CN105524978B - A method of detection extracellular protease Caseinolytic activity and heat resistance - Google Patents
A method of detection extracellular protease Caseinolytic activity and heat resistance Download PDFInfo
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Abstract
The present invention provides a kind of detection extracellular protease Caseinolytic activity and the methods of heat resistance.This method comprises: microbial culture medium is centrifuged, takes supernatant by (1)；(2) a part of supernatant is heated；(3) will by mix with reaction buffer with the supernatant without Overheating Treatment, 35-45 DEG C reaction 2-3 hour, terminate and react, be centrifuged, supernatant is taken to be transferred to porous plate, addition strong base solution, detection 450nm light absorption value；(4) 450nm light absorption value is scaled extracellular protease Caseinolytic activity；The heat resistance of extracellular protease is the ratio through the extracellular protease Caseinolytic activity in Overheating Treatment and the supernatant without Overheating Treatment.The method of the present invention can carry out quantitative test to extracellular microbial exoproteinase Caseinolytic activity and heat resistance simultaneously, difference between more accurate relatively bacterial strain, keep operation more convenient, saves culture medium, can be realized batch operation and can directly detect supernatant of bacteria solution.
Description
Technical field
The present invention relates to microorganisms technical field, more particularly to a kind of detection extracellular protease Caseinolytic activity and
The method of heat resistance.
Background technique
The extracellular thermophilic protease that microorganism secretes in cow's milk production chain process will lead to cow's milk and product generates gel,
Precipitating and change are bitter.Existing skimmed milk plate screening technology passes through the training to microorganism using skimmed milk is added into culture medium
It supports, sees whether the casein hydrolysis vigor of hydrolysis circle formed to judge microorganism, it is easy to operate, but can only detect micro-
Whether biology has casein hydrolysis vigor, and detects and compare casein hydrolysis vigor with being unable to accurate quantification.Existing azo
Casein detection technique, the degradation using microorganism to azo casein substrate discharge the production with specific UV absorbing wavelength
Object, in combination with detection thalli growth concentration, may be implemented to microorganism casein water by detecting the ultraviolet absorption value of product
The quantitative detection of solution vigor, but due to needing while detecting cell concentration, causing detection process complicated and can not achieve batch
Detection.The azo-casein detection technique reported recently directly detects culture medium supernatant, can not depend on cell concentration,
Can casein hydrolysis vigor quantitative test to extracellular protease, but fail to its casein hydrolysis vigor and heat resistance simultaneously
It carries out quantitative, batch detection and is not able to achieve detection to extracellular protein heat resistance, and in bacterial strain rejuvenation culture, culture volume
At 5-8 milliliters, needs to use Tube propagation, be unfavorable for batch operation.
Summary of the invention
The technical problem to be solved by the present invention is in order to overcome at present detection extracellular protease activity method cannot be same
When detect extracellular protease heat resistance in bulk, provide a kind of detection extracellular protease Caseinolytic activity and heat resistance
Method.The method of detection extracellular protease Caseinolytic activity of the invention and heat resistance can be with 1) can be simultaneously to microorganism
Extracellular protease Caseinolytic activity and heat resistance carry out quantitative test, pass through the extracellular protein between obtained different samples
Enzyme Caseinolytic activity and heat resistance difference can the more accurate differences compared between bacterial strain；2) all processing operations, all
It can be carried out in the centrifuge tube of 1.5ml, save test tube to the transfer step of centrifuge tube, it can be with after strain culturing and processing
It is directly centrifuged, so that operation is more convenient；3) use of culture medium is saved；4) batch can be realized by the use of ELISA Plate
Operation；5) without the concern for cell concentration, supernatant of bacteria solution can be directly detected, compares extracellular protease junket in certain volume unit
Proteolytic activity and heat resistance.
The present invention provides following technical scheme to solve above-mentioned technical problem:
One of technical solution of the present invention: it is a kind of simultaneously, batch detection extracellular microbial exoproteinase Caseinolytic activity and
The method of heat resistance, the described method comprises the following steps:
(1) microbial culture medium is centrifuged, takes supernatant；
(2) by 90-100 DEG C of heating 1-3min of a part of step (1) supernatant, the supernatant through Overheating Treatment is obtained,
Remaining step (1) supernatant is the supernatant without Overheating Treatment；
(3) by described in step (2) by with supernatant and reaction buffer by volume 1 without Overheating Treatment:
0.5-1: 2 mix respectively, and the group of the reaction buffer is divided into 2-4.5g/L azo-casein, 25-45mM N-morpholinyl
(MOPS) it is terminated instead with 1.5-3mM calcium chloride, 35-45 DEG C of reaction 2-3h, addition 1.5-3M solution of trichloroacetic acid or trifluoroacetic acid
It answers, is centrifuged, supernatant is taken to be transferred to porous plate, strong base solution is added into the supernatant to the final concentration of 0.2-0.5M of highly basic, divides
It Jian Ce not 450nm light absorption value；
(4) by 450nm light absorption value obtained by step (3) be scaled respectively step (2) it is described through Overheating Treatment with without overheat
Extracellular protease Caseinolytic activity in the supernatant of processing, the method for the conversion are the suction by sample at 450nm
Light value subtracts blank control and obtains light absorption value increment in the light absorption value of 450nm, then light absorption value increment is scaled every milliliter of sample
Product light absorption value hourly rises in value up to the extracellular protease Caseinolytic activity；The heat resistance of the extracellular protease is
Ratio of the gained through the extracellular protease Caseinolytic activity in Overheating Treatment and the supernatant without Overheating Treatment.
In the present invention, step (1) are as follows: by microbial culture medium centrifugation, take supernatant.In step (1), the microorganism training
Nutrient solution can be obtained by the method for this field routine, be obtained preferably by method comprising the following steps: 1) by microorganism
It is inoculated in 30 DEG C of cultures in rejuvenation culture medium and for 24 hours, obtains rejuvenation culture solution；2) step 1) the rejuvenation culture solution is forwarded to sterilizing
Skimmed milk, 30 DEG C continue cultivate 48h to get.In step 1), the rejuvenation culture medium can be the Bacteria Culture of this field routine
Base, preferably MPC culture medium, the group of the MPC culture medium be divided into 5.0g/L tryptone, 2.5g/L yeast extract and
1.0g/L glucose.The culture can be carried out by the method for the conventional culture bacterium in this field, preferably shaken cultivation, institute
The revolving speed for stating the culture of oscillation is preferably 150-250rpm, is more preferably 180-220rpm, is most preferably 200rpm.Step 2)
In, the sterilized non-fat cream can be the sterilized non-fat cream of this field routine, as sterilized non-fat fresh milk or sterilized non-fat restore
Cream, preferably sterilized non-fat reconstituted milk.The ratio of the switching can be the switching ratio of this field routine, preferably body
Product ratio 1: 10.The culture can be carried out by the method for the conventional culture bacterium in this field, preferably shaken cultivation, the vibration
The revolving speed for the culture swung is preferably 150-250rpm, is more preferably 180-220rpm, is most preferably 200rpm.
In the present invention, step (2) are as follows: by 90-100 DEG C of heating 1-3min of a part of step (1) supernatant, obtain by
The supernatant of heat treatment, the remaining step 1) supernatant are the supernatant without Overheating Treatment.It is described to add in step (2)
The temperature of heat is preferably 95-100 DEG C, is more preferably 100 DEG C, is most preferably 100 DEG C, the time of the heating is preferably 2-
3min is more preferably 2min.The heating can be carried out by the method for this field routine, and preferably water-bath or metal bath adds
Heat is more preferably metal bath heating.Described a part can be the conventionally referred ratio in this field, preferably 1/2.
In the present invention, step (3) are as follows: by described in step (2) by with without Overheating Treatment supernatant with react delay
Fliud flushing respectively mixes at 1: 0.5-1: 2 by volume, and the group of the reaction buffer is divided into 2-4.5g/L azo-casein, 25-
45mM N-morpholinyl (MOPS) and 1.5-3mM calcium chloride, 35-45 DEG C of reaction 2-3h, be added 1.5-3M solution of trichloroacetic acid or
Trifluoroacetic acid solution terminates reaction, and centrifugation takes supernatant to be transferred to porous plate, and strong base solution is added into the supernatant to highly basic end
Concentration is 0.2-0.5M, detects 450nm light absorption value respectively.
In step (3), it is described by with supernatant and reaction buffer by volume 1: 0.5-1: 2 without Overheating Treatment
It mixes respectively, preferably 1: 1 mixing by volume.The concentration of the azo-casein is preferably 3-4.5g/L, more preferably for
4.5g/L；The concentration of the N-morpholinyl is preferably 35-45mM, is more preferably 45mM；The concentration of the calcium chloride is preferable
Ground is 1.5-2mM, is more preferably 2mM.The temperature of the reaction is 35-45 DEG C, preferably 40 DEG C.The time of the reaction is
2-3h, preferably 2h.The termination reaction terminates reaction, the trichloroacetic acid for solution of trichloroacetic acid or trifluoroacetic acid is added
Or the concentration of trifluoroacetic acid is preferably 2M, the volume of the solution of trichloroacetic acid or trifluoroacetic acid solution preferably accounts for reaction
The 1/10 of system total volume.The final concentration of 0.2-0.5M of the highly basic, preferably 0.2M.The highly basic can be this field
Conventional highly basic, preferably sodium hydroxide or potassium hydroxide, are more preferably sodium hydroxide.The concentration of the strong base solution can be with
For the concentration of this field routine, preferably 1M.The revolving speed of the centrifugation can be the revolving speed of this field routine, preferably
10000rpm.The time of the centrifugation can be the duration of this field routine, preferably 15min.The temperature of the centrifugation can
Think the temperature of this field routine, preferably 25 DEG C.The porous plate can be the porous plate of this field routine, such as 96 orifice plates
With 384 orifice plates, preferably 96 orifice plates.Detecting the absorption value at the 450nm can carry out with the conventional methods in the field, compared with
It is detected goodly for sample batch to be placed in the porous plate with microplate reader.
In the present invention, step (4) are as follows: 450nm light absorption value obtained by step (3) is scaled step (2) described process respectively
Heat treatment and the extracellular protease Caseinolytic activity in the supernatant without Overheating Treatment, the method for the conversion are by sample
Light absorption value of the product at 450nm subtracts blank control and obtains light absorption value increment in the light absorption value of 450nm, then the light absorption value is rised in value
Every milliliter of sample light absorption value hourly is scaled to rise in value up to the extracellular protease Caseinolytic activity；The extracellular egg
The heat resistance of white enzyme is that gained is living through the extracellular protease casein hydrolysis in Overheating Treatment and the supernatant without Overheating Treatment
The ratio of property.In step (4), the blank control is preferably deionized water.
In the present invention, the heat treatment can be the heat treatment to extracellular protease of this field routine,
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: method 1 provided by the invention) it can be simultaneously to extracellular microbial exoproteinase
Caseinolytic activity and heat resistance carry out quantitative test, pass through the extracellular protease casein water between obtained different samples
Solution activity and heat resistance difference can the more accurate differences compared between bacterial strain；2) all processing operations, can be in 1.5ml
Centrifuge tube in carry out, save test tube to centrifuge tube transfer step, strain culturing and processing after can directly be centrifuged, make
It is more convenient to operate；3) use of culture medium is saved；4) batch operation can be realized by the use of ELISA Plate；5) it is not required to
Consider cell concentration, can directly detect supernatant of bacteria solution, it is living to compare extracellular protease casein hydrolysis in certain volume unit
Property and heat resistance.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
The culture medium used in the embodiment of the present invention is as follows:
MPC culture medium: 5.0g/L tryptone, 2.5g/L yeast extract and 1.0g/L glucose
MPC solid medium: skimmed milk powder is added in above-mentioned MPC culture medium to 1g/L, agar to 18g/L adjusts pH value
7.0 ± 0.1,115 DEG C of sterilizing 15min
Sterilized non-fat cream: take 10g skimmed milk powder to be dissolved in 1L water, 115 DEG C, 15min sterilizes to obtain the final product
Bacterial strain used in following embodiment of the present invention is pseudomonad (Pseudomonas sp), and number is respectively
N2-3、N5-3、N9-2、M41、M46、J937、N96、D11、D13、M13、N97、D62、D72、D81、D104、D15、D19、D44、
J435, J436, J737, D21, D22, D46, D84, D105, J935, J225 and N71.The above bacterial strain is by this laboratory from raw milk
Middle separation obtains, and separation method takes 10 the following steps are included: carry out gradient dilution with sterile water to raw milk-2~10-5Four
Each 100 μ L of dilution sample, is coated on MPC solid medium, chooses single colonie scribing line, is repeated twice, resulting bacterial strain is
Experimental strain.
The detection of the general bacterium extracellular protease Caseinolytic activity of embodiment 1 and heat resistance
(1) pseudomonad strain N2-3, N5-3 and N9-2 for freezing are taken out from -20 DEG C of refrigerators and is placed on ice chest, with
The inoculum concentration of 2% volume ratio is inoculated into the EP pipe of the culture medium containing 1mLMPC, and 30 DEG C of incubator 200rpm vibrate rejuvenation culture
For 24 hours, rejuvenation inoculum is obtained；The 100 μ L of rejuvenation inoculum is taken to be forwarded to 30 in the EP pipe of the cream of sterilized non-fat containing 1mL
25 DEG C of 12000rpm of cultured bacterium solution are centrifuged 15min, obtain supernatant by DEG C 200rpm shaken cultivation 48h；
(2) 100 μ L step (1) supernatants is taken after 100 DEG C of heating 2min, 4 DEG C of cooling 30s, separately to take 100 μ L in metal bath
Stand-by heat is not added in step (1) supernatant；
(3) step (2) are mixed with 100 μ L of reaction buffer by heating with not heated supernatant respectively, 40 DEG C
2h is reacted, the group of the reaction buffer is divided into 4.5g/L azo-casein, 45mM MOPS and 1.5mM calcium chloride.Two groups of samples
Reaction is terminated with the trichloroacetic acid of 20 μ L 2M, 25 DEG C of 10000rpm are centrifuged 15min；Supernatant 150 μ L to 96 holes after taking centrifugation
In tissue culture plate, add 50 μ L 1M sodium hydroxide solutions, surveys the light absorption value of 450nm with microplate reader immediately；
(4) the sample light absorption value that step (3) measures is subtracted into same blank sample (supernatant of bacteria solution is substituted by deionized water)
Light absorption value up to light absorption value rise in value (DA).The light absorption value increment hourly of every milliliter of sample is proteinase activity at 450nm
(ΔA h-1mL-1).The proteinase activity of bacterial strain is greater than 0.3 Δ A h-1mL-1It is considered as having proteinase activity.Test every time
Twice, be averaged.The calculation method of heat resistance is, by the proteinase activity value of sample after heat treatment divided by without heat
The proteinase activity value of the sample of reason, obtained activity of residual enzyme percentage is as thermal resistance values.The proteinase activity that measures and
The results are shown in Table 1 for heat resistance:
Table 1
Extracellular protein enzyme activity measures under the conditions of 40 DEG C by substrate of azo-casein, and experimental result is tested twice
Average deviation.There are apparent differences between the heat resistance of tri- bacterial strains of N2-3, N5-3 and N9-2 seen from table 1.
The detection of the general bacterium extracellular protease Caseinolytic activity of embodiment 2 and heat resistance
(1) pseudomonad strain M41, M46 and J937 for freezing are taken out from -20 DEG C of refrigerators and is placed on ice chest, with 2%
The inoculum concentration of volume ratio is inoculated into the EP pipe of the culture medium containing 1mLMPC, and 30 DEG C of incubator 200rpm oscillation rejuvenation cultures for 24 hours, obtain
Rejuvenation inoculum；The 100 μ L of rejuvenation inoculum is taken to be forwarded in the EP pipe of the cream of sterilized non-fat containing 1mL 30 DEG C
25 DEG C of 12000rpm of cultured bacterium solution are centrifuged 15min, obtain supernatant by 200rpm shaken cultivation 48h；
(2) 100 μ L step (1) supernatants is taken after 90 DEG C of heating 3min, 4 DEG C of cooling 30s, separately to take 100 μ L in metal bath
Stand-by heat is not added in step (1) supernatant；
(3) step (2) are mixed with 50 μ L of reaction buffer by heating with not heated supernatant respectively, 35 DEG C anti-
3h is answered, the group of the reaction buffer is divided into 2g/L azo-casein, 25mM MOPS and 2mM calcium chloride.Two groups of samples use 20
The trichloroacetic acid of μ L1.5M terminates reaction, and 25 DEG C of 10000rpm are centrifuged 15min；Take the 100 μ L of supernatant after being centrifuged to 96 hole cells
In culture plate, add 100 μ L 1M potassium hydroxide solutions, surveys the light absorption value of 450nm with microplate reader immediately；
(4) the sample light absorption value that step (3) measures is subtracted into same blank sample (supernatant of bacteria solution is substituted by deionized water)
Light absorption value up to light absorption value rise in value (DA).The light absorption value increment hourly of every milliliter of sample is proteinase activity at 450nm
(ΔA h-1mL-1).The proteinase activity of bacterial strain is greater than 0.3 Δ A h-1mL-1It is considered as having proteinase activity.Test every time
Twice, be averaged.The calculation method of heat resistance is, by the proteinase activity value of sample after heat treatment divided by without heat
The proteinase activity value of the sample of reason, obtained activity of residual enzyme percentage is as thermal resistance values.The proteinase activity that measures and
The results are shown in Table 2 for heat resistance:
Table 2
Extracellular protein enzyme activity measures under the conditions of 40 DEG C by substrate of azo-casein, and experimental result is tested twice
Average deviation.As can be seen from Table 2, the extracellular protease of bacterial strain M41 does not have heat resistance, the heat resistance of bacterial strain M46 and J937 are relatively connect
Closely.
The detection of 3 psychrophile extracellular protease Caseinolytic activity of embodiment and heat resistance
Psychrophile is that a kind of maximum growth temperature is higher than 20 DEG C, and optimum growth temperature is higher than 15 DEG C, and still may be used at 0~5 DEG C
With the microorganism of growth and breeding.The present embodiment detects the extracellular protease activity and heat resistance of 20 plants of psychrophiles, specific as follows:
(1) by 20 plants of thermophilic cold pseudomonad strain N97, D62, D72, D81, D104, D15, D19, D44, J435, J436,
J737, D21, D22, D46, D84, D105, J935, J225 and N71 take out from -20 DEG C of refrigerators and are placed on ice chest, with 2% volume
The inoculum concentration of ratio is inoculated into the EP pipe of the culture medium of MPC containing 1mL, and 30 DEG C of incubator cultures for 24 hours, obtain rejuvenation culture bacterium solution；It takes multiple
Strong culture 100 μ L of bacterium solution is forwarded to 30 DEG C of 150rpm shaken cultivation 48h in the EP pipe of the cream of sterilized non-fat containing 1mL, will be cultured
25 DEG C of 12000rpm centrifugation 15min of bacterium solution obtain supernatant；
(2) (1) 100 μ L supernatant of step 100 DEG C of heating 2min, 4 DEG C of cooling 30s in metal bath are taken；Separately take step (1)
100 μ L supernatants are spare without heating；
(3) step (2) are mixed with 100 μ L of reaction buffer by heating with not heated supernatant respectively, 40 DEG C
2h is reacted, the group of the reaction buffer is divided into 4.5g/L azo-casein, 45mM MOPS and 1.5mM calcium chloride.Two groups of samples
Reaction is terminated with the trichloroacetic acid of 20 μ L 2M, 25 DEG C of 10000rpm are centrifuged 15min；On 150 μ L after taking step (2) to be centrifuged
Clearly into 96 hole porocyte culture plates, add 50 μ L 1M sodium hydroxide solutions, surveys the light absorption value of 450nm with microplate reader immediately.
(4) 450 nanometers of light absorption value light absorption values obtained by step (3) are subtracted into same blank sample (supernatant of bacteria solution is by deionization
Water substitution) light absorption value obtain light absorption value increment (DA).The light absorption value increment hourly of every milliliter of sample is protease at 450nm
Activity (Δ A h-1mL-1).The proteinase activity of bacterial strain is greater than 0.3 Δ A h-1mL-1It is considered as having proteinase activity.Every time
Test twice, be averaged.The calculation method of heat resistance is, by the proteinase activity value of sample after heat treatment divided by without
The proteinase activity value of the sample of heat treatment, obtained activity of residual enzyme percentage is as thermal resistance values.The protease activity measured
The results are shown in Table 3 for property and heat resistance:
Table 3
Extracellular protein enzyme activity measures under the conditions of 40 DEG C by substrate of azo-casein, and experimental result is tested twice
Average deviation.Seen from table 3, the extracellular protease of bacterial strain N97 do not have heat resistance, bacterial strain D62, D72, D81, D104, D15,
The heat resistance of the extracellular protease of D19, D44, J435, J436 and J737 has a different between each other, bacterial strain D21, D22,
Extracellular protease activity is respectively less than 0.3 Δ A h to the supernatant of D46, D84, D105, J935, J225 and N71 before the heat treatment- 1mL-1, it is believed that do not have extracellular protease activity.
The detection of 4 psychrophile extracellular protease Caseinolytic activity of embodiment and heat resistance
(1) 4 plants of thermophilic cold pseudomonad strain N96, D11, D13 and M13 are taken out from -20 DEG C of refrigerators and are placed on ice chest,
It is inoculated into the inoculum concentration of 2% volume ratio in the EP pipe of the culture medium of MPC containing 1mL, 30 DEG C of incubator cultures for 24 hours, obtain rejuvenation culture
Bacterium solution；100 μ L of rejuvenation culture bacterium solution is taken to be forwarded to 30 DEG C of 150rpm shaken cultivation 48h in the EP pipe of the cream of sterilized non-fat containing 1mL, it will
25 DEG C of 12000rpm centrifugation 15min of cultured bacterium solution obtain supernatant；
(2) (1) 100 μ L supernatant of step 95 DEG C of heating 3min, 4 DEG C of cooling 30s in metal bath are taken；Separately take step (1)
100 μ L supernatants are spare without heating；
(3) step (2) are mixed with 100 μ L of reaction buffer by heating with not heated supernatant respectively, 45 DEG C
2h is reacted, the group of the reaction buffer is divided into 2g/L azo-casein, 25mM MOPS and 3mM calcium chloride.Two groups of samples are used
The trifluoroacetic acid of 20 μ L 3M terminates reaction, and 25 DEG C of 10000rpm are centrifuged 15min；150 μ L supernatants after taking step (2) to be centrifuged are extremely
In 96 hole porocyte culture plates, add 37.5 μ L 1M sodium hydroxide solutions, surveys the light absorption value of 450nm with microplate reader immediately.
(4) 450 nanometers of light absorption value light absorption values obtained by step (3) are subtracted into same blank sample (supernatant of bacteria solution is by deionization
Water substitution) light absorption value obtain light absorption value increment (DA).The light absorption value increment hourly of every milliliter of sample is protease at 450nm
Activity (Δ A h-1mL-1).The proteinase activity of bacterial strain is greater than 0.3 Δ A h-1mL-1It is considered as having proteinase activity.Every time
Test twice, be averaged.The calculation method of heat resistance is, by the proteinase activity value of sample after heat treatment divided by without
The proteinase activity value of the sample of heat treatment, obtained activity of residual enzyme percentage is as thermal resistance values.The protease activity measured
The results are shown in Table 4 for property and heat resistance:
Table 4
Extracellular protein enzyme activity measures under the conditions of 40 DEG C by substrate of azo-casein, and experimental result is tested twice
Average deviation.By table 4 as it can be seen that bacterial strain N96, D11 and D13 do not have heat resistance.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (10)
1. it is a kind of simultaneously, the method for batch detection extracellular microbial exoproteinase Caseinolytic activity and heat resistance, feature exists
In the described method comprises the following steps:
(1) microbial culture medium is centrifuged, takes supernatant；
(2) by a part of step (1) 90-100 DEG C of supernatant heating 1-3 minutes, the supernatant through Overheating Treatment is obtained, it is remaining
The step of (1) described supernatant be the supernatant without Overheating Treatment；
(3) by described in step (2) by with supernatant and reaction buffer by volume 1: 0.5-1: 2 without Overheating Treatment
It mixes respectively, the group of the reaction buffer is divided into 2-4.5g/L azo-casein, 25-45mM N-morpholinyl and 1.5-3mM
Calcium chloride, 35-45 DEG C reaction 2-3 hours, 1.5-3M solution of trichloroacetic acid or trifluoroacetic acid is added and terminates reaction, centrifugation takes
It is transferred to porous plate clearly, strong base solution is added into the supernatant to the final concentration of 0.2-0.5M of highly basic, detects 450nm respectively and inhales
Light value；
(4) by 450nm light absorption value obtained by step (3) be scaled respectively step (2) it is described through Overheating Treatment with without Overheating Treatment
Supernatant in extracellular protease Caseinolytic activity, the method for the conversion is the light absorption value by sample at 450nm
It subtracts blank control and obtains light absorption value increment in the light absorption value of 450nm, then to be scaled every milliliter of sample every by light absorption value increment
The light absorption value of hour rises in value up to the extracellular protease Caseinolytic activity；The heat resistance of the extracellular protease is gained
Ratio through the extracellular protease Caseinolytic activity in Overheating Treatment and the supernatant without Overheating Treatment.
2. method as described in claim 1, which is characterized in that in step (1), the microbial culture medium is by including following step
Rapid method obtains:
1) by microbial inoculant, 30 DEG C of cultures for 24 hours, obtain rejuvenation culture solution in rejuvenation culture medium；
2) by step 1) the rejuvenation culture solution be forwarded to sterilized non-fat cream, 30 DEG C continue cultivate 48h to get.
3. method as claimed in claim 2, which is characterized in that in step 1), the rejuvenation culture medium is MPC culture medium, described
The group of MPC culture medium is divided into 5.0g/L tryptone, 2.5g/L yeast extract and 1.0g/L glucose；And/or step 1)
Described in culture be shaken cultivation；And/or in step 2), the sterilized non-fat cream is sterilized non-fat reconstituted milk；And/or it is described
The ratio of switching is volume ratio 1: 10；And/or culture described in step 2) is shaken cultivation.
4. method as described in claim 1, which is characterized in that in step (2), the temperature of the heating is 95-100 DEG C；With/
Or, the time of the heating is preferably 2-3 minutes；And/or it is described be heated to be water-bath or metal bath heating；And/or it is described
A part is 1/2.
5. method as described in claim 1, which is characterized in that described to pass through and the supernatant without Overheating Treatment in step (3)
It is mixed at 1: 1 by volume with reaction buffer.
6. method as described in claim 1, which is characterized in that in step (3), the concentration of the azo-casein is 3-4.5 grams/
It rises；And/or the concentration of the N-morpholinyl is 35-45mM；And/or the concentration of the calcium chloride is 1.5-2mM.
7. method as described in claim 1, which is characterized in that in step (3), the temperature of the reaction is 40 DEG C；And/or institute
Stating reaction is 2 hours.
8. method as described in claim 1, which is characterized in that in step (3), the concentration of the trichloroacetic acid or trifluoroacetic acid is
2M；And/or the volume of the solution of trichloroacetic acid or trifluoroacetic acid solution accounts for the 1/10 of reaction system total volume；And/or institute
State the final concentration of 0.2M of highly basic.
9. method as described in claim 1, which is characterized in that in step (3), the highly basic is sodium hydroxide or potassium hydroxide；
And/or the concentration of the strong base solution is 1M；And/or the revolving speed of the centrifugation is 10000 revs/min；And/or it is described from
The time of the heart is 15 minutes；And/or the temperature of the centrifugation is 25 DEG C；And/or the porous plate is 96 orifice plates；And/or inspection
Sample batch is placed in the porous plate and is detected with microplate reader by the absorption value surveyed at the 450nm.
10. method as described in claim 1, which is characterized in that in step (4), the blank control is deionized water.
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| CN1649501A (en) * | 2002-04-05 | 2005-08-03 | 普瑞图斯股份有限公司 | Method and composition for the prevention or retarding of staling and its effect during the baking process of bakery products |
| CN104130995A (en) * | 2014-07-16 | 2014-11-05 | 光明乳业股份有限公司 | Paenibacillus sp. fermentation method |
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| CN1649501A (en) * | 2002-04-05 | 2005-08-03 | 普瑞图斯股份有限公司 | Method and composition for the prevention or retarding of staling and its effect during the baking process of bakery products |
| CN104130995A (en) * | 2014-07-16 | 2014-11-05 | 光明乳业股份有限公司 | Paenibacillus sp. fermentation method |
Non-Patent Citations (1)
| Title |
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| Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk;Claudia Baur等;《International Dairy Journal》;20150513;第49卷;23-29 |
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