CN104789491B - Lichem bacillus strain and its application - Google Patents
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Abstract
A kind of lichem bacillus strain and its application, the bacterial strain is lichem bacillus strain BL14 (Bacillus licheniformis), China Committee for Culture Collection of Microorganisms's common micro-organisms center was preserved on 2 28th, 2014, deposit number is CGMCC NO.8865.The application of the lichem bacillus strain, including：Tofu wastewater is collected, pH to 7.0 is adjusted, 121 DEG C of autoclaving 25min is placed in, by lichem bacillus strain BL14 with 106Cfu/mL inoculum concentration is inoculated in tofu wastewater, aeration, and 33 DEG C are cultivated 3 days, and tofu wastewater is fermented into complex enzyme liquid.Bacillus licheniformis can be using tofu wastewater as unique nutriment Fermentative growth, and produces the enzyme liquid of endoglucanase and zytase, and the production for endoglucanase and xylanase preparation provides new fermentation bacterium source.
Description
Technical field
Present invention relates particularly to a kind of lichem bacillus strain and its application.
Background technology
In order to make full use of resource, cost-effective, cellulose and high the material such as rice bran, wheat time of hemicellulose level
Powder, Cottonseed Meal, powder of straw, herbage etc. are often used as the materials such as the high corn and soybean of raw material fictitious hosts, wheat.So at high proportion
Crude fibre daily ration not only can not directly be absorbed by livestock and poultry, be also easy to cause the various problems such as livestock and poultry diarrhea, slow-growing.
To improve feed quality, feed factory and plant have been fully recognized that in feed and have added enzyme material, particularly cellulase
The importance for the non-digestive ferment that can not be synthesized with animals itself such as hemicellulases.
The main component of plant cell wall is made up of cellulose, hemicellulose and lignin, cellulose and hemicellulose phase
Mutual commissure, after cellulose hydrolyzation plant cellulose discharges free sugar so that cellulose obtains pine with hemicellulose commissure body
Speed, hemicellulose exposure is simultaneously resolved into rapidly the materials such as xylose by zytase.Because endoglucanase and zytase are
One of main hydrolase of degraded cellulose and hemicellulose.Therefore the dual of endoglucanase and zytase is passed through
Hydrolysis can be by the cellulose of high content, hemicellulose degradation Cheng Yi are absorbed by animal in feed soluble small molecule
Class carbohydrate, not only increase feed palatability, more improve feed absorption rate, be greatly facilitated livestock and poultry health into
It is long.
For feed processing industry, cellulose in degrading plant cell membrane can not only be met simultaneously by adding single enzyme preparation
With the demand of hemicellulose, and the production cost that various enzyme preparations also considerably increase enterprise is added one by one, therefore from application
Effect and the angle of the production cost all kinds of complex enzyme formulations that set out increasingly are favored by enterprise, substantial amounts of zoopery
Show to add the utilization ratio that complex enzyme formulation greatly improves feed really.The widely used excellent species selected at present
The mostly Mycophyta such as trichoderma, Penicillium, aspergillus niger, because the production cycle used in such bacterium production cellulase is grown, producing enzyme
Substantial amounts of noxious material is also produced simultaneously, thick enzyme can not be directly appended in feed caused by its metabolism, therefore such as film mistake
Filter etc. removes noxious material must can not in industrial fiber element enzyme preparation to purify that the technical program of cellulase substantially produces
Few link.Just not saying enzyme occurs largely being lost in this course, the use of this production routine and the length of production cycle
Also the production cost of enterprise is significantly greatly increased.
Tofu wastewater, is the bean curd draining that bean curd is formed in process of production, and its yield is 3-5 times of soybean dry weight.
Bean curd water solid content accounts for 1%, is mainly made up of soluble protein, oligosaccharide, vitamin, lipid, trace element etc..
China is the big country of bean curd production and consumption, and it produces a large amount of high concentrated organic wastewaters of generation and enters the surface water, sends out in the environment
Ferment causes the severe exacerbation of neighbouring water quality, serious threat environment for human survival.
The content of the invention
One of the technical problem to be solved in the present invention, it is to provide a kind of lichem bacillus strain.
What the present invention was realized in：A kind of lichem bacillus strain, the bacterial strain are lichem bacillus strain
BL14 (Bacillus licheniformis), Chinese microorganism strain preservation conservator was preserved on 2 28th, 2014
Meeting common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCC NO.8865.
The second technical problem to be solved by the present invention, it is to provide a kind of application of described lichem bacillus strain.
What the present invention was realized in：A kind of application of described lichem bacillus strain, collects during bean curd makes and gives up
The tofu wastewater abandoned, the pH to 7.0 of tofu wastewater is adjusted, be placed in 121 DEG C of autoclaving 25min in fermentation tank, by the lichens bud
Spore bacillus strain BL14 is with 106Cfu/mL inoculum concentration is inoculated in fermentation tank, aeration, and 33 DEG C are cultivated 3 days, and tofu wastewater is sent out
Ferment is into the complex enzyme liquid containing endoglucanase and zytase.
Further, the assay method of the endo-glucanase enzyme activity is as follows：
The carboxymethylcellulose sodium solution for being l% with 0.05mol/L pH 5.0 phosphate buffered saline mass fraction
1.7ml, add complex enzyme liquid described in 0.5mL, 0.3ml 0.05mol/L pH 5.5 phosphate buffer, in 50 DEG C of reactions
After 5min, add 1mL 3,5- edlefsen's reagent terminating reactions, then boil 5min in boiling water bath, dilute after
540nm colorimetrics.
Further, the assay method of the Xylanase activity is as follows：Delayed with 0.05mol/L pH 5.0 phosphate
Fliud flushing prepares the xylan solution 1.0ml that mass fraction is l%, adds complex enzyme liquid described in 0.5mL, 1.0ml 0.05mol/
L pH 5.5 phosphate buffer, after reacting 10min at 50 DEG C, 1mL 3 is added, 5- edlefsen's reagents terminate anti-
Should, 5min then is boiled in boiling water bath, is diluted after 540nm colorimetrics.
The advantage of the invention is that：Lichem bacillus strain BL14 can ferment by unique nutriment of tofu wastewater
Growth, and the enzyme liquid of endoglucanase and zytase is produced, so as to the recycling for tofu wastewater and feed manufacturing
The production of endoglucanase and xylanase preparation provides new fermentation bacterium source in industry.
Brief description of the drawings
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the colonial morphology figure of lichem bacillus strain BL14 in the present invention.
Fig. 2 is the Gram's staining figure of lichem bacillus strain BL14 in the present invention.
Fig. 3 is the JSM-6380LV ESEM thalli morphology observation figures of lichem bacillus strain BL14 in the present invention.
Fig. 4 is the pcr amplified fragment gel electrophoresis spectrum of lichem bacillus strain BL14 in the present invention.
Fig. 5 is that phylogenetic trees of the lichem bacillus strain BL14 based on 16S rRNA gene orders shows in the present invention
It is intended to.
Bacterial strain preservation
Bacterial strain in the present invention was lichem bacillus strain BL14 (Bacillus licheniformis), in 2014
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC within 28 days 2 months
NO.8865。
Embodiment
A kind of lichem bacillus strain, the bacterial strain are lichem bacillus strain BL14 (Bacillus
Licheniformis), it was preserved on 2 28th, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, deposit number are CGMCC NO.8865.
The lichem bacillus strain can be applied to be administered in tofu wastewater, while also produces endoglucanase and wood
Dextranase complex enzyme formulation, specific side are as follows：The tofu wastewater discarded during bean curd makes is collected, adjusts the pH to 7.0 of tofu wastewater,
121 DEG C of autoclaving 25min in fermentation tank are placed in, by the lichem bacillus strain BL14 with 106Cfu/mL inoculum concentration
It is inoculated in fermentation tank, is aerated, 33 DEG C is cultivated 3 days, and tofu wastewater is to be fermented into containing endoglucanase and zytase
Complex enzyme liquid.
The assay method of the endo-glucanase enzyme activity is as follows：
The carboxymethylcellulose sodium solution for being l% with 0.05mol/L pH 5.0 phosphate buffered saline mass fraction
1.7ml, add complex enzyme liquid described in 0.5mL, 0.3ml 0.05mol/L pH 5.5 phosphate buffer, in 50 DEG C of reactions
After 5min, add 1mL 3,5- edlefsen's reagent terminating reactions, then boil 5min in boiling water bath, dilute after
540nm colorimetrics.
The assay method of the Xylanase activity is as follows：With 0.05mol/L pH 5.0 phosphate buffered saline matter
The xylan solution 1.0ml that fraction is l% is measured, adds complex enzyme liquid described in 0.5mL, 1.0ml 0.05mol/L pH's 5.5
Phosphate buffer, after reacting 10min at 50 DEG C, 1mL 3 is added, 5- edlefsen's reagent terminating reactions, is then boiled
5min is boiled in water-bath, is diluted after 540nm colorimetrics.
The lichem bacillus strain BL14 (Bacillus licheniformis) of the present invention is from Nanping City in Fujian province
The separation containing rotten straw soil obtains in the periphery farmland of awns Dangshan.
1st, lichem bacillus strain BL14 acclimation and screening separation：
From soil of the Nanping City in Fujian province awns Dangshan periphery farmland collection containing the straw that rots, 7 are spontaneously dried at room temperature
My god, claim 5g soil in the triangular flask equipped with 195ml acclimating nutrient solutions, 30 DEG C of incubated domestications under the conditions of 160r/min
5d, 1g domestications bacterium solution is taken to dilute 10 respectively-5、10-6、10-7After power, it is coated in Congo red solid medium, choosing can be firm
Grown in arnotto solid medium and can produce the bacterium colony of transparent circle, flat board setting-out purifying culture, the bacterium of purifying is inoculated in point
After fermented and cultured in culture medium, bacterium solution 0.1ml is drawn in the Kong Zhongpei of the Congo red solid medium treated with card punch
Support, the formational situation of peep hole side transparent circle, choose transparent circle and form obvious strain, preserve and be named as bacillus licheniformis
Bacterial strain BL14.
The formula of wherein each culture medium is as follows：
(1) acclimating nutrient solution：(NH4)2SO4 2g/L,KH2PO4 3g/L,MgSO4·7H20 3g/L,CaCl2 3g/
L,MnSO4·H2O 0.16g/L,ZnSO4·7H2O 0.14g/L,CoCL20.2/L, rice straw powder 150g/L, okara powder：5g/L.
(2) isolation medium：(NH4)2S041.5g/L, KH2PO42.0g/L, MgSO40.3g/L, CaC120.3g/L,
FeSO40.1g/L, ZnSO40.1g/L, CMC-Na 10g/L, beech wood glycan 10g/L, pH are adjusted to 7.4.
(3) Congo red solid medium：(NH4)2SO42g/L, MgSO4·7H20 3g/L,KH2PO41g/L, NaCl
0.5g/L, CMC-Na 10g/L, xylan 10g/L, Congo red 0.2g/L, pH 7.0, agar 20g/L.
2nd, the identification of strain
The morphologic observation of 2.1 bacterium：Colony morphological observation (see Fig. 1), electronic display are carried out to lichem bacillus strain BL14
Micro mirror observes the Gram's staining form (see Fig. 2) of thalline, JSM-6380LV scanning electron microscopic observations thalli morphology (see Fig. 3).Ground
Clothing Bacillus strain BL14, bacterium colony decomposite leaf shape is opaque, wide：It is 0.6-0.8 μm, long：It is 1.5-3 μm, raw in spore, oval, leather
Lan Shi is positive, lines up chain.
The identification of 2.2 physiological and biochemical properties：
Lichem bacillus strain BL14 bacterial strain physiological and biochemical properties are shown in Table 1, as seen from table：VP, Starch Hydrolysis and peroxide
It is positive to change hydrogen experiment display, illustrates that the bacterial strain can produce pyruvic acid with decomposition glucose, the further decarboxylation of pyruvic acid forms acetyl
Carbinol methine, it is glucide that can produce amylase by Starch Hydrolysis；Gelatin can not be utilized, the experiment of production ammonia, MR experiments are negative；
It can ferment using D-Fructose production acid, aerogenesis, fermentation utilizes sucrose, not glucose production acid, aerogenesis.
The physiological and biochemical property of table 1
Note：Physiologic character：("+" represents positive；"-" represents negative)；Carbohydrate fermentation experimental result record sheet：("+,+"
Represent production acid, aerogenesis；Acid is not produced in "-,-" expression, not aerogenesis；" w-" represents weakly positive production acid, not aerogenesis.)
2.3 lichem bacillus strain BL14 molecular biology identification
(1) bacterial genomes DNA extraction:Extracted using the bacterial genomes DNA extraction kit of TIANGEN companies.
(2) the PCR amplifications and sequencing of 16S rDNA sequences：16SrDNA gene V6-V8 variable regions are expanded, the primer is
F968:5’-AAC GCG AAG CTT AC-3’(SEQ ID NO：2)；L1401:5’-CGG TGT GTA CAA GAC CC-3’
(SEQ ID NO：3).
PCR reaction systems:2*Taq PCR MasterMix:12.5ul, primer and DNA each 1.0ul, ddH20:9.5ul。
PCR amplification programs：94 DEG C of pre-degeneration 5min, then 94 DEG C are denatured 1min, 55 DEG C of annealing 1min, 72 DEG C of extensions
1min, totally 35 circulations, last 72 DEG C fully extension 10min, 4 DEG C of preservations.
(3) PCR primer detection and sequencing analysis：5ul PCR primer is taken, the gel in 1.0% agarose for adding EB
Electrophoretic separation is examined, and amplification obtains purpose fragment and is about 400bp or so (see Fig. 4), by the product commission containing target fragment
Marine growth Engineering Co., Ltd completes sequencing, and obtained actual nucleotide sequence is that (sequence is shown in SEQ ID NO to 397bp：1).
(4) Phylogenetic Analysis：Blast is carried out to each 16S rRNA sequences in NCBI data and compares analysis, obtains sequence
The sequence homology of row and Bacillus licheniformis is more than 99%, using the Maximum in MEGA 4.1
The creation analysis that Parsimony develop tree is same, as a result sees Fig. 5.
(5) acquisition of the bacterium number of logining：Gene order is submitted in ncbi database, carries out the application of the bacterium number of logining,
The accession number for applying obtaining is KJ627224.
With reference to bacterial strain morphologic observation and bio-chemical characteristics, and phylogenetic tree in homology analysis, bacterial strain is true
It is set to lichem bacillus strain BL14 (Bacillus licheniformis) strain.
3rd, lichem bacillus strain BL14 characteristic research
Prepare strain mother liquor, the ring of oese picking 1 lichem bacillus strain BL14 in 100mL basal mediums,
150r/min, 33 DEG C of culture 24h, obtains strain mother liquor；
The above-mentioned strain mother liquors of 0.1mL are taken to be inoculated in the several bottles of triangular flasks containing 100mL tofu wastewater culture mediums respectively, in
150r/min, 33 DEG C of culture 1d to 20d, using basal medium as control group, the ground is taken to 20d in 1d, 2d of fermentation respectively
Clothing Bacillus strain BL14 zymotic fluids and the activity for determining endoglucanase and zytase respectively, while determine the hair
The changes of contents situation of glucose and xylose in zymotic fluid.
The basal medium (g/L) is：Peptone 10, beef extract 5, sodium chloride 5, PH：7.6.
Tofu wastewater culture medium：Soya bean is 1 with water quality ratio:10, after making bean curd by traditional bean curd manufacture craft legal system, receive
The tofu wastewater flowed out in the last pressure bean curd program of collection, adjusts pH 7.0, is placed in 121 DEG C of autoclaving 25min, as tofu wastewater
Culture medium.
(1) prepared by complex enzyme liquid：The zymotic fluid is taken in 4 DEG C in culture 1d to 20d difference incubation times respectively, 5000r/
Min centrifuges 15min, and it is crude enzyme liquid to take supernatant.To boil 15min complex enzyme liquid as control.
(2) measure of endo-glucanase enzyme activity (CMCase)：Matched somebody with somebody with 0.05mol/L pH 5.0 phosphate buffer
Mass fraction processed is l% sodium carboxymethylcellulose (CMC-Na) solution 1.7ml, adds complex enzyme liquid described in 0.5mL,
0.3ml 0.05mol/L pH 5.5 phosphate buffer, after 50 DEG C are reacted 5min, add 1mL DNS reagents and terminate instead
Should, 5min is then boiled in boiling water bath, proper proportion is diluted after 540nm colorimetrics.
(3) measure of Xylanase activity：It is with 0.05mol/L pH 5.0 phosphate buffered saline mass fraction
L% xylan solution 1.0ml, adds complex enzyme liquid described in 0.5mL, and 1.0ml 0.05mol/L pH 5.5 phosphate delays
Fliud flushing, after reacting 10min at 50 DEG C, 1mL 3 is added, 5- edlefsen's reagent terminating reactions, is then boiled in boiling water bath
5min is boiled, is suitably diluted after 540nm colorimetrics.
Above enzyme activity deducts the sugared content of zymotic fluid and substrate, makees standard liquid with glucose/xylose, with every milliliter
Enzyme liquid generation 1ug glucose/xylose per minute is as an enzyme-activity unit (U).
(4) in zymotic fluid glucose/Xylose Content measure：Take complex enzyme liquid 0.5ml (using 0.5ml sterilized water as pair
According to), 2ml 0.05mol/L pH 5.5 phosphate buffer is added, adds in 1mL DNS reagent boiling water baths and boils 5min,
Suitably dilute after 540nm colorimetrics.
The change of glucose in 3.1 endoglucanases and zymotic fluid
With base culture base lichem bacillus strain BL14, caused maximum inscribe glucanase vigor appears in hair
The 3rd day of ferment, vigor are 64.73 ± 7.45 (U/ml), then rapid to decline, and enzyme activity is can't detect when fermenting to 6d.With
When tofu wastewater is culture medium, maximum enzyme activity appears in the 3d of fermentation, and vigor is 213.80 ± 9.74, is cultivated compared to basis
Base improves 2.30 times, and when fermentation is to 4d, enzyme activity is 112.63 ± 24.03 (U/ml), then starts rapid decline and (is shown in Table
2), illustrate that lichem bacillus strain BL14 can not only be grown by direct nutriment of tofu wastewater, and and base
Basal culture medium is compared, and the inducing action of nutriment makes it produce endoglucanase ability to reach and significantly carry in tofu wastewater
Height, the retention time of endo-glucanase enzyme activity also extend to 10d from the 4d of basal medium, i.e., should using tofu wastewater culture
Endo-glucanase caused by bacterium is easier to preserve.
Glucose content situation of change in culture medium is detected simultaneously, as known from Table 2：In basal medium, general trend
On, drop to 192.84 ± 7.68 (μ g/ml) from 263.836 ± 7.31 (μ g/ml) before fermentation, small size consumption occur and decline
Situation.In tofu wastewater culture medium, compared with before fermentation, fermentation 1d glucose contents by 1283.06 before fermenting ±
31.54 (μ g/ml) rise to 3204.68 ± 39.61, illustrate that lichem bacillus strain BL14 is induced a large amount of interior of secretion
Cut dextranase and the macromolecular carbohydrate in tofu wastewater is resolved into glucose.With longer fermentation times, zymotic fluid
The content of middle glucose starts to be gradually reduced, and when fermenting to 20d, drops to 303.00 ± 9.15 (μ g/ml), illustrates the lichens bud
Spore bacillus strain BL14 can use glucose as the energy and nutriment is grown.
The change of glucose in the endo-glucanase enzyme activity of table 2 and zymotic fluid
Note：ND is not detect
The change of xylose in 3.2 zytases and zymotic fluid
As shown in Table 3, with basal medium and tofu wastewater medium culture lichem bacillus strain BL14, produce most
High xylanase activity force value is both present in the 3rd day of fermentation, and vigor is respectively 424.48 ± 9.24,487.13 ± 11.06 (U/
Ml), it is gradually reduced thereafter, ferments to also detectable enzyme activity during 20d, respectively 93.31 ± 10.76,199.540 ±
25.32(U/ml).As shown in Table 3, generally lichem bacillus strain BL14 caused zytases in tofu wastewater
Vigor improves 15% obviously higher than basal medium, highest enzyme activity.
Detect Xylose Content situation of change in culture medium simultaneously：The content of xylose is presented under straight line substantially in basal medium
The trend of drop, fermentation 20d 1074.64 ± 94.70 (μ are gradually decreased to from 5519.64 ± 223.96 (μ g/ml) before fermentation
g/ml)；And in tofu wastewater culture medium, xylose slightly rises when fermenting 1d, by 8169.84 ± 194.02 (μ g/ before fermenting
Ml 9330.02 ± 276.72 (μ g/ml)) are risen to, illustrate that zytase is to beans caused by lichem bacillus strain BL14
Macromolecular hemicelluloses in rotten waste water are degraded, and are gradually reduced afterwards, are fermented by the 20th day and are dropped to
2215.73 ± 105.95 (μ g/ml), illustrate that lichem bacillus strain BL14 has carried out metabolism to xylose.
The change of xylose in the xylose enzyme activity of table 3 and zymotic fluid
In summary, the lichem bacillus strain BL14, can be using tofu wastewater as unique battalion after screening is tamed
Material is supported, while high yield endoglucanase and zytase, the vigor of caused two kinds of enzymes are obvious compared to basal medium
Improve, illustrate that lichem bacillus strain BL14 is more suitable for growing in tofu wastewater culture, given birth to by culture medium of tofu wastewater
The most suitable fermentation time for producing endoglucanase and zytase is fermentation 3d.
Claims (2)
- A kind of 1. lichem bacillus strain, it is characterised in that：The bacterial strain is lichem bacillus strain（Bacillus licheniformis）BL14, China Committee for Culture Collection of Microorganisms's commonly micro- life was preserved on 2 28th, 2014 Thing center, deposit number are CGMCC NO. 8865.
- A kind of 2. application of lichem bacillus strain as claimed in claim 1, it is characterised in that：Collect during bean curd makes and give up The tofu wastewater abandoned, the pH to 7.0 of tofu wastewater is adjusted, be placed in 121 DEG C of autoclaving 25min in fermentation tank, by the lichens bud Spore bacillus strain BL14 is with 106Cfu/mL inoculum concentration is inoculated in fermentation tank, aeration, and 33 DEG C are cultivated 3 days, and tofu wastewater is sent out Ferment is into the complex enzyme liquid containing endoglucanase and zytase.
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| CN106591264B (en) * | 2017-03-02 | 2019-07-23 | 福建省农业科学院农业工程技术研究所 | A kind of endoglucanase promotive factor |
| CN107475233B (en) * | 2017-09-30 | 2021-01-12 | 南京工业大学 | Method for producing nattokinase by using bean curd yellow serofluid |
| CN108271785B (en) * | 2018-01-30 | 2021-08-06 | 南京工业大学 | Biopesticide produced by fermenting bean curd yellow water and preparation method thereof |
| CN114480180B (en) * | 2022-01-04 | 2023-08-04 | 山东蔚蓝生物科技有限公司 | Bacillus licheniformis for straw degradation and application thereof |
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| KR100882280B1 (en) * | 2007-11-22 | 2009-02-06 | 건국대학교 산학협력단 | Method for mass production of isoflavone aglycone from soybean curd residues |
| CN104307014A (en) * | 2014-09-26 | 2015-01-28 | 任杰 | Microbial deodorant and preparation method thereof |
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|---|---|---|---|---|
| KR100882280B1 (en) * | 2007-11-22 | 2009-02-06 | 건국대학교 산학협력단 | Method for mass production of isoflavone aglycone from soybean curd residues |
| CN104307014A (en) * | 2014-09-26 | 2015-01-28 | 任杰 | Microbial deodorant and preparation method thereof |
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