CN104450571A - Bacillus thuringiensis strain for efficient degradation of fly larvae protein - Google Patents
Bacillus thuringiensis strain for efficient degradation of fly larvae protein Download PDFInfo
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- CN104450571A CN104450571A CN201410729336.1A CN201410729336A CN104450571A CN 104450571 A CN104450571 A CN 104450571A CN 201410729336 A CN201410729336 A CN 201410729336A CN 104450571 A CN104450571 A CN 104450571A
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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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- 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
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a bacillus thuringiensis strain KF228911 for efficient degradation of fly larvae protein, and belongs to the technical field of agricultural microorganisms. The strain is classified and named as bacillus thuringiensis serovar thuringienesis which is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.9789 on October 17, 2014. The strain is inoculated to a seed culture medium, and is cultivated under 30 DEG C at 150r/min for 28hr, so that a KF228911 liquid culture is obtained. The strain can generate a transparent hydrolysis ring on a fly larvae protein powder culture medium and the fly larvae protein is degraded in a fly larvae protein fermentation medium, and the content of generated soluble amino acid can reach 1.744mg/mL. The strain disclosed by the invention can be used for degrading the fly larvae protein by virtue of microorganism fermentation, and is suitable for the production of high-quality protein feed. The strain is applicable to the development and utilization of fly larvae protein resource, and in particular applicable to transformation of the fly larvae protein into feed, so that the nutritive value of the fly larvae protein is improved.
Description
Technical field
The present invention relates to a kind of fly maggot protein efficient degrading bacteria, belong to field of agricultural microbial technology, be the method degraded fly maggot protein utilizing fermentable, be applicable to the production of high-quality protein feed.
Background technology
In recent years, along with the fast development of China's fodder industry, the development of protein feed there is lack of raw materials problem seriously limits China's fodder industry.At present, China mainly leans on imported fish meal to solve the problem of the deficiency of protein resource, so develop the hot subject that new protein resource becomes scientific research.Soybean is the protein raw materials of high-quality, but its higher price is undoubtedly by limiting feed industrial expansion.Therefore, developing, seek and promote protein feed raw material that is novel, safety is as early as possible current top priority.Housefly is the most common a kind of insect in district from all parts of the world, and its quantity is huge, and fecundity is strong, and reproduction speed is fast.Fly maggot is the Larva Morpho. Logy of housefly, containing multiple lipid acid, amino acid, VITAMIN and mineral substance.Can breed at China's fly maggot, there is higher protein content, can develop.Make Flyblow albumen powder at present.In fly maggot, protein content is about 60.53%, amino acid classes is complete, and contain 8 kinds of indispensable amino acids and the Histidine of needed by human body, its essential amino acids content accounts for 42.14% of total amino acid content, and the fine protein indispensable amino acid well beyond FAO/WHO suggestion should account for the standard of total amino acid content 40%.Fly maggot protein is not a halfpenny the worse compared with other high-quality animal proteinum, and its nutritive value is even higher than other animal proteinum, therefore the protein resource of its a kind of high-quality especially.If but fly maggot protein Direct-fed, but there is the problem that fly maggot protein nutritional utilization is low, and fly maggot protein matter is at hydrolysis oligopeptides, after polypeptide and amino acid whose mixture, the nutritive value of feed can be improved, be conducive to the absorption of animal; Certainly enzyme process can be adopted to degrade, but cost is high, and institute's proteinase activity of purchasing is low, hydrolysis degree is little, if adopt fly maggot protein degradation bacteria strains, both cost-saved, can improve hydrolysis degree again.Current studies in China is less, is the developing direction of fly-maggot protein fodder.
Summary of the invention
The object of this invention is to provide a kind of fly maggot protein efficient degrading bacterial strain, for the degraded of fly maggot protein, thus develop a kind of useful protein resource newly, solve the problem that current fly maggot protein nutritional utilization is low.
The invention provides a kind of bacillus thuringiensis bacterial strain of efficient degradation fly maggot protein, strain classification called after serum anomaly bacillus thuringiensis (Bacillus thuringiensis serovar thuringiensis), on October 17th, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.9789.Main Biological is, colonial morphology is circular, oyster white, and surface wettability is typical ne ar, and through gramstaining staining and the qualification of spore staining method, this bacterial strain is Gram-negative genus bacillus.Serum anomaly bacillus thuringiensis is accredited as through gene sequencing.This bacterial strain Sep-7S ribosomal RNA gene accession number is KF228911.
Fly maggot protein degradation bacteria strains can grow and produce hydrolysis on Flyblow albumen powder substratum, and its culture medium prescription is: containing 2.0g Flyblow albumen powder in 100mL distilled water, 0.5g sodium-chlor, 2.0g agar powder, heating for dissolving, regulates pH value 7.0,121 DEG C of autoclaving 30min.Described Flyblow albumen powder, after being dried by fly maggot, carries out pulverizing and form, cross 100 mesh sieves.
The enlarged culturing method of above-mentioned bacterial strains KF228911 is:
(1) formula of seed culture medium is: contain in 1L distilled water: 3g extractum carnis, 10g peptone, 5g sodium-chlor, heating for dissolving, regulates pH value 7.0,121 DEG C of autoclaving 30min.
(2) be inoculated in the Erlenmeyer flask of the 250mL that 100mL seed culture medium is housed by described bacterial strain KF228911, under temperature 30 DEG C of conditions, shaking table cultivate 24 hours with the rotating speed of 150r/min, obtain bacteria suspension, bacterium liquid quantity is 4 × 10
10cFU/ml.
Fly maggot protein degradation bacteria fermentative medium formula is: contain in 1L distilled water: 20g Flyblow albumen powder, 20g glucose, 5g sodium-chlor, regulates pH value 7.0,121 DEG C of autoclaving 30min.
With fly maggot protein degradation bacteria KF228911 bacteria suspension with 5% inoculum size inoculate, after fermentation, can degrade to fly maggot protein, culture temperature 30 DEG C, 150r/min cultivates 48 hours, after completing cultivation, carry out centrifugal 20 minutes to fermented liquid 4000r/min, collect supernatant liquor, can produce soluble Amino acid content is 1.744mg/mL.
Beneficial effect
The invention provides the bacillus thuringiensis bacterial strain KF228911 of efficient degradation fly maggot protein.Fly maggot protein degradation bacteria can grow on Flyblow albumen powder substratum, produces hydrolysis.Provide the enlarged culturing method of fly maggot protein degradation bacteria KF228911.Cultivate fly maggot protein fermention medium with fly maggot protein degradation bacteria strains, the aminoacids content that in fermented liquid, fly maggot protein degraded produces is 1.744mg/mL.Fly-maggot protein fodder can improve the content of its soluble amino acid as inoculation fly maggot protein degradation bacteria KF228911, improves fly maggot protein as the nutritive value of feed, has vast potential for future development.
The bacillus thuringiensis bacterial strain of energy efficient degradation fly maggot protein of the present invention, its name is called serum anomaly bacillus thuringiensis (Bacillus thuringiensis serovar thuringiensis) KF228911, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCC NO.9789.Depositary institution address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and preservation date is on October 17th, 2014.
Embodiment
Embodiment 1 high efficient strain is separated
Take fly maggot dead in physical environment to drive 1 gram, body sample, add 100mL stroke-physiological saline solution, vibrate 10 minutes, leave standstill 5 minutes, coat on screening plate culture medium after supernatant liquor 10 times dilution, 37 DEG C of growths; Fly maggot protein degradation bacteria strains screening culture medium consists of: 2.0g Flyblow albumen powder, 0.5g sodium-chlor, 2.0g agar powder, water 100mL, and pH value is 7.0,121 DEG C of sterilizing 30min.
Substratum compound method of the present invention is as follows:
Accurately take Flyblow albumen powder 2.0g, add water 100mL, and heating makes protein dissolution, adds 0.5g sodium-chlor, 2.0g agar powder, adjust ph, 121 DEG C of sterilizing 30min.
Fly maggot protein degradation bacteria strains screening embodiment is as follows:
1. primary dcreening operation: sampling liquid, after suitably being diluted, is got 0.1mL and is coated on fly maggot protein degradation bacteria strains screening culture medium flat board, cultivate 24 ~ 48h at 37 DEG C of constant incubators by conventional 10 times of dilution methods.Measure single bacterium colony hydrolysis transparent circle and colony diameter, carry out primary dcreening operation according to transparent circle diameter and colony diameter ratio (H/C) size, therefrom choose 12 larger strain bacterial strains of ratio and carry out preservation and shake flask fermentation screening.
Beef extract-peptone inclined-plane Storaged media: in 100mL water, adds extractum carnis 0.3g, peptone 1.0g, NaCl 0.5g, agar 2.0g, regulates pH value 7.0.
Fermentation minimum medium: in 100mL water, wheat bran 3.0g, peptone 1.0g, NaCl 0.5g, pH nature, 121 DEG C of sterilizing 30min.
Shake flask fermentation: by transfering loop picking strain inoculation in fermentation minimum medium, leavening temperature 36 DEG C, shaking speed is 180r/min, fermentation culture 48h, measures enzyme and lives, and chooses and produces the maximum bacterial strain of enzyme activity;
(1) enzyme activity determination method:
The drafting of table 1 tyrosine typical curve:
Get 18 test tubes and be divided into 6 groups, often organize 3, parallel and number, standard tyrosine, deionized water, the sodium carbonate of 0.4mol/L and Folin-Phenol reagent is added respectively by table 1, after mixing, put into 40 DEG C of water bath heat preservation 20min, then on 721 type spectrophotometers, carry out colorimetric estimation (wavelength 660nm), do blank with deionization.With OD
660value is X-coordinate, and tyrosine amount (μm ol) is ordinate zou, obtains equation of linear regression; Y=109.34 × OD
660-1.9026, linearly dependent coefficient R
2=0.9983
(2) mensuration of sample enzyme activity
Get 4 centrifuge tube numberings (No. 0 is blank), add crude enzyme liquid 1mL respectively, No. 0 pipe adds 0.4mol/L TCA solution 2mL immediately, make enzyme deactivation, another 3 samples add 1% casein solution 1mL, rapid mixing, and put into 40 DEG C of accurate timing of water bath with thermostatic control immediately, after insulation 10min, add 0.4mol/L TCA solution 2mL immediately, termination reaction, in No. 0 blank tube, add 1% casein solution 1mL simultaneously, shake up, continue to be placed in water-bath and be incubated 20min, take out centrifugal removing residue casein and zymoprotein throw out, then each pipe centrifugate 1mL is got, move in another 4 10mL scale test tubes respectively, add the Folin-Phenol reagent 1mL of 0.4mol/L sodium carbonate solution 5mL and oneself dilution again, shake up, after insulation colour developing 20min, carry out colorimetric estimation (wavelength 660nm).Tyrosine growing amount A is calculated by tyrosine equation of linear regression.
Proteinase activity calculates: proteinase activity: A × 4.0 × N/ (l0 × 1.0).
In formula: A represents the tyrosine growing amount obtained by equation of linear regression; 4.0 represent reaction solution cumulative volume (mL); N represents the extension rate of enzyme liquid; 10 represent the reaction times (min); 1.0 represent enzyme liquid long-pending (mL); Protease activity defines: under certain pH value (pH7.0) and 40 DEG C of temperature condition, the enzyme amount that every min protease hydrolysis casein produces 1 μ g tyrosine is defined as a protease activity unit of force.
2. multiple sieve: bacterial strain selected after primary dcreening operation is pressed the step after primary dcreening operation, and again carry out cultivating and answer and sieve, utilize fly maggot protein degradation bacteria strains screening culture medium, the method same by primary dcreening operation carries out multiple sieve.
3. preservation: adopt beef extract-peptone inclined-plane Storaged media to carry out preservation by producing the high bacterial strain of enzyme activity.
4. strain identification
(1) 10 times of dilution method is cultivated and is observed colonial morphology, color, feature;
(2) by gram staining method, at oily Microscopic observation, qualification Gram-negative or the positive;
(3) whether the kind of spore staining method qualification bacterial strain is genus bacillus;
Apply method of the present invention, the bacterial strain obtaining a strain energy efficient degradation fly maggot protein can be screened, this strain classification called after serum anomaly bacillus thuringiensis (Bacillus thuringiensis serovar thuringiensis), on October 17th, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.9789.Main Biological is, colonial morphology is circular, oyster white, and surface wettability is typical ne ar, and through gram staining method and the qualification of spore staining method, this bacterial strain is Gram-negative genus bacillus.The Genbak of the DNA of this bacterial strain, what similarity was the highest is: Bacillus thuringiensis serovar thuringiensis, the number of logging in is: KF228911
The concrete zymologic property research of embodiment 2 fly maggot protein degrading enzyme is as follows:
The source of enzyme: fly maggot protein degradation bacteria strains is inoculated in fermentation minimum medium (wheat bran 3.0%, peptone 1.0%, NaCl0.5%, pH nature, 121 DEG C of sterilizing 30min) in, 36 DEG C, rotating speed 180r/min cultivates 48 hours, the centrifugal 10min of nutrient solution 4000r/min, and supernatant liquor is fly maggot protein degrading enzyme liquid.
(1) optimum temperuture of enzyme: the vigor measuring degrading enzyme under differing temps (35,40,45,50,55,60 DEG C), temperature when enzyme activity is the highest is the optimum temperuture of enzyme
(2) enzyme is to the stability of temperature: degrading enzyme is incubated 1h under differing temps (35,40,45,50,55,60 DEG C), then measures the vigor of enzyme, thus knows that enzyme is stable at which temperature.
(3) optimum pH of enzyme: the vigor measuring degrading enzyme under different pH (5.5,6.0,6.5,7.0,7.5,8.0), pH when enzyme activity is the highest is the optimum pH of enzyme;
(4) enzyme is to the stability of pH: degrading enzyme keeps 1h under different pH (5.5,6.0,6.5,7.0,7.5,8.0), then measures the vigor of enzyme, thus knows that enzyme is stable under which pH value.
Interpretation of result
Apply method of the present invention, can obtain fly maggot protein efficient degradation enzyme zymologic property as follows: the optimal reactive temperature of enzyme is 50 DEG C, 35 DEG C of insulations are after 1 hour, and enzyme is lived still can retain 76.9%, and living more than 35 DEG C of enzymes declines rapidly; The optimum pH of enzyme is 7.5), be 6.0,6.5 in pH value, keep after 1 hour in the damping fluid of 7.0, the phenomenon that the vigor of enzyme is all improved.
Embodiment 3 fly maggot protein biological degradation effect:
Culture medium prescription: glucose 2.0g, fly maggot protein 2.0g, NaCl 0.5g; Water 100mL;
1) take each component respectively according to substratum composition, use tap water to dissolve;
2) substratum that configures is loaded in triangular flask, stirring and evenly mixing, 121 DEG C of sterilizings 30 minutes, cooling;
3) inoculate fly maggot protein degradation bacteria, cultivate 48h;
4) after nutrient solution 4000r/min, supernatant liquor measures amino acid growing amount, to determine its palliating degradation degree.
Amino acid whose mensuration
The drafting of leucine typical curve
Prepare 100 μ g/mL standard leucine sample liquids, get 6 test tube numberings, standard leucine is added respectively, without ammonia distilled water, ninidrine and 0.1% xitix by table 2, put into boiling water bath after mixing and heat 15min, frequent shake, make the redness that formed during heating gradually oxidation by air and fade to blue purple time, the ethanol constant volume with 60% is to 20mL, mixing, then measures O.D under 570nm wavelength
570value.
The drafting of table 2 leucine typical curve
| Guan Hao | 0 | 1 | 2 | 3 | 4 | 5 |
| Standard leucine (mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 |
| Without ammonia distilled water (mL) | 2.0 | 1.8 | 1.6 | 1.4 | 1.2 | 1.0 |
| Ninidrine (mL) | 3.0 | 3.0 | 3.0 | 3.0 | 3.0 | 3.0 |
| 0.1％Vc | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| O.D 570Value | ||||||
| Leucine amount y (μ g) | 20 | 40 | 60 | 80 | 100 |
With O.D
570for X-coordinate (x), leucine amount (y) is ordinate zou, and drawing standard curve obtains its equation of linear regression: y=220.51-0.1994, R
2=0.9912.
Get centrifuge tube four, No. 0 get heating for dissolving but the substratum 2.0mL not inoculating degraded as blank, another three draw 2.0mL culture supernatant, respectively add the TCA aqueous solution 2.0mL of 0.4mol/L, mixing, leave standstill 20min, then centrifugal 10min under 4000r/min, removing albumen precipitation thing, then gets each pipe centrifugate 0.1mL, moves in 4 20mL scale test tubes (reference numeral) respectively, add without ammonia distilled water 1.9mL, below operate same Specification Curve of Increasing, with No. 0 as blank, colorimetric estimation O.D under 570nm wavelength
570, calculate amino acid growing amount (y) by equation of linear regression.
Interpretation of result
Apply substratum of the present invention and fermentation process thereof, fermented liquid produces aminoacids content 1.744mg/mL.Therefore can improve the content of soluble amino acid in fly maggot protein in feed as inoculated fly maggot protein degradation bacteria, improving fly maggot protein as the nutritive value of feed, also can be used for amino acid whose suitability for industrialized production.
Claims (5)
1. the bacillus thuringiensis bacterial strain of a strain energy efficient degradation fly maggot protein, it is characterized in that: this bacterial classification is called serum anomaly bacillus thuringiensis (Bacillus thuringiensis serovar thuringiensis) KF228911, on October 17th, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.9789.
2. the bacillus thuringiensis bacterial strain of energy efficient degradation fly maggot protein according to claim 1, it is characterized in that: can grow in Flyblow albumen powder screening culture medium and produce hydrolysis, required Flyblow albumen powder screening and culturing based formulas is: containing 2.0g Flyblow albumen powder in 100mL water, 0.5g sodium-chlor, 2.0g agar powder, regulate pH7.0,121 DEG C of autoclaving 30min.
3. the bacillus thuringiensis bacterial strain of energy efficient degradation fly maggot protein according to claim 1, is characterized in that: its enlarged culturing method is:
(1), the configuration of seed culture medium: contain 3g extractum carnis in 1L water, 10g peptone, 5g sodium-chlor, adjust ph 7.0,121 DEG C of autoclaving 30min;
(2), bacterial strain KF228911 described in claim 1 is inoculated in the Erlenmeyer flask of the 250mL that 100mL seed culture medium is housed, under temperature 30 DEG C of conditions, shaking table cultivates 24 hours with the rotating speed of 150r/min.
4. the bacillus thuringiensis bacterial strain of energy efficient degradation fly maggot protein according to claim 3, it is characterized in that: the fly maggot protein bacteria suspension of cultivation is inoculated in fermention medium with the inoculum size of 5%, under temperature 30 DEG C of conditions, 150r/min cultivates 48 hours, and producing soluble Amino acid content is 1.744mg/mL; Fermentative medium formula is: containing 20g Flyblow albumen powder in 1L water, 20g glucose, 5g sodium-chlor, adjust ph 7.0, and 121 DEG C of autoclaving 30min, carry out centrifugal 20 minutes to nutrient solution 4000r/min after completing cultivation, collects supernatant liquor.
5. the application of bacillus thuringiensis bacterial strain in degraded fly maggot protein of energy efficient degradation fly maggot protein as claimed in claim 1.
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| CN105533136A (en) * | 2015-12-25 | 2016-05-04 | 广东鑫肽生物科技股份有限公司 | Preparation method of novel fermented feed and worm enzyme |
| CN106190904A (en) * | 2016-07-17 | 2016-12-07 | 湖南农业大学 | Amino nitrogen feed process is prepared in fly maggot protein biology solid-state degraded |
| GB2561348A (en) * | 2017-04-07 | 2018-10-17 | Entomics Biosystems Ltd | Process for converting invertebrates into feedstock |
| CN112608871A (en) * | 2021-01-12 | 2021-04-06 | 江南大学 | Method for producing probiotic active substances by high-density fermentation of bacillus thuringiensis |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105533136A (en) * | 2015-12-25 | 2016-05-04 | 广东鑫肽生物科技股份有限公司 | Preparation method of novel fermented feed and worm enzyme |
| CN105533136B (en) * | 2015-12-25 | 2019-11-05 | 广东鑫肽生物科技股份有限公司 | A kind of preparation method and worm ferment of novel fermentation feed |
| CN106190904A (en) * | 2016-07-17 | 2016-12-07 | 湖南农业大学 | Amino nitrogen feed process is prepared in fly maggot protein biology solid-state degraded |
| CN106190904B (en) * | 2016-07-17 | 2018-06-19 | 湖南农业大学 | Fly-maggot protein biology solid-state degradation prepares ammonia nitrogen feed process |
| GB2561348A (en) * | 2017-04-07 | 2018-10-17 | Entomics Biosystems Ltd | Process for converting invertebrates into feedstock |
| GB2561348B (en) * | 2017-04-07 | 2020-01-15 | Entomics Biosystems Ltd | Process for converting invertebrates into feedstock |
| CN112608871A (en) * | 2021-01-12 | 2021-04-06 | 江南大学 | Method for producing probiotic active substances by high-density fermentation of bacillus thuringiensis |
| CN112608871B (en) * | 2021-01-12 | 2022-11-01 | 江南大学 | Method for producing probiotic active substances by high-density fermentation of bacillus thuringiensis |
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