CMC 20.0g, peptone 10.0g, magnesium chloride 0.3g, sodium chloride 10.0g,
Dipotassium hydrogen phosphate 1.0g, distilled water 1000ml, 121 DEG C of sterilizing 15min.
5.MRS culture medium: MRS culture medium 65.25g, distilled water are settled to 800mL, 121 DEG C of 15~20min of sterilizing.
6.YPD culture medium: yeast powder 10g, peptone 20g, glucose 20g, distilled water are settled to 1000mL, and 121 DEG C go out
15~20min of bacterium.
Embodiment 1: the separation and screening of bacillus licheniformis
It takes 10g Shanxi Fenjiu yeast sample in 90mL sterile saline, is placed on shaking table, turn in 30 DEG C, 200rpm
Speed is lower to vibrate 30min, then 10 times of gradient dilutions to 10-8.Take 1mL10-8Dilution into 6cm plate, be added 20mL 80
DEG C holding melting state bacillus licheniformis solid medium, mixing is cooled to room temperature, and 30 DEG C of perseverances are inverted in after to be solidified
It is cultivated for 24 hours in warm incubator.After for 24 hours, it can observe that multiple bacterium colonies generate on plate, colonial morphology is circular colonies, side
Edge is irregular, there is fold on surface, is positive through Gram's staining；100 times of optical microscopy oil under the microscope, thalli morphology at
Elongated rod shape, single, pairs of or catenation；This feature is identical as the morphological feature of bacillus licheniformis.
Picking individual colonies (concentration 109CFU/mL it) is seeded on CMC screening and culturing medium plate, is cultivated in 30 DEG C of inversions
24h.After for 24 hours, the Congo red solution of 5mL 1w/v% is added into plate, discards Congo red solution after standing 30min at room temperature.To
1mol/L sodium chloride solution is added in plate, discards sodium chloride solution after standing 30min at room temperature.At this point, part bacterium on plate
It falls around and a circle transparent circle occurs, as shown in Figure 2.The single colonie that wherein transparent circle is relatively large in diameter (diameter is about 4.0cm) is connect
Kind is into bacillus licheniformis culture medium, and shaken cultivation for 24 hours, then carries out scribing line culture and obtains single bacterium under 30 DEG C, 200rpm
It falls.
The single colonie that scribing line obtains is repeated the above steps 3 times.Transparent loop diameter is bigger to illustrate bacterial strain cellulase-producing energy
Power is stronger, and according to transparent circle size caused by bacterium colony on CMC screening and culturing medium, having filtered out one plant can be compared with good utilisation CMC's
Bacterial strain is named as bacillus licheniformis 50.
Embodiment 2: the identification to bacillus licheniformis 50
The identification of 2.1 colonial morphologies
To the bacterial strain separated in embodiment 1 by Observation of biological characteristics, carrying out further Morphological Identification (includes: to see
Examine form and edge, the Gram's staining etc. of bacterium colony), above-mentioned standard of perfection is referring to " Berger bacterial identification manual ", scientific publication
Society, 1984 and " common bacteria system identification handbook ", Science Press, 2001.By the bacterial strain of the separation in lichens bud
On spore bacillus solid medium after 30 DEG C of cultures for 24 hours, colonial morphology is circular colonies, edge is irregular, there is fold on surface, is passed through
Gram's staining is positive；In 100 times of oil microscopic observations of optical microscopy, thalli morphology is at elongated rod shape, single, pairs of or chain
Arrangement；This feature is identical as the morphological feature of bacillus licheniformis.
The identification of 2.2 taxology
The identification on taxology is carried out to bacillus licheniformis 50 by 16S rDNA sequence.Use hundred Tyke bacterium high passes
Amount DNA extraction kit (hundred Tyke Bioisystech Co., Ltd of Beijing, AU46111-96) extracts 50 bacterial strain of bacillus licheniformis
Genomic DNA.With bacterial universal primers (upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID NO.:2)；
Downstream primer: 5 '-AAGGAGGTGATCCAGCCGCA-3 ' (SEQ ID NO.:3)) carry out 16S rDNA PCR amplification.PCR is anti-
Answer condition are as follows: 95 DEG C of 4min；94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1.5min, 30 circulations；72 DEG C of extension 8min, 4 DEG C of preservations.
Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced amplified production, gained sequence results (SEQ as shown in Figure 1
ID NO.:1).Sequencing result is subjected to BLAST comparison, the bacterial strain and Bacillus licheniformis HT- in NCBI
Z58 (GeneBank:KJ526871.1 homology highest, homology 100%.Therefore, the bacillus licheniformis 50 filtered out exists
Belong to bacillus licheniformis on taxology.
The identification of 2.3 cellulase activities
By isolated bacillus licheniformis A1 and bacillus licheniformis according to the present invention from Xiangshan's soil
50 are seeded in the 50mL cellulase-producing test media in 250mL triangular flask, are placed on shaking table, 30 DEG C and 200rpm of turn
Speed is lower to cultivate 48h.The enzyme activity of their cellulase-producings is measured using CMC enzyme activity determination method, specific method is referring to following document
Record: " separation of one plant of cellulose-degrading bacteria, identification and the degradation characteristic to corn stover ", Wu Wentao, Ju Meiting, Liu Jin
Roc etc., microbiology notification, 2013,40 (4): 712-719.After measured, the cellulose of the 48h fermentation liquid of bacillus licheniformis A1
The enzyme activity of enzyme is only 36.65U/mL, and the corresponding enzyme activity of bacillus licheniformis 50 is up to 114U/mL.
The preparation of 3 microbe leaven of embodiment
Obtain bacillus licheniformis 50, lactobacillus plantarum (separating from commercially available fermentation cow's milk) respectively by following steps
Bacterium solution after being cultivated with candida utili (being separated from commercially available fermented feed):
(1) bacillus licheniformis: the bacillus licheniformis 50 that embodiment 1 obtains is seeded to ground with the inoculum concentration of 1v/v%
It in clothing bacillus culture medium, is placed on constant-temperature table, 30 DEG C, overnight incubation under 200rpm revolving speed.
(2) lactobacillus plantarum: lactobacillus plantarum is seeded in MRS culture medium with the inoculum concentration of 1v/v%, is placed in constant temperature
On shaking table, 30 DEG C, overnight incubation under 200rpm revolving speed.
(3) candida utili: candida utili is seeded in YPD culture medium with the inoculum concentration of 1v/v%, is placed in
On constant-temperature table, 30 DEG C, overnight incubation under 200rpm revolving speed.
The viable count that each bacterium in above-mentioned bacterium solution is measured using GB4789.2-2006 method, by the bacillus licheniformis of acquisition,
Lactobacillus plantarum and candida utili bacterium solution are mixed with 2: 2: 1 ratio, wherein so that bacillus licheniformis content is about 1
Hundred million CFU/mL, lactobacillus plantarum content are about 100,000,000 CFU/mL, and candida utili content is about 100,000,000 CFU/mL, are prepared into micro- life
Object fermentation preparation.
Embodiment 4 is fermented using the microbial fermentation preparation of embodiment 3
By 1.4 parts by weight Corn starch slags, 0.08 parts by weight beet molasses, 0.72 parts by weight DDGS, 0.6 parts by weight DDS,
0.24 parts by weight Corn powder, the whitewashing maize peel mixing of 0.4 parts by weight are used as feedstuff, add water, water content is adjusted to
35wt%, is distributed into 400g/ bags, spare.Microbial fermentation preparation prepared by embodiment 3 is inoculated with the inoculative proportion of 4v/w%
Extremely experimental group is used as in above-mentioned solid feed raw material.Add same amount of microbial fermentation preparation containing bacillus licheniformis A1
(the bacterium solution in addition to the bacterium solution of bacillus licheniformis 50 to be substituted for bacillus licheniformis A1, according to the identical method system of embodiment 3
) as a control group.Experimental group and control group are left to ferment at 30 DEG C, the pH value of real-time monitoring feed, when pH value is 4.5
Stop fermentation (at this point, common fermentation 6 days).The titratable acidity of test experience group and control group fermentation front and back, thick according to the following method
Albumen, crude fibre, neutral detergent fiber, acid detergent fiber and viable count: titratable acidity measurement: GB/T 12456-2008；Slightly
Protein determination: Kjeldahl's method (GB6432-94)；Neutral detergent fiber, acid detergent fiber, crude fibre measurement: normal form fiber
Measuring method (Van Soest etc., 1991).Testing result is shown in Table 1.
The testing result of 1 experimental group of table and control group fermentation front and back
As shown in table 1, experimental group makes microbial fermentation preparation prepared with embodiment 3 (containing lichens gemma of the invention
Bacillus 50) fermented after, before fermentation, the ratio that crude fibre in feedstuff reduces is about 24.66%, neutral
The ratio that washing fiber reduces is about 16.73%, and the ratio that acid detergent fiber reduces is about 18.06%；And control group uses
After fermentation preparation (containing bacillus licheniformis A1) is fermented, before fermentation, the ratio of the crude fibre reduction in feedstuff
Example is only about 13.33%, and the ratio that neutral detergent fiber reduces is about 4.71%, and the ratio that acid detergent fiber reduces is about
5.56%, therefore, the microbial fermentation of the invention being made of bacillus licheniformis 50, lactobacillus plantarum, candida utili
Agent can be effectively reduced using corn processing by-product as the content of cellulose in feed made of raw material, improve the product of such feed
Matter is conducive to fowl poultry kind digestion and absorption.
In addition, (before fermentation, being increased 1.7%), present invention experiment compared to the feed after the fermentation of control group
There is feed after the fermentation of group higher titratable acidity (before fermentation, to increase 2.8%), therefore, using the present invention
The microbial fermentation preparation makes the degree of feed fermentation higher.
Meanwhile the feed of bacillus licheniformis A1, lactobacillus plantarum and candida utili after fermentation in control group
In viable count respectively reach 6.7 × 107CFU/mL、1.2×107CFU/mL、4.3×106CFU/mL, in contrast, experimental group
In bacillus licheniformis 50, lactobacillus plantarum and candida utili feed after fermentation in viable count respectively reach
5.6×108CFU/mL、4.8×108CFU/mL、3.4×108CFU/mL, therefore, microbial fermentation preparation of the present invention exist
The viable count of considerably higher content can be generated after fermentation.The beneficial bacterium of high-content is determined after entering animal intestinal tract with fermented feed
It grows in animal intestinal tract, can more strongly adjust animal intestinal micro-ecology balance, better play and ensure animal body health
Effect.
Sequence table
<110>Jilin COFCO Biochemical Co., Ltd.
Cofco Nutrition And Health Research Institute Co., Ltd.
COFCO Biochemical (Anhui) Co., Ltd.
<120>bacillus licheniformis of cellulase-producing, its microbial fermentation preparation and its application
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ggaaaccggg gctaataccg gatgcttgat tgaaccgcat ggttcaatca taaaaggtgg 180
cttttagcta ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaacggc 240
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ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt 660
agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg 720
taactgacgc tgaggcgcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780
acgccgtaaa cgatgagtgc taagtgttag agggtttccg ccctttagtg ctgcagcaaa 840
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aggtcttgac atcctctgac aaccctagag atagggcttc cccttcgggg gcagagtgac 1020
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg 1140
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200
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Claims (10)
1. the bacillus licheniformis (Bacillus licheniformis) 50 of one plant of cellulase-producing, deposit number CGMCC
No.15012。
2. bacillus licheniformis 50 as described in claim 1, wherein the cellulose of the fermentation liquid of the bacillus licheniformis 50
Enzyme enzyme activity is up to 114U/mL.
3. a kind of microbial fermentation preparation, wherein the microbial fermentation preparation includes lichens bud of any of claims 1 or 2
Spore bacillus 50.
4. microbial fermentation preparation as claimed in claim 3, wherein the microbial fermentation preparation further comprises feed science
Upper or veterinarily acceptable additive；Preferably, the additive is selected from antibiotic, antioxidant, mould inhibitor, coheres
Agent, colorant and/or flavoring agent.
5. microbial fermentation preparation as described in claim 3 or 4, wherein the microbial fermentation preparation also includes feed science
Upper or veterinarily beneficial other microorganisms；
Preferably, beneficial other microorganisms are selected from yeast, bacillus subtilis, bifidobacterium bifidum and/or lactobacillus；
Preferably, the yeast is candida utili；Additionally preferably, the lactobacillus is lactobacillus plantarum；
It is further preferred that the microbial fermentation preparation further includes lactobacillus plantarum and/or candida utili.
6. the microbial fermentation preparation as described in any one of claim 3-5, wherein the content of the bacillus licheniformis 50
It is at least 1 × 108CFU/mL, preferably 1 × 108CFU/mL-10×108CFU/mL；
Preferably, the content of the candida utili is 1 × 108CFU/mL-10×108CFU/mL；
Additionally preferably, the content of the lactobacillus plantarum is 1 × 108CFU/mL-10×108CFU/mL。
7. microbial fermentation described in any one of bacillus licheniformis 50 of any of claims 1 or 2 or claim 3-6
Purposes of the preparation in fermenting to the feed comprising the corn processing by-product containing cellulose.
8. purposes as claimed in claim 7, wherein the corn processing by-product containing cellulose be selected from corn bran,
Whitewashing maize peel, DDGS, DDS, corn flour or corn starch residue or their any combination.
9. purposes as claimed in claim 7 or 8, wherein the temperature of the fermentation is 28-37 DEG C, preferably 30 DEG C；In addition preferably
Ground, the pH value of real-time monitoring fermentation system during the fermentation stop fermentation when pH value reaches 4.5 or less；Into one
Preferably, the time of the fermentation is 3-6 days to step.
10. purposes as claimed in any one of claims 7-9, wherein the lichens after carrying out the fermentation, in the feed
The total viable count of bacillus 50 is 1 × 108CFU/mL-10×108CFU/mL。
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| CN109913388A (en) * | 2019-03-20 | 2019-06-21 | 中粮集团有限公司 | Improve composite bacteria agent and its application of corn soaking effect |
| CN110024910A (en) * | 2019-05-17 | 2019-07-19 | 河南农业大学 | The method for improving wormwood tunning quality with cellulase and composite probiotics ferment |
| CN110157636A (en) * | 2019-04-01 | 2019-08-23 | 成都市农林科学院 | A kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis |
| CN111621432A (en) * | 2020-04-10 | 2020-09-04 | 四川轻化工大学 | Bacillus licheniformis, screening method and application |
| CN112980740A (en) * | 2021-04-12 | 2021-06-18 | 江苏省农业科学院 | Bacillus licheniformis and application thereof |
| CN113388535A (en) * | 2020-03-13 | 2021-09-14 | 中粮集团有限公司 | Staple food leavening agent and preparation method and application thereof |
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| CN110157636A (en) * | 2019-04-01 | 2019-08-23 | 成都市农林科学院 | A kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis |
| CN110024910A (en) * | 2019-05-17 | 2019-07-19 | 河南农业大学 | The method for improving wormwood tunning quality with cellulase and composite probiotics ferment |
| CN113388535A (en) * | 2020-03-13 | 2021-09-14 | 中粮集团有限公司 | Staple food leavening agent and preparation method and application thereof |
| CN111621432A (en) * | 2020-04-10 | 2020-09-04 | 四川轻化工大学 | Bacillus licheniformis, screening method and application |
| CN111621432B (en) * | 2020-04-10 | 2022-04-12 | 四川轻化工大学 | Bacillus licheniformis, screening method and application |
| CN112980740A (en) * | 2021-04-12 | 2021-06-18 | 江苏省农业科学院 | Bacillus licheniformis and application thereof |
| CN112980740B (en) * | 2021-04-12 | 2021-09-28 | 江苏省农业科学院 | Bacillus licheniformis and application thereof |
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