CN105820980B - A kind of bacillus of yielding lipase and its application - Google Patents
A kind of bacillus of yielding lipase and its application Download PDFInfo
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Abstract
The invention discloses a kind of bacillusBacillus Sp.HFE722 and its application, the bacterial strain are preserved in China typical culture collection center, deposit number on December 10th, 2015 are as follows: CCTCC NO:M 2015735.The bacterial strain of institute's breeding of the present invention can high yield lipase using gutter oil as raw material, so that refuse reclamation, protects environment, realize sustainable development；The bacterial strain can significantly improve feed conversion rate as feed addictive, improve livestock growth performance, adjusts intestinal microbiota system and reduce feed-weight ratio etc., and to reduce livestock and poultry cultivation cost, increasing economic efficiency provides important theory and practice basis.
Description
Technical field
The invention belongs to microorganisms technical fields, in particular to one plant of stronger bacillus of yielding lipase ability
(Bacillus sp.) and its application.
Background technique
Lipase is a kind of special ester bond hydrolase, is one of biocatalyst well sold and in short supply at present, is generally used for catalytic water
Solution and synthetic reaction.The catalytic activity of lipase essentially consists in its protein structure, and on oil-water interfaces, it is sweet that it is catalyzed trigalloyl
The hydrolysis of the ester bond of oil, hydrolysis generate monoglyceride, diester or directly generate glycerol and fatty acid.
Microbe-derived lipase content is the abundantest, including bacterium, mould and yeast sources.Due to microbial species
Class is more, breeding is fast, and the lipase of generation has pH value more wider array of than andvegetable fats enzyme effect, and microbe-derived fat
Enzyme is typically all the ectoenzyme of secretory, is suitable for industrialized production and sample cleanup, and therefore, microbial lipase is industry
It is one of the most widely used enzyme in biotechnology and organic chemistry application with the important sources of lipase.
Microbial lipase is broadly divided into 3 major class: on the basis of i.e. non-specific, specific selectivity and substrate specificity
Lipase.Nonspecific lipid enzyme chance mechanism is fatty acid and glycerol by complete hydrolysis in triglycerides molecule, triglycerides.
In contrast, specific lipase is l, 3 specific lipases, hydrolyzing triglyceride C1 and C3 ester bond, to generate trip
From fatty acid, 1,2 one diglycerides, 2,3 one diglycerides and 2 one monoglycerides.Bacterium extracellular lipase is specific fat
Enzyme.Third quasi-lipase includes fatty acid specific lipase, it shows as apparent fatty acid selective hydrolysis.
Lipase is widely used in all trades and professions, and lipase is added in feed can be improved the digestion benefit of grease
With rate, more energy are provided for animal body；The fat that the chain released after lipase action-reaction is shorter is utilized in food
Acid can increase and improve the flavor and fragrance of food；Lipase is added in paper industry can directly decompose the oil on waste paper
Ink, coating and colorant, reach deinking efficiency, moreover it is possible to remove the glutinous mud of resin in paper pulp, paper grade (stock) plain boiled water and cooling water.In skin
In leather processing, a kind of degreasing agent of the lipase as efficient pollution-free is especially easier to fat from skin under alkaline condition
It removes；In detergent industry, lipase, which is added, can make triglycerides resolve into the fatty acid and glycerol for being easy to dispel, and can mention significantly
The clean effect of high detergent, and lipase is biological product, is easily degraded, it is free from environmental pollution；In brewed spirit, fat
Enzyme can be used for promoting the balance of rouge in white wine, acid, alcohol；In addition lipase acts in nonaqueous phase catalysis, can catalyze and synthesize rouge
One of substance, that is, the biodiesel described in us, and the hot spot currently studied.
The microbial resources for generating lipase in nature are very rich, according to incompletely statistics, micro- life of yielding lipase
Object up to 65 categories, wherein bacterium accounts for 28 categories, and actinomyces account for 4 categories, and saccharomycete accounts for 10 categories, and fungi accounts for 23 categories.Therefore,
Microbial lipase is the important sources of industrial enzyme catalyst.Wherein bacillus has stronger resistance, easy to produce, easy to maintain
The advantages that, it is a kind of good additive for microbe feedstuff, and produce protease, produce the function stems such as amylase, anti-Escherichia coli
Aquaculture is had been used to, but yielding lipase bacillus application study is also fewer.Excellent microorganism formulation must store up for a long time
Hide non-inactivation.Common thallus poor resistance, storage phase is short, easily dead, is not ideal microorganism formulation.Gemma is to produce gemma
The resistance hypopus for the round or ellipse that bacterium was formed into the cell in its growth and development later period has extremely strong heat resistanceheat resistant, anti-spoke
It penetrates, some special properties such as anti-chemicals and hydrostatic pressure resistant, it is very good that these biological properties have bacillus
Application prospect, powerful vitality is especially shown in the biological products using active bacteria formulation.
Summary of the invention
In view of the deficiencies of the prior art, the present inventor sieves the bacillus of yielding lipase using butchering field waste
Choosing and optimization, do further breeding to the bacillus of acquisition, are finally obtained the bacillus of one plant of yielding lipase
(Bacillus sp.HFE722) is preserved in China typical culture collection center on December 10th, 2015, address: in
Wuhan Wuhan University, state, deposit number are as follows: CCTCC NO:M 2015735.
Bacillus Sp.HFE722 is using gutter oil caused by dining room as raw material, and ferment yielding lipase, then will be fatty
Enzyme is applied to feed addictive.The bulk processing process of gutter oil are as follows: the waste of feeding hall is first removed by filtration waste residue, then centrifuging and taking
Supernatant is spare.By comparing under identical additive amount, the shadow to lipase activity such as above-mentioned gutter oil and olive oil, soybean oil
It rings, show that gutter oil can replace other oil kind completely, promote bacterial strain yielding lipase as culture medium additive.In this way, not only
It solves the problems, such as refuse reclamation, and also promotes the significantly promotion of enzyme activity, microbial cells are subsequent to be added as feed
Agent effect is added also to be improved.
The bacillus that the present invention is obtained by screeningBacillus Sp.HFE722 has the following advantages that and marked improvement:
(1) bacterial strain of institute's breeding of the present invention is by optimization, and lipase activity reaches 12.6U/mL in gained fermentation liquid after fermentation.(2) originally
The bacterial strain of institute's breeding is invented, using gutter oil as raw material yielding lipase, so that refuse reclamation, protects environment, realizing can be held
Supervention exhibition.(3) the present inventor's bacterial strain obtained is not only only capable of High yielding fat enzyme, it is often more important that thallus is as feed addictive
Feed conversion rate can be significantly improved, improve livestock growth performance, adjust intestinal microbiota system and reduce feed-weight ratio etc., to reduce
Livestock and poultry cultivation cost, increasing economic efficiency provides important theory and practice basis.
Detailed description of the invention
Fig. 1 is influence curve figure of the fermentation period to enzyme activity.
Fig. 2 is influence column diagram of the fermentation temperature to enzyme activity.
Fig. 3 is influence column diagram of the inoculum concentration to enzyme activity.
Fig. 4 is influence column diagram of the initial pH to enzyme activity.
Fig. 5 is influence column diagram of the liquid amount to enzyme activity.
Fig. 6 is influence column diagram of the different oils to enzyme activity.
Specific embodiment
Form is described in further detail above content of the invention again by the following examples, but should not manage this
For solution for the scope of the above subject matter of the present invention is limited to the following embodiments, all technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Embodiment 1: the breeding of strain
(1) used medium and solution in test
Every 1000g enriched medium contains: gutter oil 10mL, yeast extract 2g, NaCl 0.5g, K2HPO4 1g、 MgSO4
· 7H2O 0.1g, (NH4)2SO4 1g, surplus are water, and pH is natural；
Every 1000g Pseudonocardia contains: tryptone 10g, yeast extract 5g, NaCl 10g, three butyric acid are sweet
Grease 2mL, agar 20g, surplus are water, and pH is natural；
Every 1000g shake-flask seed culture medium contains: glucose 20g, K2HPO4 1g、MgSO4 · 7H2O 0.5g、
(NH4)2SO45g, peptone 25g, gutter oil 10mL, surplus are water, and pH is natural；
Every 1000g solid slope culture medium contains: tryptone 10g, yeast extract 5g, NaCl 10g, agar 20g, remaining
Amount is water, and pH is natural；
Secondary screening fermentation medium contains in every 1000g shaking flask: glucose 5g, K2HPO4 1g、MgSO4 · 7H2O
0.5g、(NH4)2SO41g, yeast powder 20g, gutter oil 10mL, surplus are water, pH7.0.
Sterile saline: taking 8.5g sodium chloride to be dissolved in the distilled water of 1L and dissolve, 0.1MPa sterilizing 20min.
(2) experimental procedure
15 parts of soil sample are acquired from slaughterhouse, takes certain sample from every part of sample first, with sterile saline by sample
It breaks up；30 DEG C of culture 1d of enriched medium are used again；Then 0.1mL is taken at different soil supensions with physiological saline gradient dilution
It is coated under the conditions of Selective Separation plate sets 37 DEG C, cultivates 2-3 d, picking transparent circle is big and apparent bacterial strain is as primary dcreening operation
Bacterial strain；Choose the single colonie on plate, access inclined-plane solid medium preservation is spare.
By shake flask fermentation verification experimental verification, obtain one plant of stronger bacillus of yielding lipase ability (Bacillus
Sp. HFE722), China typical culture collection center, deposit number are preserved on December 10th, 2015 are as follows: CCTCC NO:
M 2015735。
Embodiment 2: bacillusBacillus Sp. the training systern of HFE722
(1) bacterial strain: the strain bacillus that embodiment 1 filters outBacillus sp. HFE722。
(2) method and step:
It is investigated using single factor experimentBacillus Sp. the fermentation temperature of HFE722, initial pH, fermentation period, inoculation
Amount, influence of the liquid amount to fatty production of enzyme.
Fermentation medium is initial medium (glucose 5g, K2HPO4 1g、MgSO4 · 7H2O 0.5g、(NH4)2SO4
1g, yeast powder 20g, gutter oil 10mL, surplus are water).Initial fermentation condition are as follows: natural pH, inoculum concentration 1%(v/v), liquid amount
100 mL/250 mL, 30 DEG C, 160r/min shake flask fermentation 36h.
A. the optimization test of fermentation period
Other fermentation conditions determine bacillus every 6h sampling and measuring lipase activity for initial fermentation conditionBacillus Sp. the optimal fermentation period of HFE722 yielding lipase.As shown in Figure 1, the enzyme activity highest when fermentation is to 36h is
4.7U/mL, therefore select fermentation period for 36h.
B. the optimization test of fermentation temperature
The optimal result that fermentation time takes above-mentioned test to determine, other fermentation conditions are initial fermentation condition, respectively 26
DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, carry out fermented and cultured in 36 DEG C and 38 DEG C of shaking tables after measure lipase activity, investigate temperature
To bacillusBacillus Sp. the influence of HFE722 yielding lipase.As shown in Figure 2, when fermentation temperature is 30 DEG C, enzyme activity
Highest is 5.2 U/mL, therefore selects fermentation temperature for 30 DEG C.
C. the optimization test of inoculum concentration
The optimal result that fermentation time and fermentation temperature take above-mentioned test to determine, other fermentation conditions are Preliminary fermentation item
Part, inoculum concentration are respectively 0.5%, 1%, 1.5%, 2%, 2.5% and 3%(v/v) when, inoculum concentration is investigated to bacillusBacillus
Sp. the influence of HFE722 yielding lipase.Known by Fig. 3, when inoculum concentration is 1%, enzyme activity highest is 4.3U/mL, therefore selects inoculation
Amount 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 conditions are Preliminary fermentation
Condition, adjusting initial pH is respectively 4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 and 8.5, is measured after shake flask fermentation culture
Lipase activity investigates pH to bacillusBacillus Sp. the influence of HFE722 yielding lipase.As shown in Figure 4, work as fermentation
When initial pH is 7.0, enzyme activity highest 4.8U/mL, therefore selecting initial pH is 7.0.
E. the optimization test of fermentation shake flask liquid amount
The optimal result that fermentation time, temperature and inoculum concentration and initial pH take above-mentioned test to determine, other fermentation conditions are
Initial fermentation condition fills liquid using 250 mL shaking flasks, when shaking flask liquid amount is respectively 25mL, 50mL, 75mL, 100mL, 125mL,
Liquid amount is investigated to bacillusBacillus Sp. the influence of HFE722 yielding lipase.As shown in Figure 5, when shaking flask liquid amount
When for 50mL (Flask volume 250mL), enzyme activity 5.8U/mL, therefore select liquid amount for 50mL (Flask volume 250mL).
Embodiment 3: the influence of gutter oil, olive oil, soybean oil, peanut oil, cottonseed oil to bacterial strain HFE722 producing enzyme
It changes the gutter oil in initial medium into olive oil, soybean oil, peanut oil and cottonseed oil respectively, investigates identical
Additive amount under, influence of the various oil additives to strain enzyme-producing.
Fig. 6 control group refers to being not added with any oils, and as seen from the figure, gutter oil, olive oil and cottonseed oil are to enzyme activity
It is little to influence difference, wherein enzyme activity is up to 6.3U/mL when additive is gutter oil.From the point of view of cost and environmental protection,
Gutter oil perfect can replace other oil kind, the enzyme-producing agent as culture medium.
Application of the embodiment 4:HFE722 bacterial strain on feed addictive
By in-vitro simulated artificial human gastro intestinal tract, bacterial strain HFE722 is to simulated person's work gastrointestinal toleration ratio for verifying discovery
It is relatively strong.Safety detection is passed through, has illustrated that it does not have toxic action to animal.It then is effect with laying hen by HFE722 microbial inoculum
Object, the study found that the activity of lipase significantly improves in the duodenum of chicken, residue drop of each nutriment in excrement
It is low, while the content of the content and reduction Escherichia coli of lactobacillus and Bifidobacterium in caecum can be significantly improved.It illustrates
HFE722 microbial inoculum can significantly improve feed conversion rate as feed addictive, improve livestock growth performance, adjusting enteric microorganism
Group is and reduces feed-weight ratio etc., and to reduce livestock and poultry cultivation cost, increasing economic efficiency provides important theory and practice basis.
Embodiment 5: fermentation verification test
With bacillusBacillus Sp. 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 250mL)；Medium component are as follows: glucose 5g, K2HPO4
1g、MgSO4 · 7H2O 0.5g、(NH4)2SO41g, yeast powder 20g, gutter oil 10mL, carry out fermentation verification test, after fermentation
Lipase activity is 12.6U/mL in gained fermentation liquid.
Claims (1)
1. a kind of bacillus of yielding lipase (BacillusSp.) HFE722, it is characterised in that: the bacterial strain is in 2015
December 10 was preserved in China typical culture collection center, deposit number are as follows: CCTCC NO:M 2015735, which can
Using gutter oil as fermenting raw materials yielding lipase, it is also applicable in feed addictive.
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