CN105820980A - Bacillus for generating lipase and application of bacillus - Google Patents
Bacillus for generating lipase and application of bacillus Download PDFInfo
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Abstract
The invention discloses Bacillus sp.HFE722 and application thereof. The strain was collected in the China Center for Type CultureCollection on December 10, 2015, and the preservation number is CCTCC NO:M 2015735. The strain bred in the invention can generate lipase with a high yield, andas swill-cooked dirty oil is taken as raw material, the waste can be reused, the environment is protected, and sustainable development is achieved; when the strain is used as a feed additive, the feed conversion rate can be remarkably increased, the growth property of livestock can be improved, the enteric microorganism formation can be adjusted, the feed-weight ratio can be reduced, the livestock breeding cost can be reduced, the economic benefits can be increased, and significant theoretical and practice basis can be provided.
Description
Technical field
The invention belongs to microbial technology field, in particular to the stronger bacillus cereus of a strain yielding lipase ability (Bacillussp.) and application.
Background technology
Lipase is a class special ester bond hydrolytic enzyme, is one of the wellst sold and in short supply biocatalyzer, is generally used for catalyzing hydrolysis and synthetic reaction.The catalysis activity of lipase essentially consists in its protein structure, and on oil-water interfaces, the hydrolysis of the ester bond of its catalysis triacylglycerol, hydrolysis generates monoglyceride, diester or directly generates glycerol and fatty acid.
Microbe-derived lipase content is the abundantest, including antibacterial, mycete and yeast sources.Owing to microbe species is many, breeding fast, the lipase produced has the ratio andvegetable fats wider array of pH value of enzyme effect, and microbe-derived lipase is typically all the exoenzyme of secreted, it is suitable for industrialized great production and sample cleanup, therefore, microbial lipase is the important sources of industrial lipase, is one of the most widely used enzyme in biotechnology and organic chemistry application.
Microbial lipase is broadly divided into 3 big classes: the lipase on the basis of the most non-specific, specific selectivity and substrate specificity.Nonspecific lipid enzyme chance mechanism is fatty acid and glycerol in triglyceride molecule, triglyceride by complete hydrolysis.In contrast, specific lipase is l, 3 specific lipases, hydrolyzing triglyceride C1 and C3 ester bond, thus produces free fatty, 1,2 one diglycerides, 2,3 one diglycerides and 2 one monoglycerides.Antibacterial extracellular lipase is specific lipase.3rd quasi-lipase comprises fatty acid specific lipase, and it shows as obvious fatty acid selective hydrolysis.
Lipase is all widely used in all trades and professions, adds lipase and can improve the digestive utilization ratio of oils and fats, provide more energy for animal body in feedstuff；The fatty acid that the chain that discharges after utilizing lipase action-reaction in food is shorter, can increase and improve local flavor and the fragrance of food；In paper industry, add ink, coating and the colorant on lipase energy Direct Resolution waste paper, reach deinking efficiency, moreover it is possible to remove the resin in paper pulp, paper grade (stock) plain boiled water and the glutinous mud of cooling water.In leather processing, lipase, as the degreasing agent of a kind of efficient pollution-free, makes fat be more easy to from skin remove the most in the basic conditions；In detergent industry, add lipase and triglyceride can be made to resolve into the fatty acid and glycerol easily dispelled, be greatly improved the clean effect of detergent, and lipase is biological product, is easily degraded, free from environmental pollution；In brewed spirit, lipase can be used for promoting fat, acid, the balance of alcohol in Chinese liquor；During additionally lipase acts on nonaqueous phase catalysis, lipid material can be catalyzed and synthesized, namely our described biodiesel, also be one of focus of currently being studied.
The microbial resources producing lipase in nature are the abundantest, and according to incompletely statistics, up to 65 genus of the microorganism of yielding lipase, wherein antibacterial accounts for 28 genus, and actinomycetes account for 4 genus, and yeast accounts for 10 genus, and fungus accounts for 23 genus.Therefore, microbial lipase is the important sources of industrial enzyme catalyst.Wherein bacillus cereus has the advantages such as stronger resistance, easily production, easy preservation, it it is a kind of well additive for microbe feedstuff, and produce protease, produce the function stem such as amylase, anti-escherichia coli and have been used to aquaculture, but yielding lipase bacillus cereus applied research is the most fewer.Excellent microorganism formulation must long-term storage non-inactivation.Common thalline poor resistance, the storage phase is short, easily dead, is not preferable microorganism formulation.Spore is the spore production bacteria circle in its growth promoter later stage intracellular formation or the resistance hypopus of ellipse, there are some special character such as extremely strong heat resistanceheat resistant, radioprotective, anti-chemicals and hydrostatic pressure resistant, these biological properties make bacillus cereus have the best application prospect, particularly show powerful vitality in the biological product using active bacteria formulation.
Summary of the invention
For the deficiencies in the prior art, the present inventor utilizes slaughterhouse garbage screen the bacillus cereus of yielding lipase and optimize, the bacillus cereus obtained is done further selection-breeding, it is finally obtained the bacillus cereus (Bacillussp.HFE722) of a strain yielding lipase, within 10th, it is preserved in China typical culture collection center in December in 2015, address: Wuhan, China Wuhan University, deposit number is: CCTCCNO:M2015735.
BacillusSp.HFE722 is with waste oil produced by dining room as raw material, and ferment yielding lipase, then lipase is applied to feed additive.The bulk processing process of waste oil is: the garbage taking food hall is first removed by filtration waste residue, then centrifuging and taking supernatant is standby.By comparing under identical addition, above-mentioned waste oil and the impact on lipase activity such as olive oil, soybean oil, show that waste oil can replace other oil completely and plant, promote bacterial strain yielding lipase as culture medium additive.So, the problem not only solving refuse reclamation, and also promote the significantly lifting that enzyme is lived, microbial cells is follow-up have also been obtained improvement as feed additive effect.
The bacillus cereus that the present invention is obtained by screeningBacillusSp.HFE722 has the advantage that and marked improvement: the bacterial strain of (1) institute of the present invention selection-breeding is by optimizing, and after fermentation, in gained fermentation liquid, lipase activity reaches 12.6U/mL.(2) bacterial strain of institute of the present invention selection-breeding, with waste oil for raw material yielding lipase so that refuse reclamation, protects environment, it is achieved that sustainable development.(3) bacterial strain that the present inventor is obtained not only is only capable of High yielding fat enzyme, the more important thing is that thalline can significantly improve feed conversion rate as feed additive, improves domestic animal growth performance, regulating intestinal canal microbial population and reduce feed-weight ratio etc., for reducing livestock and poultry cultivation cost, increasing economic efficiency provides important theory and practice basis.
Accompanying drawing explanation
Fig. 1 is the influence curve figure that enzyme is lived by fermentation period.
Fig. 2 is that fermentation temperature affects bar diagram to what enzyme was lived.
Fig. 3 is that inoculum concentration affects bar diagram to what enzyme was lived.
Fig. 4 is that initial pH affects bar diagram to what enzyme was lived.
Fig. 5 is that liquid amount affects bar diagram to what enzyme was lived.
Fig. 6 is that different oils affects bar diagram to what enzyme was lived.
Detailed description of the invention
The foregoing of the present invention is described in further detail by form more by the following examples, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to below example, and all technology realized based on foregoing of the present invention belong to the scope of the present invention.
Embodiment 1: the selection-breeding of strain
(1) used medium and solution in test
Every 1000g enrichment medium contains: waste oil 10mL, yeast extract 2g, NaCl0.5g, K2HPO41g、MgSO4·7H2O0.1g, (NH4)2SO41g, surplus are water, and pH is natural；
Every 1000g Pseudonocardia contains: tryptone 10g, yeast extract 5g, NaCl10g, tributyrin 2mL, agar 20g, and surplus is water, and pH is natural；
Every 1000g shake-flask seed culture medium contains: glucose 20g, K2HPO41g、MgSO4·7H2O0.5g、(NH4)2SO45g, peptone 25g, waste oil 10mL, surplus are water, and pH is natural；
Every 1000g solid slant culture base contains: tryptone 10g, yeast extract 5g, NaCl10g, agar 20g, surplus are water, and pH is natural；
Sieve fermentation medium again in every 1000g shaking flask to contain: glucose 5g, K2HPO41g、MgSO4·7H2O0.5g、(NH4)2SO41g, yeast powder 20g, waste oil 10mL, surplus are water, pH7.0.
Physiological saline solution: take 8.5g sodium chloride and be dissolved in the distilled water of 1L dissolving, 0.1MPa sterilizing 20min.
(2) experimental procedure
Gather soil sample 15 parts from slaughterhouse, from every part of sample, first take certain sample, with physiological saline solution, sample is broken up；1d is cultivated again with enrichment medium 30 DEG C；Then becoming different soil supensions with normal saline gradient dilution, take 0.1mL and coat under the conditions of Selective Separation flat board puts 37 DEG C, cultivate 2-3d, picking transparent circle is big and obvious bacterial strain is as primary dcreening operation bacterial strain；Choose the single bacterium colony on flat board, access inclined-plane solid medium preservation standby.
By shake flask fermentation verification experimental verification, it is thus achieved that bacillus cereus that a strain yielding lipase ability is stronger (BacillusSp.HFE722), within 10th, being preserved in China typical culture collection center in December in 2015, deposit number is: CCTCCNO:M2015735.
Embodiment 2: bacillus cereusBacillusThe training systern of sp.HFE722
(1) bacterial strain: the strain bacillus cereus that embodiment 1 filters outBacillussp.HFE722。
(2) method step:
Employing single factor experiment is investigatedBacillusThe fermentation temperature of sp.HFE722, initial pH, fermentation period, inoculum concentration, the liquid amount impact on lipase yield.
Fermentation medium is initial medium (glucose 5g, K2HPO41g、MgSO4·7H2O0.5g、(NH4)2SO41g, yeast powder 20g, waste oil 10mL, surplus are water).Initial fermentation condition is: natural pH, inoculum concentration 1%(v/v), liquid amount 100mL/250mL, 30 DEG C, 160r/min shake flask fermentation 36h.
A. the optimization test of fermentation period
Other fermentation condition is initial fermentation condition, every 6h sampling and measuring lipase activity, determines bacillus cereusBacillusThe optimum fermentation period of sp.HFE722 yielding lipase.As shown in Figure 1, when fermentation to 36h, enzyme is lived the highest, and for 4.7U/mL, therefore selecting fermentation period is 36h.
B. the optimization test of fermentation temperature
Fermentation time takes the optimal result that above-mentioned test determines, other fermentation condition is initial fermentation condition, measures lipase activity in 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C and 38 DEG C of shaking tables after carrying out fermentation culture respectively, investigates temperature to bacillus cereusBacillusThe impact of sp.HFE722 yielding lipase.As shown in Figure 2, when fermentation temperature is 30 DEG C, enzyme is lived the highest, and for 5.2U/mL, therefore selecting fermentation temperature is 30 DEG C.
C. the optimization test of inoculum concentration
Fermentation time and fermentation temperature take the optimal result that above-mentioned test determines, other fermentation condition is initial fermentation condition, and inoculum concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% and 3%(v/v) time, investigate inoculum concentration to bacillus cereusBacillusThe impact of sp.HFE722 yielding lipase.Being known by Fig. 3, when inoculum concentration is 1%, enzyme is lived the highest, and for 4.3U/mL, therefore selecting inoculum concentration is 1%.
D. the optimization test of initial pH
Fermentation time, temperature and inoculation measure the optimal result that above-mentioned test determines, other fermentation condition is initial fermentation condition, adjust initial pH and be respectively 4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 and 8.5, shake flask fermentation measures lipase activity after cultivating, and investigates pH to bacillus cereusBacillusThe impact of sp.HFE722 yielding lipase.As shown in Figure 4, when the initial pH that ferments is 7.0, enzyme the highest 4.8U/mL alive, therefore selecting initial pH is 7.0.
E. the optimization test of fermentation shake flask liquid amount
Fermentation time, temperature and inoculum concentration and initial pH take the optimal result that above-mentioned test determines, other fermentation condition is initial fermentation condition, use 250mL shaking flask dress liquid, when shaking flask liquid amount is respectively 25mL, 50mL, 75mL, 100mL, 125mL, investigate liquid amount to bacillus cereusBacillusThe impact of sp.HFE722 yielding lipase.As shown in Figure 5, when shaking flask liquid amount is 50mL (Flask volume 250mL), it is 5.8U/mL that enzyme is lived, therefore selecting liquid amount is 50mL (Flask volume 250mL).
Embodiment 3: bacterial strain HFE722 is produced the impact of enzyme by waste oil, olive oil, soybean oil, Oleum Arachidis hypogaeae semen, Oleum Gossypii semen
Waste oil in initial medium is changed into respectively olive oil, soybean oil, Oleum Arachidis hypogaeae semen and Oleum Gossypii semen, investigates under identical addition, the impact on strain enzyme-producing of the various oil additives.
Fig. 6 matched group refers to be not added with any oils, and as seen from the figure, it is little that what enzyme was lived by waste oil, olive oil and Oleum Gossypii semen affects difference, and wherein when additive is waste oil, enzyme is lived and is up to 6.3U/mL.From the point of view of cost and environmental protection, waste oil perfect can replace other oil and plant, as the product enzyme accelerator of culture medium.
The application on feed additive of the embodiment 4:HFE722 bacterial strain
By in-vitro simulated artificial human gastro intestinal tract, checking finds that bacterial strain HFE722 is stronger to simulated person's work gastrointestinal toleration.Pass through safety detection, illustrated that it does not has toxic action to animal.Then by HFE722 microbial inoculum with laying hen as effective object, research finds, in the duodenum of chicken, the activity of lipase significantly improves, and each nutrient substance residue in feces reduces, and can significantly improve in caecum lactobacillus and the content of bacillus bifidus simultaneously and reduce colibacillary content.Illustrating that HFE722 microbial inoculum can significantly improve feed conversion rate as feed additive, improves domestic animal growth performance, regulating intestinal canal microbial population and reduce feed-weight ratio etc., for reducing livestock and poultry cultivation cost, increasing economic efficiency provides important theory and practice basis.
Embodiment 5: fermentation checking test
With bacillus cereusBacillusSp.HFE722 is fermentation strain；Condition of culture is fermentation time 36h, temperature 30 DEG C, inoculum concentration 1%, initial pH7.0, liquid amount 50mL (shaking flask is 250mL)；Medium component is: glucose 5g, K2HPO41g、MgSO4·7H2O0.5g、(NH4)2SO41g, yeast powder 20g, waste oil 10mL, carry out fermentation checking test, and after fermentation, in gained fermentation liquid, lipase activity is 12.6U/mL.
Claims (3)
1. the bacillus sp.HFE722 of a yielding lipase, it is characterized in that: this bacterial strain Bacillussp.HFE722 is preserved in Chinese Typical Representative culture on the 10th in December in 2015 and contains center, and Classification And Nomenclature is that Bacillussp.HFE722 deposit number is: CCTCCNO:M2015735.
2. the application of the bacillus sp.HFE722 of a yielding lipase, it is characterised in that: this bacterial strain can be with waste oil for fermenting raw materials yielding lipase.
3. the application of the bacillus sp.HFE722 of a yielding lipase, it is characterised in that: this bacterial strain can be applicable in feed additive.
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| WO2021056683A1 (en) * | 2019-09-27 | 2021-04-01 | 常熟理工学院 | Strain for producing lipase and application thereof |
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