CN104911126B - Bacillus amyloliquefaciens Z16-1 - Google Patents
Bacillus amyloliquefaciens Z16-1 Download PDFInfo
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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Abstract
Bacillus amyloliquefaciens Z16 1, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.10727.The fermentation enzyme solution of bacillus amyloliquefaciens Z16 1 is applied in aqueous enzymatic extraction camphor tree seeds oil technique, the yield of camphor tree seeds oil may make to reach 95%, 15% is improved than blank group camphor tree seeds oil yield, and acid value increase rate is less than 1, there is stronger application value in carbochain grease field in aqueous enzymatic extraction camphor tree seeds oil etc..
Description
Technical field
The invention belongs to biotechnologies, are related to a kind of bacillus amyloliquefaciens (Bacillus
amyloliquefaciens)Z16-1。
Background technology
The middle chain triglyceride content of camphor tree seeds oil is higher than 94%, can be used as excellent function edible oil, together
When be also production parenteral nutrition agent of new generation, middle carbochain food additives, cosmetic emulsifier etc. good raw material.China camphor tree
Resource very abundant has a large amount of plantations, at present nearly 30,000 tons of China's Camphor Tree Seeds annual output, camphor tree seeds oil in the Yellow River areas to the south
Annual output is up to 10,000 tons or more, however these resources are not developed and used reasonably yet so far.
Up to the present, the technology of the vegetable fat of production extraction both at home and abroad is still with squeezing method and organic solvent lixiviation process
Based on.Squeezing method extracts vegetable fat yield and is less than 90%, and oil content is up to 7% or more in the dregs of rice, while vegetable oil material albuminous degeneration
Seriously it is difficult to be extracted utilization.Organic solvent lixiviation process extraction vegetable fat needs toxic, inflammable using n-hexane, petroleum ether etc.
Organic solvent, there are the security risks such as inflammable and explosive in production process, product the problems such as there are organic solvent residuals.
Aqueous enzymatic extraction vegetable fat is that the biological enzyme such as protease are added and are sufficiently destroyed oil by oil plant after Mechanical Crushing
Expect that cell tissue discharges grease, is finally centrifuged vegetable fat and aminosal.The method not only carry oil quality it is good, extraction
Mild condition, low energy consumption, process safety, and oil yield is higher (being more than 90%), can obtain vegetable protein simultaneously, embody
The developing direction of grease production and research and development field.However there is also greases to be easily hydrolyzed, the biological enzyme cost mistake that uses for the method
The shortcomings of high, can not be applied in industrialized production so far.The neutral proteinase used in aqueous enzymatic extraction grease is substantially
What the bacterial strains such as bacillus subtilis AS1.398, aspergillus terricola 3942, actinomyces 166 were produced.The study found that using these bacterium
The aqueous enzymatic extraction camphor tree seeds oil of the produced neutral protein enzyme solution of strain, the yield of camphor tree seeds oil are less than 83%, acid value and are more than
5mgKOH/g.It is the medium-chain fatty acid glyceride in camphor tree seeds oil to cause the reason that camphor tree seeds oil yield is low, acid value is high
There is stronger inhibiting effect, bacillus subtilis to the gram-positive bacterias such as bacillus subtilis AS1.398, mould and biological enzyme
Bacterium AS1.398 yielding lipase vigor is higher (yielding lipase vigor is in 2U/mL or more).
Screen resistance to medium-chain fatty acid (Medium Chain Fatty Acids, MCFA), production neutral proteinase ability it is strong,
The very weak bacterial strain of yielding lipase ability is to improve aqueous enzymatic extraction camphor tree seeds oil yield, aqueous enzymatic extraction plant is greatly reduced
The production cost of grease technology realizes the key of the industrialized production and application of aqueous enzymatic extraction vegetable fat technology.The existing nothing in the country
Relevant bacterial strain screening technology and example for the problem reports that the present invention is enriched with using Camphor Tree Seeds benevolence dregs of rice powder tablet, degreasing
Milk flat band method, grease flat band method primary dcreening operation, the methods of Forint phenol method, Camphor Tree Seeds benevolence culture media shaking vase secondary screening are given birth to from camphor tree seeds oil
Higher resistance to MCFA, production neutral protease vigor are filtered out in production waste residue, fat-free enzyme activity, are suitable for aqueous enzymatic extraction Camphor Tree Seeds
The bacterial strain of carbochain grease in benevolence oil etc..
Invention content
The object of the present invention is to provide a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Z16-
1CGMCC No.10727, it is resistant to, and MCFA, production neutral protease vigor be higher and not yielding lipase, is suitable for aqueous enzymatic extraction
Carbochain grease in camphor tree seeds oil etc..
The present invention is achieved by the following technical solutions.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Z16-1, in preservation on April 21 in 2015
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica), it is referred to as CGMCC, deposit number is CGMCC No.10727.
After the bacillus amyloliquefaciens Z16-1 is cultivated for 24 hours on nutrient agar, bacterium colony is rounded, size
5.0~10.0mm, thickness about 2mm, surface are light yellow, have irregular wavy protrusion, edge-smoothing.Gram's staining is sun
Property, it is rod-shaped, 0.7~0.9 3~5 μm of μ m to observe cellular morphology under the microscope, and endogenous spore, no parasporal crystal, gemma is normal
It sees in the middle part of cell, sporangium is without apparent expansion.The colonial morphology and cellular morphology of the bacterial strain are shown in Fig. 1 and Fig. 2.
The bacillus amyloliquefaciens Z16-1 catalases are tested, V.P is tested, glucose production acid is tested, gelatin liquefaction reality
Test, Starch Hydrolysis experiment is the positive, oxidizing ferment experiment, indoles experiment, glucose aerogenesis, nitrate reductase are feminine gender, and energy is sharp
With xylose, L-arabinose, mannitol, citric acid cannot be utilized.
The seed culture based formulas of the bacillus amyloliquefaciens Z16-1 is：Beef extract 3g, peptone 10g, NaCl
5g, water 1000mL, pH 7.0-7.2,121 DEG C of sterilizing 20min.Seed culture condition is：37 DEG C, 220r/min, shaking flask culture
24h。
The enzymatic production medium optimization formula of the bacillus amyloliquefaciens Z16-1 is：Corn flour 45g, wheat bran
25g, dregs of beans 40g, CaCl23g, KH2PO40.3g, Na2HPO4·12H2O 4g, water 1000mL, pH 7.5, inoculum concentration 2%.Fermentation
The optimal conditions of producing enzyme culture are：37 DEG C, 220r/min shaking flask cultures.
After fermented culture, zymotic fluid, 8000r/min is taken to centrifuge 10min, gained supernatant is crude enzyme liquid.
Method is surveyed using GB/T 23527-2009 protease preparation enzyme activities, measures the prolease activity of crude enzyme liquid.Albumen
The definition of enzyme activity：1mL enzyme solutions, under the conditions of 40 DEG C, pH are 7.0,1min caseinhydrolysates generate 1 μ g tyrosine, are defined as
One protease activity unit of force.
Using iodimetric titration specified in light industry standard QB1502-92, the pectinase activity of crude enzyme liquid is measured.Pectin enzyme activity
Power defines：1mL enzyme solutions, under the conditions of 40 DEG C, pH are 7.0, it is a pectase enzyme that 1h, which decomposes pectin and generates 1mg galacturonic acids,
Unit of activity.
Using iodimetric titration specified in national standard GB 8276-2006, the saccharifying enzymic activity of crude enzyme liquid is measured.Saccharifying enzymic activity
Definition：1mL enzyme solutions, under the conditions of 40 DEG C, pH are 7.0,1h hydrolyzes soluble starch and generates 1mg glucose, as 1 carbohydrase
Unit of activity.
Using filter paper enzyme specified in national standard GB 23881-2009, the cellulase activity of crude enzyme liquid is measured.Cellulose
Enzyme activity defines：1mL enzyme solutions, under the conditions of 40 DEG C, pH are 7.0, hydrolysis filter paper per minute generates the glucose of 1.0 μ g, i.e.,
For 1 cellulase activity unit.
Using iodine colorimetry specified in national standard GB 24401-2009, the amylase activity of crude enzyme liquid is measured.Diastatic activity
Power defines：1mL enzyme solutions, under the conditions of 40 DEG C, pH are 7.0,1h liquefaction 1g soluble starches, as 1 diastatic activity unit of force.
Using acid-base titration specified in national standard GB 23535-2009, the lipase activity of crude enzyme liquid is measured.Lipase
Vigor defines：1mL enzyme solutions, under the conditions of 40 DEG C, pH are 7.0,1min hydrolysis substrate 1 μm of titratable aliphatic acid of ol of generation, as 1
A enzyme activity unit.
The bacillus amyloliquefaciens Z16-1 is under Optimal Medium and optimum culture condition, when fermented and cultured 40h,
Produced neutral protease vigor is 5734 ± 2%U/mL；Produced pectinase activity is 1411 ± 2%U/mL；Produce saccharification enzyme activity
Power is 10727 ± 2%U/mL；Produced amylase activity is；29 ± 2%U/mL；Institute's cellulase-producing vigor is 6 ± 2%U/mL；
Lipase activity fails to detect.
The bacillus amyloliquefaciens Z16-1 is under Optimal Medium and optimum culture condition, when fermented and cultured 44h,
Produced neutral protease vigor is 6984 ± 2%U/mL；Produced pectinase activity is 1506 ± 2%U/mL；Produce saccharification enzyme activity
Power is 8535 ± 2%U/mL；Produced amylase activity is；15 ± 2%U/mL；Institute's cellulase-producing vigor is 3 ± 2%U/mL；
Lipase activity fails to detect.
The bacillus amyloliquefaciens Z16-1 is under Optimal Medium and optimum culture condition, when fermented and cultured 48h,
Produced neutral protease vigor is 6765 ± 2%U/mL；Produced pectinase activity is 580 ± 2%U/mL；Produced saccharifying enzymic activity
For 6066 ± 2%U/mL；Produced amylase activity is；13 ± 2%U/mL；Institute's cellulase-producing vigor is 3 ± 2%U/mL；Fat
Fat enzyme activity fails to detect.
Using Optimal Medium and optimal conditions, the bacillus amyloliquefaciens Z16-1 fermentation production neutral proteinases are bent
Line is as shown in Figure 3.
The optimum temperature of the produced neutral proteinases of bacillus amyloliquefaciens Z16-1 is 40 DEG C, most suitable work
It is 7.0, Mn with pH2+There is apparent activation to its enzyme activity, the MnCl of final concentration of 0.01mol/mL is added2It can make described
The produced neutral protease vigors of bacillus amyloliquefaciens Z16-1 improve 46.5%.
The beneficial effects of the invention are as follows：It is applied to aqueous enzymatic extraction using the fermentation enzyme solution of bacillus amyloliquefaciens Z16-1
In camphor tree seeds oil technique, the yield of camphor tree seeds oil may make to reach 95%, than blank group (being not added with any enzyme solution) camphor tree
Seed benevolence oil yield improves 15%, and acid value increase rate is less than 1, the carbochain grease field in aqueous enzymatic extraction camphor tree seeds oil etc.
There is stronger application value.
Description of the drawings
Fig. 1 is the colonial morphology figure of bacillus amyloliquefaciens Z16-1 of the present invention.
The cellular morphology figure (200 times of amplification) that Fig. 2 is bacillus amyloliquefaciens Z16-1 of the present invention.
Fig. 3 is bacillus amyloliquefaciens Z16-1 of the present invention fermentation production neutral proteinase curves.
Specific implementation mode
With reference to specific example, the present invention will be further described.
Embodiment 1.The screening technique of bacillus amyloliquefaciens Z16-1.
(1) experiment material prepares.
1, separation source is derived from camphor tree seeds oil production waste residue (cinnamomum camphora woods High Seience Technology Co., Ltd. of Nanchang City).
2, culture medium.
Broth bouillon：Peptone 1%, beef extract 0.3%, 7.0-7.2,121 DEG C of sterilizings of NaCl 0.5%, pH
20min。
Nutrient agar：The agar that whole mass concentration is 2% is added on the basis of broth bouillon.
Protease primary dcreening operation culture medium：1.5% skimmed milk power, broth bouillon and skimmed milk power are added in broth bouillon
Separately sterilizing, 115 DEG C of sterilizing 25min.
Lipase primary dcreening operation culture medium：Peptone 1%, beef extract 0.5%, NaCl 0.5%, camphor tree seeds oil 1%, 1.6%
Neutral red aqueous solution 0.1%, agar 2%, 7.2,121 DEG C of sterilizing 20min of pH.
The preparation of Camphor Tree Seeds benevolence dregs of rice powder：The Camphor Tree Seeds benevolence for taking a certain amount of drying, after pulverizer crushes, with solid-liquid ratio 1:
4 add water mixing, and are added to wet pulverizing 4min in colloid mill, will obtain white color emulsion 4000r/min and centrifuge
25min, it is Camphor Tree Seeds benevolence dregs of rice powder to take bottom slag phase, crushed after being dried.
Camphor Tree Seeds benevolence dregs of rice powder primary dcreening operation culture medium：Camphor Tree Seeds benevolence dregs of rice powder 2%, glucose 0.7%, KH2PO40.03%,
Na2HPO412H2O 0.4%, agar 2%, 7.0~7.2,115 DEG C of sterilizing 25min of pH.
Fermentation medium：Corn flour 4.5%, wheat bran 2.5%, dregs of beans 4%, CaCl20.2%, KH2PO40.03%,
Na2HPO4·12H27.5,121 DEG C of sterilizing 20min of O 0.4%, pH.
Camphor Tree Seeds benevolence dregs of rice powder secondary screening culture medium：Camphor Tree Seeds benevolence dregs of rice powder 5%, glucose 0.7%, yeast powder 0.3%,
KH2PO40.03%, Na2HPO412H27.0~7.2,115 DEG C of sterilizing 25min of O 0.4%, pH.
3, experimental facilities.
Constant temperature oscillator, biochemical cultivation case, superclean bench, high-pressure sterilizing pot, pH meter, colloid mill, 721 type ultraviolet spectrometries
Photometer.
(2) bacterial strain screening.
(1) type of resistance to medium-chain fatty acid bacterial strain is enriched with：1g or so separation source samples are weighed, with sterile saline and 10 times
Dilution method prepares different dilutions (10-1~10-7) dilution, from three appropriate dilutions draw 100uL dilutions it is equal
It is even to be applied on Camphor Tree Seeds benevolence culture medium A tablet.37 DEG C of constant temperature incubations for 24 hours, it is spare to choose the tablet with 10 or so bacterium colonies.
(2) high proteinase yield bacterial strain primary dcreening operation：Using dibbling method, the bacterium colony dibbling obtained in previous step is picked with sterile toothpick
Onto defatted milk culture medium flat plate, 37 DEG C of constant temperature incubations for 24 hours, according to the transparent loop diameter of bacterium colony in culture medium and colony diameter ratio
(H/C) size carries out bacteria produced proteinase strain primary dcreening operation, and it is spare to choose the larger bacterial strains of H/C.
(3) low yield lipase strain primary dcreening operation：Using dibbling method, by bacterial strain dibbling larger H/C in previous step to camphor tree
On seed benevolence oil culture medium flat plate, for 24 hours, whether observation periphery of bacterial colonies generates red hydrolysis circle to 37 DEG C of constant temperature incubations.Choose redfree
The bacterial strain of grease hydrolysis circle carries out shaking flask secondary screening.
(4) high yield neutral proteinase, low yield lipase strain secondary screening.
In the inoculation to broth bouillon that primary dcreening operation is obtained, 37 DEG C, the culture of 220r/min shaking flasks for 24 hours, seed is made
Liquid.Seed liquor is added in fermentation medium with 2% inoculum concentration, 37 DEG C, 220r/min shaking flask culture 44h take zymotic fluid,
8000r/min centrifuges 10min, and supernatant is crude enzyme liquid.National standard GB/T23527-2009 is respectively adopted《Protease preparation
Enzyme activity determination method》Specified in Forint phenol method and national standard GB/T 23535-2009《Lipase preparation enzyme activity determination method》
Specified in determination of acid-basetitration crude enzyme liquid prolease activity and lipase activity, choose that production prolease activity is high, fat
The low bacterial strain of enzyme activity.
(5) it is used for the bacterial strain screening of carbochain grease in aqueous enzymatic extraction.
In the inoculation to broth bouillon that secondary screening in previous step is obtained, 37 DEG C, the culture of 220r/min shaking flasks for 24 hours,
Seed liquor is made.Seed liquor is added in Camphor Tree Seeds benevolence culture medium B with 6% inoculum concentration, 37 DEG C, 220r/min shaking flask cultures
After 40h, 90 DEG C heat preservation 10min inactivation, colloid mill wet pulverizing emulsify 4min, by obtained white " milky " liquid 4000r/min from
The heart detaches 25min, collects bottom slag phase, obtains Camphor Tree Seeds benevolence residue after dry, weighing and measuring it using soxhlet extraction contains
Oil mass.Selection can most make the bacterial strain that Camphor Tree Seeds benevolence level of residue is few, oil content is low, as bacillus amyloliquefaciens Z16-1.
(3) bacterial strain colony morphology characteristic and physiological and biochemical property.
After bacterial strain Z16-1 is cultivated for 24 hours on nutrient agar, bacterium colony is rounded, 5.0~10.0mm of size, thickness
About 2mm, surface are light yellow, have irregular wavy protrusion, edge-smoothing.Gram's staining is the positive, is observed under the microscope
Cellular morphology is rod-shaped, 0.7~0.9 3~5 μm of μ m, endogenous spore, no parasporal crystal, and gemma is common in the middle part of cell, spore
Capsule is without apparent expansion.Catalase experiment, V.P experiments, the sour experiment of glucose production, gelatin liquefaction experiment, Starch Hydrolysis experiment are sun
Property, oxidizing ferment experiment, indoles experiment, glucose aerogenesis, nitrate reductase are feminine gender, can be using xylose, L-arabinose, sweet
Reveal alcohol, citric acid cannot be utilized.
The 16S rDNA gene orders of bacterial strain Z16-1 are shown in " specification nucleotide and amino acid sequence table ".
The 16S rDNA gene orders of bacterial strain Z16-1 are found with GenBank database comparative analysis using BLAST methods,
The homology of bacterial strain Z16-1 and bacillus amyloliquefaciens is up to 99%.
The Physiology and biochemistry and Molecular Identification of comprehensive bacterial strain are as a result, bacterial strain Z16-1 is named as bacillus amyloliquefaciens Z16-1
(Bacillus amyloliquefaciens Z16-1)。
Data of literatures is consulted, high proteinase yield, low yield lipase and the type of resistance to medium-chain fatty acid solution starch bud are there is no
The screening of spore bacillus and the report of application aspect.Bacillus amyloliquefaciens Z16-1 is new bacterial strain, on April 21st, 2015
It hands over in China General Microbiological Culture Collection Center (CGMCC) preservation, preserving number 10727.
The present invention is screened up to arrives 6984U/ very much with the bacillus amyloliquefaciens Z16-1 production neutral protease vigors isolated
ML, fat-free enzyme activity and resistance to medium-chain fatty acid, fermenting enzyme liquid energy significantly improve aqueous enzymatic extraction camphor tree seeds oil grease
And yield of aminosal.For aqueous enzymatic extraction vegetable fat provide for bacterium source and technology, have stronger practical application
Value.
Claims (1)
1. a kind of bacillus amyloliquefaciens Z16-1 for extracting camphor tree seeds oil is deposited in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, deposit number are CGMCC No. 10727.
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| CN1952116A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillusamyloliquefaciens strain and application thereof |
| CN102191200A (en) * | 2011-03-31 | 2011-09-21 | 国家粮食局科学研究院 | Bacillus amyloliquefaciens and application thereof |
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| CN1952116A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillusamyloliquefaciens strain and application thereof |
| CN102191200A (en) * | 2011-03-31 | 2011-09-21 | 国家粮食局科学研究院 | Bacillus amyloliquefaciens and application thereof |
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| Title |
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| 解淀粉芽孢杆菌BF7658中性蛋白酶的生理作用;耿运琪 等;《工业微生物》;19921231(第5期);6-9 * |
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Inventor after: Zeng Zheling Inventor after: Wang Jianyu Inventor after: Xiao Yanjun Inventor after: Zeng Cheng Inventor after: Yu Ping Inventor after: Wen Xuefang Inventor before: Zeng Zheling Inventor before: Xiao Yanjun Inventor before: Zeng Cheng Inventor before: Yu Ping |
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