CN103614313A - Strain for producing high temperature resistance beta-galactosidase through fermentation, and screening method thereof - Google Patents
Strain for producing high temperature resistance beta-galactosidase through fermentation, and screening method thereof Download PDFInfo
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Abstract
The present invention relates to a strain for producing high temperature resistance beta-galactosidase through fermentation, and a screening method thereof. The strain F69 has a classification name of Bacillussubtilis, is preserved in the China General Microbiological Culture Collection Center on January 9, 2012, and has a preservation number of CGMCC No.5699. The screening method specifically and sequentially comprises: enrichment culture, primary screening, strain purification, re-screening, strain preservation, and beta-galactosidase activity determination. The strain has characteristics of short beta-galactosidase production fermentation period, rapid beta-galactosidase production, good genetic stability, good pH value stability, strong catalytic activity, simple culture condition and high production safety, and has broad application prospects in industrial fields of food, feeds and the like.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly relate to a kind of bacterial strain and screening method thereof that produces high temperature resistant beta-galactosidase enzymes.
Background technology
Beta-galactosidase enzymes (β-galactosidase, EC3.2.1.23), formal name used at school is β-D-galactoside galactohydrolase, commodity are called Sumylact L, can catalysis β-
d-synthesis hydrolysis or turn glycosylation, be usually used in being hydrolyzed the lactose in cow's milk and other milk-product, also can be used to generate oligomeric galactose, be widely used in the production of low-lactose milk and non-crystalline type enriching milk, solve the lactose intolerance problem that world's population over half exists, beta-galactosidase enzymes also has galactoside transferance, for the production of bifidus factor-oligomeric galactose, thus widespread use in probiotic food is produced.The significant application value of beta-galactosidase enzymes in food causes the extensive concern of people to it.Beta-galactosidase enzymes is mainly derived from animal, plant, bacterium, yeast, fungi or mould.Due to advantages such as the Fast Growth of microorganism, efficient metabolism and separation and purification are relatively simple, make microorganism beta-galactosidase enzymes become the main of industrialization zymin and come
source.How dairy industry is extracted and is obtained after enzyme is produced in induction from yeast, aspergillus tubigensis or milk-acid bacteria for the beta-galactosidase enzymes reducing lactose at present, still all has the shortcoming of poor heat stability, has seriously limited its application in milk-product.
If adopt high temperature resistant beta-galactosidase enzymes can overcome the defect of poor heat stability, enzyme, at more than 60 ℃ temperature hydrolyzes lactose, can contribute to suppress miscellaneous bacteria microbial growth.And enzyme reaction rate is fast, enzyme dosage is low, and the reaction times is short, can carry out with pasteurize or cooling stages insulation hydrolysis in production simultaneously, in pasteur sterilizing process, carries out lactose hydrolysis simultaneously, saves time, saving of labor and can prevent living contaminants.Although current many bibliographical informations the multiple beta-galactosidase strain of screening and separating, as the scheme of the serial bacterial strains such as yeast, genus lactubacillus, bacillus, Eurotium, but produce at present the most optimum temperature of beta-galactosidase enzymes used 30 ℃ of left and right, enzyme activity is at high temperature all very low, and cost is high, is difficult to meet market and produces required.How to overcome the deficiencies in the prior art, accelerate the resistant to elevated temperatures beta-galactosidase enzymes product of research and development and become one of emphasis difficult problem urgently to be resolved hurrily in current technical field of bioengineering.
Summary of the invention
The object of the invention is provides a kind of fermentation to produce bacterial strain and the screening method thereof of high temperature resistant beta-galactosidase enzymes for overcoming the deficiencies in the prior art, this bacterial strain produce beta-galactosidase enzymes fermentation period short, produce that beta-galactosidase enzymes speed is fast, genetic stability good, pH good stability and catalysis activity strong, and culture condition is easy and production security is high.
A kind of fermentation proposing according to the present invention produced the bacterial strain of high temperature resistant beta-galactosidase enzymes, the Classification And Nomenclature that it is characterized in that described bacterial strain F69 be subtilis (
bacillus subtilis), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on January 9th, 2012, deposit number is: CGMCC No.5699, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Bacterial strain F69(CGMCC No.5699 of the present invention) there is following characteristic:
(1) growth conditions: subtilis (
bacillus subtilis) 55～75 ℃ of the growth temperatures of beta-galactosidase enzymes that F69 produces, 65 ℃ of optimum growth temperatures; The beta-galactosidase enzymes vigor of surveying all more than 90%, show that this enzyme catalytic activity in higher temperature range is very high, thermal adaptability is strong;
(2) enzymic activity: be incubated 90min at 50 ℃～80 ℃, remnant enzyme activity, more than 80%, is incubated 60min at 80 ℃ and 90 ℃, and remnant enzyme activity is respectively 85% and 65%; Show that subtilis beta-galactosidase enzymes that F69 produces has very strong thermostability;
(3) slightly acidic: in enzymic activity between pH5.5～7.5 more than 80%; Between pH 4.5～7.5, keep more than 81% enzymic activity, the suitableeest action pH is 6.5; Show that beta-galactosidase enzymes is highly stable under slightly acidic and neutral environment.
The screening method of the bacterial strain of a kind of high temperature resistant beta-galactosidase enzymes of product that ferments proposing according to the present invention, is characterized in that comprising successively following concrete steps:
Step 1, enrichment culture: soil sampling 1g porphyrize, add in the 250ml triangular flask that 80ml sterilized water is housed, add sterile glass beads, the 20min that vibrates in 150r/min shaking table then shakes 30 min in 80 ℃ of shaking water bath shaking tables, gets 1-2 ml in 50 mL enrichment mediums, 40 ℃ of water bath with thermostatic control shaking culture 36h-48h, concussion frequency is 150-180r/min;
Step 2, primary dcreening operation: it is 10 that the nutrient solution after enrichment is carried out to 10 times of gradient dilutions to extension rate
-8, get respectively 10
-6, 10
-7, 10
-8the diluent 0.2ml of three gradients is coated on primary dcreening operation isolation medium flat board, and flat board dries in the air while there is no fluxion to surface, in 40-42 ℃ of constant incubator, is inverted and cultivates 24-36h, then culture temperature is dropped to 32～35 ℃, continues to cultivate 24～72h; Picking blue colonies is to another primary dcreening operation isolation medium, and culture condition is: temperature 40-42 ℃, incubation time 30-36h; After cultivating, bacterium colony presents deep mixed blueness, further chooses blue darker bacterial strain line purifying;
Step 3, bacterial strain purifying: by primary dcreening operation bacterial strain out, in the flat lining out of beef-protein medium, be placed in 40-42 ℃ of constant incubator and cultivate 48h, repeat streak culturely more than three times, as continuous several times, examine under a microscope thalline that form is single and think bacterial strain purifying;
The present invention compared with prior art its remarkable advantage is: the one, the growth temperature of beta-galactosidase enzymes that the present invention produces is 55～75 ℃, optimal reactive temperature is 65 ℃, the enzyme activity of surveying 90% left and right of all living at the highest enzyme; Enzyme is incubated 90min at 50 ℃～80 ℃, and remnant enzyme activity, more than 80%, has good thermostability; The 2nd, enzyme is between pH 4.5～7.5, enzyme is lived stable, in view of the pH value of most of milk-product and beverage is also within the scope of this, therefore having given subtilis F69 beta-galactosidase enzymes that bacterial strain produces, the biochemical characteristic of this enzyme will have stronger application advantage in milk-product processing; The 3rd, the bacterial strain genetic stability that the present invention screens is good, can continuous passage more than 10 generations, and the synthesis capability of beta-galactosidase enzymes is constant; The 4th, beta-galactosidase enzymes of the present invention at high temperature has satisfactory stability, at industrial circles such as food and feed, has broad application prospects.
Accompanying drawing explanation
Fig. 1 is for producing the dull and stereotyped schematic diagram of beta-galactosidase strain primary dcreening operation.
Fig. 2 be take 16S rDNA sequence as basic F69 bacterium phylogenetic tree schematic diagram.
Fig. 3 high temperature resistant beta-galactosidase enzymes optimum temperature schematic diagram of the present invention.
The thermostability schematic diagram of Fig. 4 high temperature resistant beta-galactosidase enzymes of the present invention.
The suitableeest action pH schematic diagram of Fig. 5 high temperature resistant beta-galactosidase enzymes of the present invention.
Fig. 6 high temperature resistant beta-galactosidase enzymes pH stability schematic diagram of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.
Subtilis of the present invention (
bacillus subtilis) method of F69 fermentative production beta-galactosidase enzymes, be the fermention medium forming with carbon source, nitrogenous source and inorganic salt, carry out shake flask fermentation, produce beta-galactosidase enzymes, shake flask fermentation output can reach 182U/m l, and embodiment is as follows:
Embodiment 1: the screening of producing high temperature resistant beta-galactosidase strain.
The screening method of the bacterial strain of a kind of high temperature resistant beta-galactosidase enzymes of product that ferments that the present invention proposes, is characterized in that comprising successively following concrete steps:
(1) enrichment culture: soil sampling 1g porphyrize, add in the 250ml triangular flask that 80ml sterilized water is housed, add sterile glass beads, 20min vibrates in 150r/min shaking table, then in 80 ℃ of shaking water bath shaking tables, shake 30 min, get 1-2 ml in 50 mL enrichment mediums, 40 ℃ of water bath with thermostatic control shaking culture 36h-48h, concussion frequency is 150-180r/min; Wherein, enrichment medium is: lactose 2g, yeast powder 0.5g, peptone 1g, NaCl 0.5g and distilled water 100ml, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20min;
(2) primary dcreening operation: it is 10 that the nutrient solution after enrichment is carried out to 10 times of gradient dilutions to extension rate
-8, get respectively 10
-6, 10
-7, 10
-8the diluent 0.2ml of three gradients is coated on primary dcreening operation isolation medium flat board, and flat board dries in the air while there is no fluxion to surface, in 40-42 ℃ of constant incubator, is inverted and cultivates 24-36h, then culture temperature is dropped to 32～35 ℃, continues to cultivate 24～72h; Picking blue colonies is to another primary dcreening operation isolation medium, and culture condition is: temperature 40-42 ℃, incubation time 30-36h, and as shown in Figure 1, after cultivating, bacterium colony presents deep mixed blueness to result, further chooses blue darker bacterial strain line purifying; Wherein, primary dcreening operation isolation medium is: lactose 2g, yeast powder 0.5g, peptone 1g, NaCl 0.5g, distilled water 100ml and agar 1.5g, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20 min, be cooled to 60 ℃, add the X-Gal(30mg/L with the micro-filtrate membrane filtration degerming of 0.25 μ m) 40 μ l, mix, be down flat plate;
(3) bacterial strain purifying: by primary dcreening operation bacterial strain out, in the flat lining out of beef-protein medium, be placed in 40-42 ℃ of constant incubator and cultivate 48h, repeat streak culturely more than three times, as continuous several times, examine under a microscope thalline that form is single and think bacterial strain purifying;
(4) multiple sieve: after primary dcreening operation by purified inoculation in seed culture medium, capacity is that the triangular flask liquid amount of 250ml is 30ml, in temperature, be 40-45 ℃, concussion frequency is under 160-180r/min, isothermal vibration shaking table is cultivated 18-22h, then according to the inoculum size of 4-6%, be inoculated in multiple sieve fermention medium shaking table concussion fermentation culture; Fermentation condition is: in 250ml triangular flask, fill substratum 30-50ml, shaking table concussion frequency is 160-200r/min, leavening temperature 40-45 ℃, fermentation time is 36-48h, after fermentation ends, the centrifugal 10min of fermented liquid 8000r/min, supernatant liquor is crude enzyme liquid, measures the beta-galactosidase enzymes enzymic activity of each crude enzyme liquid; Wherein bacterial strain F69 product betagalactosidase activity is the strongest, and vigor is up to 182U/ml; Wherein, seed culture medium is: peptone 1g, extractum carnis 0.5g, yeast powder 0.5g, NaCl 0.5g, K2HPO4 0.5g and distilled water 100mL, condition: pH6.5,121 ℃ of sterilising temps, sterilization time 20 min; Sieving again fermention medium is: lactose 2g, yeast powder 0.5g, peptone 1g, NaCl 0.5g and distilled water 100ml, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20 min;
(5) culture presevation: the bacterial classification that multiple sieve is obtained is rule on slant medium, cultivates 3 days, is then placed in 4 ℃ of Refrigerator stores for 40-45 ℃; Wherein, slant medium is: glucose 1g, peptone 1g, NaCl 0.5g, agar 1.5g and distilled water 100ml, condition: pH6.5～6.8,121 ℃ of sterilising temps, sterilization time 20min;
(6) beta-galactosidase enzymes enzyme assay: get 500 μ L enzyme liquid to be measured and join and be preheating in the 0.1 mol/L phosphoric acid buffer (pH 6.5) that 1.5 mL of 65 ℃ contain 2 mmol/L ONPG, after 65 ℃ of water bath heat preservation 10 min, the sodium carbonate solution termination reaction that adds 1mL 1mol/L
a 405measure absorbance value, calculate the content of hydrolysate o-nitrophenol and the activity of enzyme, simultaneously with 0.1 mol/L pH 6.5 phosphoric acid damping fluids, replace enzyme liquid in contrast, the definition of lactase activity: the activity of the Sumylact L of 1 unit be that per minute decomposes the required enzyme amount of ONPG generation l μ mol o-nitrophenol (ONP) at 6.5,65 ℃ of pH.
Embodiment 2: produce high temperature resistant beta-galactosidase strain F69 Morphology and physiology biochemical character.
The product beta-galactosidase strain that screening method of the present invention obtains, after the dull and stereotyped cultivation of beef-protein medium 18-24h, is observed bacterium colony and thalli morphology, and surface is more coarse, and edge is irregular, the dirty white of bacterium colony, and gramstaining is positive; The form of thalline is that quarter butt is bar-shaped; Thalline size is 0.65-0.7 μ m * 2.5-3 μ m; At thalline, central authorities form endogenous spore, and after sporulation, thalline does not expand; Thalline is without pod membrane, peritrichous, and thalline has mobility; Gram-positive; In liquid medium within, grow, form wrinkle mould; Cellulose decomposition experiment is negative; In carbohydrate fermentation experiment, this bacterial strain can utilize Zulkovsky starch, glucose, fructose, lactose, wood sugar, maltose and sucrose to do uniqueness carbon source for growth; In other Physiology and biochemistries detect, this bacterial strain catalase is positive, V.P measures and is positive, energy hydrolyzed starch, casein, liquefy gelatin, can utilize Citrate trianion, nitrate reduction is positive, and litmus milk reduction is positive, urea utilizes negative, does not produce fluorochrome, and methyl red experiment is positive; The morphology that bacterial strain F69 is concrete and physiological and biochemical property are in Table 1, subtilis in the outstanding bacterium mirror handbook > > of bacterium colony, dyeing characteristic and physiological and biochemical property and < < uncle (the 8th edition) approaches, can determine bacterial strain F69 be subtilis (
bacillus subtilis).
Table 1 bacterial strain F69 morphology and physio-biochemical characteristics
| Characteristic | Result | Characteristic | Result |
| Thalline size/μ m | 0.65-0.7×2.5-3 | 2% NaCl growth | + |
| | 40 | 5% NaCl growth | － |
| Gramstaining | + | D-Glucose | + |
| Optimal pH | 7.0 | Urea utilizes | － |
| Catalase test | + | Lactose | + |
| V.P test | + | Fructose | + |
| Indole test | － | Wood sugar | + |
| Reduction nitrate | + | Maltose | + |
| Gelatine liquefication | + | Sucrose | + |
| Methyl red test | + | Citrate trianion utilizes | + |
| Caseinhydrolysate | + | Cellulose hydrolysis | － |
| Aerobism | + | Starch Hydrolysis | + |
Note: "+" and "-" represents respectively positive and negative
Embodiment 3: the 16S rDNA sequential analysis of producing beta-galactosidase strain F69.
Extract bacterial strain F69 genome of the present invention, according to the most conservative primers in bacterial 16 S rDNA, wherein forward primer is F:5'-AGAGTTTGATCCTGGCTCAG-3'; Reverse primer is R:5'-AAGGAGGTGATCCAGCCGCA-3'; PCR reaction system 50 μ L; 94 ℃ of denaturation 5min of thermal circulation parameters, 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, circulate 35 times, and 72 ℃ are extended 7min.Recording sequence adopts Clustal X1.83 to carry out Multiple Sequence Alignment with the 16S rDNA sequence of comparing the close bacterium of acquisition from NCBI, then adopt MEGA4.0 biological software constructing system evolutionary tree, result as shown in Figure 2, the 16S rDNA nucleotide sequence that shows bacterial strain F69 with
bacillus subtilis(AJ276351) 16S rDNA nucleotide sequence homology is the highest, and maximum comparability (max ident) reaches 99%; Morphological feature, physiological and biochemical property and the 16S rDNA nucleotide sequence homology analytical results of comprehensive F69 bacterial strain, by bacterial strain F69 be accredited as subtilis (
bacillus subtilis).
Embodiment 4: the enzymatic property of bacterial strain high temperature resistant beta-galactosidase enzymes that F69 produces.
1, the optimum temperature of beta-galactosidase enzymes:
Enzyme liquid after fermentation, centrifugal going after thalline, gets supernatant as crude enzyme liquid, for zymologic property preliminary study; The mensuration of enzyme optimum temperuture is carried out under differing temps (40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 80 ℃), reaction pH7.0, the high enzymatic activity of take is 100%, calculate the enzyme activity under differing temps, as shown in Figure 3, enzyme optimal reactive temperature is 65 ℃ to result, but between 55～75 ℃ of temperature, the beta-galactosidase enzymes vigor of surveying also all in 90% left and right, therefore this enzyme catalytic activity in higher temperature range is all very high, strong adaptability.
2, the thermostability of beta-galactosidase enzymes:
Beta-galactosidase enzymes crude enzyme liquid is placed in respectively under the condition that differing temps (50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃), pH are 7.0, insulation 180min, every 30min sampling, surveying enzyme lives, calculate residual enzyme activity separately, the enzyme activity of initial enzyme liquid is decided to be to 100%, result is as Fig. 4, at 50 ℃～80 ℃, be incubated 90min, remnant enzyme activity is more than 80%, at 80 ℃ and 90 ℃, be incubated 60min, remnant enzyme activity is respectively 85% and 65%, shows that subtilis beta-galactosidase enzymes that F69 produces has strong thermostability.
3, the suitableeest action pH of beta-galactosidase enzymes:
Beta-galactosidase enzymes crude enzyme liquid carries out enzymatic reaction to measure its optimal pH under different pH (4.0～10.0), damping fluid used is: the sodium-acetate buffer of pH 4.0～5.5, the sodium phosphate buffer of pH 6.0～7.5, the Tris-HCl damping fluid of pH 8.0～9.0, glycine-sodium hydrate buffer solution of pH 9.0～10.5, measuring temperature is 65 ℃, the high enzymatic activity of take is 100%, calculate the enzyme activity under different pH, result as shown in Figure 5, the suitableeest action pH is 6.5, in enzymic activity between pH5.5～7.5 more than 80%.
4, the ph stability of beta-galactosidase enzymes:
The mensuration of crude enzyme liquid pH stability is that enzyme liquid is hatched to 1h in the damping fluid of different pH values at 40 ℃, measures residual enzyme activity separately; Will be wherein high enzymatic activity is decided to be 100%, and result shows, keeps more than 81% enzymic activity between pH 4.5～7.5, and more than pH7.5, enzymic activity faster declines, and shows that beta-galactosidase enzymes is highly stable under slightly acidic and neutral environment, and result as shown in Figure 6; In view of the pH value of most dairy beverages is also within the scope of this, therefore having given subtilis F69 beta-galactosidase enzymes that bacterial strain produces, these biochemical characteristics of this enzyme have larger application advantage in milk-product processing.
In the present invention, the technique means of all not specified (NS)s is method known in those skilled in the art.The present invention, through validation trial, has obtained satisfied effect.
Above embodiment and embodiment are a kind of concrete supports of producing the bacterial strain of high temperature resistant beta-galactosidase enzymes and the technological thought of screening method thereof that the present invention is proposed; can not limit protection scope of the present invention with this; every technological thought proposing according to the present invention; the change of any equivalent variations of doing on the technical program basis or equivalence, all still belongs to the scope that technical solution of the present invention is protected.
Claims (7)
1. ferment and produce a bacterial strain for high temperature resistant beta-galactosidase enzymes, the Classification And Nomenclature that it is characterized in that described bacterial strain F69 be subtilis (
bacillus subtilis), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on January 9th, 2012, deposit number is: CGMCC No.5699.
2. a kind of fermentation according to claim 1 produced the bacterial strain of high temperature resistant beta-galactosidase enzymes, it is characterized in that described bacterial strain F69(CGMCC No.5699) there is following characteristic:
(1) growth conditions: subtilis (
bacillus subtilis) 55～75 ℃ of the growth temperatures of beta-galactosidase enzymes that F69 produces, 65 ℃ of optimum growth temperatures;
(2) enzymic activity: be incubated 90min at 50 ℃～80 ℃, remnant enzyme activity, more than 80%, is incubated 60min at 80 ℃ and 90 ℃, and remnant enzyme activity is respectively 85% and 65%;
(3) slightly acidic: more than 80%, keep more than 81% enzymic activity in enzymic activity between pH5.5～7.5 between pH 4.5～7.5, the suitableeest action pH is 6.5.
3. the screening method based on a kind of bacterial strain of producing high temperature resistant beta-galactosidase enzymes of fermenting claimed in claim 1, is characterized in that comprising successively following concrete steps:
Step 1, enrichment culture: soil sampling 1g porphyrize, add in the 250ml triangular flask that 80ml sterilized water is housed, add sterile glass beads, the 20min that vibrates in 150r/min shaking table then shakes 30 min in 80 ℃ of shaking water bath shaking tables, gets 1-2 ml in 50 mL enrichment mediums, 40 ℃ of water bath with thermostatic control shaking culture 36h-48h, concussion frequency is 150-180r/min;
Step 2, primary dcreening operation: it is 10 that the nutrient solution after enrichment is carried out to 10 times of gradient dilutions to extension rate
-8, get respectively 10
-6, 10
-7, 10
-8the diluent 0.2ml of three gradients is coated on primary dcreening operation isolation medium flat board, and flat board dries in the air while there is no fluxion to surface, in 40～42 ℃ of constant incubators, is inverted and cultivates 24～36h, then culture temperature is dropped to 32～35 ℃, continues to cultivate 24～72h; Picking blue colonies is to another primary dcreening operation isolation medium, and culture condition is: 40～42 ℃ of temperature, incubation time 30～36h; After cultivating, bacterium colony presents deep mixed blueness, further chooses blue darker bacterial strain line purifying;
Step 3, bacterial strain purifying: by primary dcreening operation bacterial strain out, in the flat lining out of beef-protein medium, be placed in 40～42 ℃ of constant incubators and cultivate 48h, repeat streak culturely more than three times, as continuous several times, examine under a microscope thalline that form is single and think bacterial strain purifying;
Step 4, multiple sieve: after primary dcreening operation by purified inoculation in seed culture medium, capacity is that the triangular flask liquid amount of 250ml is 30ml, in temperature, it is 40～45 ℃, concussion frequency is under 160～180r/min, isothermal vibration shaking table is cultivated 18-22h, then according to 4～6% inoculum size, be inoculated in multiple sieve fermention medium, shaking table concussion fermentation culture, fermentation condition is: in 250ml triangular flask, fill substratum 30～50ml, shaking table concussion frequency is 160-200r/min, 40～45 ℃ of leavening temperatures, fermentation time is 36～48h, after fermentation ends, the centrifugal 10min of fermented liquid 8000r/min, supernatant liquor is crude enzyme liquid, measure the beta-galactosidase enzymes enzymic activity of each crude enzyme liquid, wherein bacterial strain F69 product betagalactosidase activity is the strongest, vigor is up to 182U/ml,
Step 5, culture presevation: the bacterial classification that multiple sieve is obtained is rule on slant medium, cultivates 3 days, is then placed in 4 ℃ of Refrigerator stores for 40～45 ℃;
Step 6, beta-galactosidase enzymes enzyme assay: get 500 μ L enzyme liquid to be measured and join and be preheating in the 0.1 mol/L phosphoric acid buffer (pH 6.5) that 1.5 mL of 65 ℃ contain 2 mmol/L ONPG, after 65 ℃ of water bath heat preservation 10 min, the sodium carbonate solution termination reaction that adds 1mL 1mol/L
a 405measure absorbance value, calculate the content of hydrolysate o-nitrophenol and the activity of enzyme, simultaneously with 0.1 mol/L pH 6.5 phosphoric acid damping fluids, replace enzyme liquid in contrast, the definition of lactase activity: the activity of the Sumylact L of 1 unit be that per minute decomposes the required enzyme amount of ONPG generation l μ mol o-nitrophenol (ONP) at 6.5,65 ℃ of pH.
4. the screening method of a kind of bacterial strain of producing high temperature resistant beta-galactosidase enzymes of fermenting according to claim 3, it is characterized in that the enrichment medium described in step 1 is: lactose 2g, yeast powder 0.5g, peptone 1g, NaCl 0.5g and distilled water 100ml, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20 min.
5. the screening method of a kind of bacterial strain of producing high temperature resistant beta-galactosidase enzymes of fermenting according to claim 3, it is characterized in that the primary dcreening operation isolation medium described in step 2 is: lactose 2g, yeast powder 0.5g, peptone 1g, NaCl 0.5g, distilled water 100ml and agar 1.5g, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20 min, then be cooled to 60 ℃, add the X-Gal(30mg/L with the micro-filtrate membrane filtration degerming of 0.25 μ m) 40 μ l, mix, be down flat plate.
6. the screening method of a kind of bacterial strain of producing high temperature resistant beta-galactosidase enzymes of fermenting according to claim 3, is characterized in that the seed culture medium described in step 4 is: peptone 1g, extractum carnis 0.5g, yeast powder 0.5g, NaCl 0.5g, K
2hPO
40.5g and distilled water 100mL, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20 min; Sieving again fermention medium is: lactose 2g, yeast powder 0.5g, peptone 1g, NaCl 0.5g and distilled water 100ml, condition: pH 6.5,121 ℃ of sterilising temps, sterilization time 20 min.
7. the screening method of a kind of bacterial strain of producing high temperature resistant beta-galactosidase enzymes of fermenting according to claim 3, it is characterized in that the slant medium described in step 5 is: glucose 1g, peptone 1g, NaCl 0.5g, agar 1.5 g and distilled water 100ml, condition: pH6.5～6.8,121 ℃ of sterilising temps, sterilization time 20min.
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| CN109182307A (en) * | 2018-10-11 | 2019-01-11 | 山东隆科特酶制剂有限公司 | A kind of method that bacillus coagulans liquid fermentation produces beta galactosidase |
| CN109207402A (en) * | 2018-10-11 | 2019-01-15 | 山东隆科特酶制剂有限公司 | One bacillus coagulans and its liquid fermentation enzyme producing method |
| CN109628340A (en) * | 2018-12-20 | 2019-04-16 | 量子高科(中国)生物股份有限公司 | A kind of Bacillus circulans bacterial strain and its selection producing high vigor beta galactosidase |
| CN111321086A (en) * | 2020-04-01 | 2020-06-23 | 玉林市容县奇昌种猪养殖有限公司 | Screening method of tannase-producing candida |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400728A (en) * | 2015-12-15 | 2016-03-16 | 重庆大学 | Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof |
| CN107182554A (en) * | 2017-06-30 | 2017-09-22 | 上海市农业科学院 | A kind of method for screening agaricus bisporus responsive to temperature type bacterial strain |
| CN109182307A (en) * | 2018-10-11 | 2019-01-11 | 山东隆科特酶制剂有限公司 | A kind of method that bacillus coagulans liquid fermentation produces beta galactosidase |
| CN109207402A (en) * | 2018-10-11 | 2019-01-15 | 山东隆科特酶制剂有限公司 | One bacillus coagulans and its liquid fermentation enzyme producing method |
| CN109207402B (en) * | 2018-10-11 | 2021-07-23 | 山东隆科特酶制剂有限公司 | Bacillus coagulans and liquid fermentation enzyme production method thereof |
| CN109182307B (en) * | 2018-10-11 | 2021-07-23 | 山东隆科特酶制剂有限公司 | Method for producing beta-galactosidase by liquid fermentation of bacillus coagulans |
| CN109628340A (en) * | 2018-12-20 | 2019-04-16 | 量子高科(中国)生物股份有限公司 | A kind of Bacillus circulans bacterial strain and its selection producing high vigor beta galactosidase |
| CN109628340B (en) * | 2018-12-20 | 2022-06-07 | 量子高科(广东)生物有限公司 | Bacillus circulans strain for producing high-activity beta-galactosidase and breeding method thereof |
| CN111321086A (en) * | 2020-04-01 | 2020-06-23 | 玉林市容县奇昌种猪养殖有限公司 | Screening method of tannase-producing candida |
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