CN116410882A - Strain for producing high-temperature-resistant protease and preparation method of protease - Google Patents
Strain for producing high-temperature-resistant protease and preparation method of protease Download PDFInfo
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- CN116410882A CN116410882A CN202111661014.4A CN202111661014A CN116410882A CN 116410882 A CN116410882 A CN 116410882A CN 202111661014 A CN202111661014 A CN 202111661014A CN 116410882 A CN116410882 A CN 116410882A
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- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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Abstract
A strain for producing high temperature resistant protease and a preparation method of the protease belong to the technical field of dairy processing. In order to obtain the bacillus cereus secreting the heat-resistant protease, 50 existing bacillus cereus separated from raw milk in a laboratory are screened, and the result shows that bacillus cereus (bacillus cereus) C58 can produce the heat-resistant protease, the activity of the protease produced by the bacillus cereus is about 24.6U/mL, and the relative enzyme activities in heat-resistant experiments at 70 ℃ for 30min and 100 ℃ for 10min are 81.97% and 40.98%, respectively. The protease is shown to be ProteaseHhoA after purification and identification. The bacillus cereus provided by the invention has the important characteristics of bacillus in raw milk, and the source of the bacillus cereus is raw milk, so that the bacillus cereus can be used for researching harm and control methods of bacillus cereus on milk and dairy products.
Description
Technical Field
The invention belongs to the technical field of dairy processing, and particularly relates to a strain for producing high-temperature-resistant protease and a preparation method of the protease.
Background
The method is a large country for producing and consuming dairy products, ensures the quality safety of liquid milk, and has important strategic significance for improving the national economy and health level of China. The bacillus cereus is one of food-borne pathogenic bacteria with higher detection rate in liquid milk, and besides the characteristics of strong heat resistance, toxin production and the like, part of bacterial strains can secrete metabolites such as protease and the like, and the enzyme substances can continuously act on the liquid milk, so that quality defects such as bitter taste, rancid taste, layering, protein precipitation and the like appear in the shelf life of the liquid milk, hidden danger is brought to the quality safety of the liquid milk, the shelf life of the liquid milk is shortened, and serious economic loss is caused. Although UHT milk has higher sterilization temperature, the UHT milk still cannot survive the breeding of bacillus with higher heat resistance and the decomposition and deterioration of thermostable enzymes.
Bacillus cereus is an important reason for jeopardizing the quality of milk and dairy products, and it is particularly important to study the enzymatic properties of Bacillus cereus protease and its influence on the quality of liquid milk.
Disclosure of Invention
The invention provides a strain for producing high-temperature resistant protease, which is bacillus cereus C58 for screening bacillus cereus producing the high-temperature resistant protease and researching the enzymatic property of the protease.
The invention also provides a preparation method of the high-temperature resistant protease, which comprises the following steps of grafting the bacillus cereus C58Culturing in primary LB seed culture medium at 37deg.C for 12 hr at rotation speed of 2 Xg, inoculating into 50mL secondary LB seed culture medium according to 2% inoculum size, and diluting the bacterial suspension after culturing for 12 hr to 10 6 CFU/mL, inoculating to protease and lipase agar culture medium, culturing at 37deg.C for 24 hr to obtain bacterial suspension containing protease, centrifuging to obtain supernatant, and purifying to obtain the high temperature resistant protease.
Further defined, the composition of the LB seed medium is 8% of skim milk powder, 1.5% of agar powder and the balance of water in percentage by weight.
Further defined, the purification steps are ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography in that order.
Further defined, the protease is a disulfide bond containing metal dependent alkaline serine protease.
Further defined, the protease has an optimum pH of 9 and an optimum temperature of 60 ℃,2mM and 5mM Ca 2+ And Fe (Fe) 2+ Has a significant improvement effect on the protease activity and Mg 2+ Has no obvious influence on the activity of the protease and Cu 2+ Has remarkable inhibition effect on the protease activity; the D value of the protease at the heat treatment temperature of 110-130 ℃ is 195.13-677.23s, t 1/2 58.74-203.87s.
The invention has the beneficial effects that:
the invention uses precipitation and chromatography to purify the heat-resistant Protease produced by the C58 strain through screening bacillus cereus producing Protease, and identifies the Protease produced by the C58 strain through SDS-PAGE and mass spectrum, and discovers that the Protease is named Protease HhoA, has serine endopeptidase activity, and performs enzymatic property research on the Protease produced by the C58 strain.
The heat-resistant protease secreted by the bacillus cereus C58 strain provided by the invention can harm the quality of raw milk. The protease existing in the milk often causes bad phenomena of gelation, bitter and the like of the liquid milk, which shortens the shelf life of the liquid milk and causes serious economic loss.
Drawings
FIG. 1 is a diagram showing hydrolysis circles of proteases produced by Bacillus cereus strains according to the first embodiment;
FIG. 2 is a scatter plot of the number of strains and their hydrolysis index of protease producing ability of Bacillus cereus strain according to one embodiment;
FIG. 3 is a bar graph showing the enzyme activity of proteases produced by Bacillus cereus strains according to the first embodiment;
FIG. 4 is a bar graph showing the relative enzyme activities of proteases produced by Bacillus cereus strains of the first embodiment under heat treatment conditions of 70℃for 30 min;
FIG. 5 is a bar graph showing relative enzyme activities of proteases produced by Bacillus cereus strains of the first embodiment under heat treatment conditions of 100℃for 10 min;
FIG. 6 is a DEAE Sepharose F.F ion exchange chromatography diagram of a protease produced by Bacillus cereus strain C58 of the second embodiment;
FIG. 7 is a Sephadex G-100 gel filtration chromatography of a protease produced by Bacillus cereus strain C58 of the second embodiment;
FIG. 8 is a diagram showing the SDS-PAGE identification of proteases produced by the purified Bacillus cereus C58 strain according to the second embodiment;
FIG. 9 is a graph showing the change in the effect of pH III on the activity of protease produced by Bacillus cereus strain C58 according to the present embodiment;
FIG. 10 is a graph showing the change in the effect of temperature III on the activity of protease produced by Bacillus cereus strain C58 according to the present embodiment;
FIG. 11 is a bar graph showing the effect of trimetallic ions on protease activity produced by Bacillus cereus strain C58 according to the embodiment;
FIG. 12 is a bar graph showing the effect of a triple enzyme inhibitor of an embodiment on the activity of a protease produced by Bacillus cereus strain C58;
FIG. 13 is a graph showing the change in the thermal inactivation kinetics of a protease produced by the Bacillus cereus C58 strain of the present embodiment;
FIG. 14 is a graph showing changes in inactivation and activation energy of protease produced by Bacillus cereus strain C58 according to the present embodiment;
FIG. 15 is a diagram of a liquid milk sample hydrolyzed at 28℃with a protease produced by Bacillus cereus strain C58 according to the embodiment;
FIG. 16 is a diagram of a liquid milk sample hydrolyzed at 10℃with protease produced by Bacillus cereus strain C58 according to the preferred embodiment;
FIG. 17 is a graph showing the hydrolysis of liquid milk by proteases produced by Bacillus cereus strain C58 at 28℃according to a fourth embodiment;
FIG. 18 is a graph showing the hydrolysis of liquid milk by proteases produced by Bacillus cereus strain C58 at 10℃according to a fourth embodiment.
Detailed Description
The first embodiment is as follows:
the 55 bacillus cereus strains in this embodiment were isolated from raw milk in the region of black longjiang.
The screening process of the protease-producing bacillus cereus according to the embodiment comprises the following steps:
bacillus cereus is purified and inoculated into LB medium (skimmed milk powder addition ratio is 8% (w/v), agar powder addition ratio is 1.5% (w/v)), and shake-cultured at 37deg.C for 12 hr at rotation speed of 2 Xg. Inoculating into 50mL of LB culture medium according to 2% inoculum size, and diluting the bacterial suspension cultured for 12h to 10 6 CFU/mL, inoculated into prepared protease and lipase agar medium, and cultured in a 37 ℃ incubator for 24 hours. The protease activity of 55 bacillus cereus strains is evaluated by an oxford cup method, the size of the diameter of a hydrolysis circle is measured by a vernier caliper, and the ratio of the difference value of the diameter of the hydrolysis circle and the diameter of a colony to the diameter of the colony is used as a hydrolysis index.
Results: the hydrolysis circle diagram of the protease produced as shown in FIG. 1 and the hydrolysis index distribution scatter diagram of the number of Bacillus cereus strains having protease producing ability as shown in FIG. 2 were obtained, and it can be seen from FIGS. 1 and 2 that the number of strains having protease and lipase producing ability was 25 and the hydrolysis index distribution of the protease producing strain was concentrated at 2.00-2.50.
The protease activity of the bacillus cereus protease is studied, and the specific process is as follows:
the strain with protease producing ability is activated and inoculated into whole sterilized milk according to the inoculum size of 2%, and is respectively cultivated for 24 hours at 37 ℃ and the rotation speed is 1 Xg, after the cultivation is finished, the sample is centrifuged for 20 minutes at the low temperature of 4 ℃ and 15000 Xg, and the supernatant is filtered by a filter membrane of 0.45 mu m, and the protease activity of the supernatant is measured.
The reaction system: 1.5% azocasein solution (50 mM PBS solution with pH 7.2 dissolved, the solution was thoroughly mixed and left to stand for 2-3 days), 20% TCA solution.
Reaction conditions: 100 mu L of supernatant, 500 mu L of PBS buffer solution and 100 mu L of azo casein substrate solution, reacting for 60min at 40 ℃, adding 500 mu L of TCA to terminate the reaction, standing for 30min at room temperature, centrifuging for 5min at 10000 Xg, taking 200 mu L of supernatant, and measuring OD by an enzyme-labeled instrument 366 Absorbance at nm. Definition of protease activity: the absorbance value per ml of protease solution at 366nm was varied by 0.01 as one enzyme activity unit (1U) per hour.
Results: the column diagram of the protease production shown in figure 3 is obtained, and as can be seen from figure 3, the protease production activity of 12 bacillus cereus is greater than 20U/mL, the protease production activity of 10 bacillus cereus is between 10 and 20U/mL, the protease production activity of 3 bacillus cereus is less than 10U/mL, the protease production ability of L76 strain is strongest, reaching 29.20U/mL, the second is C57 strain, the protease activity is 27.6U/mL, the strain with the weakest protease production activity is K36 strain, and the protease activity is 2.4U/mL.
The heat resistance of the bacillus cereus protease of the present embodiment was studied, and the specific procedure was as follows:
the strain with stronger enzyme activity is inoculated into sterilized milk according to the proportion of 2 percent, cultured for 48 hours at 28 ℃, centrifuged for 20 minutes at 15000 Xg under the low temperature condition of 4 ℃, and filtered by a filter membrane with 0.45 mu m, and 5mL of the supernatant is taken and placed in a sterile test tube to determine the protease activity.
A sterile test tube containing 5mL of the supernatant was heated at 70℃for 30min and at 100℃for 10min, and immediately after the completion of the treatment, cooled to room temperature in ice water, and the residual activity of the protease was measured. The relative enzyme activity (RA) was expressed as a percentage of the enzyme activity (Ct) remaining after the heat treatment and the enzyme activity (C0) of the control group using the protease not subjected to the heat treatment as a control.
Results: the relative enzyme activity bar graph of the heat treatment condition at 70 ℃ for 30min shown in fig. 4 and the relative enzyme activity bar graph of the heat treatment condition at 100 ℃ for 10min shown in fig. 5 are obtained, and as can be seen from fig. 4 and 5, the protease produced by 23 bacillus cereus has thermal stability under the heat treatment condition at 70 ℃ for 30min, the protease produced by the C58 strain has the highest thermal stability, the relative enzyme activity reaches 81.97%, the heat resistance of the protease produced by the L76 strain is the weakest, and the relative enzyme activity is 9.59%. Under the heat treatment condition of 100 ℃ and 10min, the protease produced by 20 bacillus cereus has heat resistance, the heat resistance of the protease produced by the C58 strain is strongest and 40.98 percent, the heat resistance of the protease produced by the K57 strain is weakest, and the relative enzyme activity is 4.00 percent.
The second embodiment is as follows:
the bacillus cereus C58 strain of the present embodiment was multiplex-screened from 55 bacillus cereus.
In this embodiment, the purification and mass spectrum identification of the thermostable protease produced by bacillus cereus strain C58 are studied, and the specific process is as follows:
ammonium sulfate precipitation: the activated bacillus cereus is inoculated into 1L of BHI culture medium according to the proportion of 2 percent, and is cultured for 48 hours at 37 ℃. The culture was dispensed into 50mL centrifuge tubes, centrifuged at 10000 Xg for 20min at low temperature of 4℃and the supernatant was filter sterilized by a 0.45 μm filter. Ammonium sulfate was added to 80% saturation and the ice water bath conditions were stirred slowly until the ammonium sulfate was completely dissolved. Separating precipitate by centrifugation at 4deg.C and 10000 Xg for 20min, washing centrifuge tube with 50mL sterile 0.01M PBS buffer solution, mixing, measuring protease activity and protein content, dialyzing with 8000 dialysis bag at 4deg.C for 48 hr, changing dialysate every 6 hr, and collecting the precipitate with BaCl solution 2 Solution detection of SO4 2- Whether or not to goFreeze drying protease solution after dialysis without precipitation in ion water, and storing at-20deg.C for use.
Ion exchange chromatography: the protein lyophilized powder was completely dissolved in 10mM PBS solution at pH7.3 and then passed through an ion exchange chromatography column DEAE-Sepharose F.F (1.5X12 cm). The equilibration buffer contained 4mM CaCl 2 And 50. Mu.M ZnCl 2 Is sterilized by passing through a 0.22 μm filter, washing the ion exchange column with 100mL of equilibration buffer to remove unadsorbed protein, eluting with a linear gradient of NaCl (0-0.8M) eluent to obtain protease at a flow rate of 12mL/h, dispensing 3mL of the protease solution per tube, and detecting the concentration of the protease solution at OD 280nm The absorbance value of (2) is calculated by taking the protease activity in the solution before ion exchange chromatography as a control, the relative enzyme activity of the protease is calculated, and the components with activity after detection are freeze-dried and stored at-20 ℃ for standby.
Gel filtration chromatography: redissolving the initially purified protease powder in 10mM PBS solution with pH of 7.3, passing through Sephadex G-100 (1.6X10 cm) gel filtration chromatographic column, performing chromatographic separation at a flow rate of 0.3mL/min, balancing buffer solution flow rate of 11mL/h, collecting 3mL per tube, and detecting protease solution at OD 280nm The relative enzyme activity of the protease is calculated by taking the protease activity in the solution before gel filtration chromatography as a control, and the part with the enzyme activity is freeze-dried and stored at the temperature of minus 20 ℃ for standby.
SDS-PAGE electrophoresis method for identifying the purity and molecular weight of the enzyme: taking out the refrigerated sample, redissolving in 10mM PBS solution with pH of 7.3, uniformly mixing the sample with the protein electrophoresis loading buffer solution, heating in a boiling water bath for 5min, centrifuging at 12000r/min for 5min, and loading. The loading amount of protease is 20 mu L, the concentration of separation gel is 12%, the concentration of concentrated gel is 5%, the initial voltage is 80V, and after bromophenol blue indication line enters the separation gel, the voltage is adjusted to 120V. After the electrophoresis, the sample was stained in coomassie brilliant blue R250 staining solution for 2 hours, and then decolorized on a shaker with a decolorizing solution (methanol: acetic acid: water=4:1:5) until the background was clear, and photographed using an Image Quant TL gel imaging system.
Enzymolysis and identification of protease: to the protease powder was added 100mM Tris-HCl solution (pH 8.0), and vortexed to dissolve the protein sufficiently. 12000 Xg was centrifuged for 15min, the supernatant was taken, dithiothreitol (DTT) was added to a final concentration of 10mM, and incubated at 37℃for 1h for reduction to open disulfide bonds, and then Iodoacetamide (IAA) was added to a final concentration of 40mM, and alkylation reaction was performed at room temperature in the dark to block thiol groups. Adding appropriate amount of 100mM Tris-HCl solution (pH 8.0), adding trypsin, incubating at 37deg.C for 24 hr, adding trifluoroacetic acid to stop digestion, adjusting pH to 6.0, centrifuging at 12000 Xg for 15min, and desalting with C18 column. And pumping the desalted peptide solution by a concentrator, freezing at the temperature of-20 ℃ and waiting for detection by an on-machine.
Mass spectrometry: the peptide fragment sample was inhaled by an autosampler and bound to a C18 capture column followed by elution to an analytical column for separation. The flow rate of the liquid phase was set at 300mL/min. In mass spectrum IDA mode analysis, each scan cycle comprises a full MS scan (m/z range 350-1500, ion accumulation time 250 MS) followed by 40 MS/MS scans (m/z range 100-1500, ion accumulation time 50 MS). The MS/MS acquisition condition is that the parent ion signal is more than 120cps, and the charge number is +2- +5. The exclusion time for ion repeat collection was set to 18s.
Results: a DEAE Sepharose F.F ion exchange chromatography as shown in FIG. 6 and a Sephadex G-100 gel filtration chromatography as shown in FIG. 7 were obtained. As can be seen from FIGS. 6, 7, 8, table 1 and 2, 2.74mg of Protease was obtained by purification and identification, the purification factor was 78.81 and the Protease yield was 11.63%, and the identification showed that the Protease was named Protease HhoA, had a molecular weight of 43.907kDa and had serine endopeptidase activity.
TABLE 1 purification results of protease produced by Bacillus cereus C58 strain
Table 2 table of mass spectrometry results for proteases produced by bacillus cereus strain C58
And a third specific embodiment:
the protease secreted by the bacillus cereus C58 strain in the embodiment is obtained through purification and identification.
The enzymatic properties of the protease secreted by the bacillus cereus C58 strain of this embodiment were studied, as follows:
effect of pH on protease activity produced by C58 strain: protease activity was measured by incubating 1h of protease solubilized in buffers at pH 5, 6, 7, 8, 9, and 10, the buffers at different pH were sodium acetate buffer (pH 4.0-5.0), potassium phosphate buffer (pH 6.0), tris-HCl buffer (pH 7.0-9.0), and glycine-NaOH buffer (pH 10.0), and relative enzyme activity was calculated by taking protease activity at pH8 as a control.
Effect of temperature on protease activity produced by C58 strain: the optimal temperature is determined by placing the protease solution in a reaction system with different temperatures of 10-70 ℃ and incubating for 1h to determine the protease activity, and the relative enzyme activity is calculated by taking the protease activity of the protease at 40 ℃ as a control.
Influence of metal ions on the protease activity produced by the C58 strain: protease was added to MgCl containing 5mM and 10mM 2 、CaCl 2 、FeSO 4 、CuCl 2 The protease activity was measured by incubating the sample in 50mM PBS (pH 7.3) buffer at 2℃for 30 minutes, and the relative enzyme activity of the protease was calculated by using the protease activity of the protease in 50mM PBS (pH 7.3) without adding metal ions as a control.
Effect of protease inhibitors on protease activity produced by C58 strain: the protease was added to 50mM PBS (pH 7.3) solution containing 5mM and 10mM phenylmethylsulfonyl fluoride (PMSF), dithiobisnitrobenzoic acid (DTNB), ethylenediamine tetraacetic acid (EDTA) and mercaptoethanol (DTT), respectively, and the relative enzyme activities were calculated by taking the protease activities of the reaction system without protease inhibitors as a control, under incubation conditions of 20℃for 30 minutes.
Kinetics of thermal inactivation of protease produced by strain C58: taking 5mL protease solution in a test tube, respectively performing heat treatment in oil bath at constant temperature of 110deg.C and 120deg.C and 130deg.C for 2min, taking samples every 20s, cooling in the ice water bath, measuring protease activity, calculating the percentage of residual enzyme activity after heat treatment, adopting a second formula if the heat inactivation of protease accords with a first order dynamics model, and calculating t 1/2 Values and D values.
lnC t ＝-kt+lnC 0 (2)
Wherein RA is residual enzyme activity; ct is the enzyme activity of the protease solution at the t second; c (C) 0 Is the initial enzyme activity of the protease solution; k is the thermal deactivation rate constant.
Results: the change of the effect of pH on the protease activity produced by Bacillus cereus C58 strain as shown in FIG. 9 was obtained, and it was found from FIG. 9 that the protease showed an upward trend between pH4 and 9, and that the protease showed a downward trend in enzyme activity when pH was greater than 9, and that the relative enzyme activity of the protease increased significantly to (105.30.+ -. 4.10)% (p < 0.05) when pH was 9, with the protease activity at pH8 as a control. Thus, the protease is an alkaline protease.
The change of the effect of the temperature on the protease activity produced by the Bacillus cereus C58 strain shown in FIG. 10 was obtained, and it was found from FIG. 10 that the relative enzyme activity of the protease was in an ascending trend between 10 and 60℃and in a descending trend at a temperature of more than 60℃with the protease activity at 40℃as a control, and calpain had the highest relative enzyme activity (126.56.+ -. 0.58%) at 60 ℃. At 10℃the protease still had a relative enzyme activity (41.25.+ -. 6.32%), which indicated that the protease was still able to catalyze the chemical reaction at low temperatures, whereas at 80℃the relative enzyme activity of the protease was reduced to (75.63.+ -. 0.98%), which also indicated that the protease had thermostability.
A histogram showing the effect of metal ions on the protease activity produced by Bacillus cereus C58 strain as shown in FIG. 11 was obtained, and it can be seen from FIG. 11 that Mg was present at metal ion concentrations of 2mM and 5mM, respectively 2+ Has no obvious influence on the activity of protease, ca 2+ And Fe (Fe) 2+ The enzyme activity of the protease is obviously improved, and the relative enzyme activities of the protease are 111.11 percent and 105.21 percent, 126.35 percent and 119.52 percent respectively. And Cu is 2+ The relative enzyme activity of the protease is obviously inhibited, and is obviously reduced to 92.06 percent and 85.71 percent respectively.
A bar graph of the effect of the enzyme inhibitor on the protease activity produced by bacillus cereus C58 strain as shown in fig. 12 was obtained, and it can be seen from fig. 12 that DTNB had no significant inhibitory effect on the protease activity, whereas DTT, EDTA and PMSF significantly inhibited the protease activity with increasing concentration. When the concentration of the inhibitor was 2mM and 5mM, the relative enzyme activities of the protease solution to which DTT was added was 35.26% and 0, respectively, the relative enzyme activities of the protease solution to which EDTA was added was 82.54% and 42.86%, respectively, and the relative enzyme activities of the protease solution to which PMSF was added was 6.34% and 0, respectively. DTNB is a thiol reactant and DTT is able to break disulfide bonds, so the protease contains disulfide bonds. In addition, because PMSF acts on serine protease, EDTA is a metal ion chelating agent, and can chelate metal ion cofactor of enzyme active center, the protease is serine protease containing metal ion and disulfide bond.
The thermodynamic parameters of the protease produced by the Bacillus cereus C58 strain shown in FIG. 13, the thermal inactivation kinetics curves of the protease produced by the Bacillus cereus C58 strain shown in FIG. 14, and the protease produced by the Bacillus cereus C58 strain shown in Table 3 were obtained, and it was found that the thermodynamic parameters were calculated at 110% in FIGS. 13, 14 and 3The D value of the catalyst at 130 ℃ heat treatment temperature is 195.13-677.23s, t 1/2 58.74 to 203.87s.
TABLE 3 thermodynamic parameters of proteases produced by Bacillus cereus strain C58
The specific embodiment IV is as follows:
the protease secreted by the bacillus cereus C58 strain in the embodiment is obtained through purification and identification.
The hydrolysis of liquid milk proteins by the protease secreted by bacillus cereus strain C58 of the present embodiment was studied as follows:
the protease solid powder is added into liquid milk to make the protease concentration in the liquid milk be 0.1mg/mL, and sodium azide is added at the ratio of 0.02% to prevent bacteria from breeding. Samples stored at 28℃were sampled every 4h and samples stored at 10℃were sampled every 24h, and the hydrolysis of UHT milk proteins by proteases was analysed by SDS-PAGE after the end of the storage period.
Results: a liquid milk sample graph of the protease produced by the Bacillus cereus C58 strain shown in FIG. 15, a liquid milk sample graph of the protease produced by the Bacillus cereus C58 strain shown in FIG. 16, a liquid milk sample graph of the protease produced by the Bacillus cereus C58 strain at 10℃shown in FIG. 17, a liquid milk hydrolysis graph of the protease produced by the Bacillus cereus C58 strain at 28℃shown in FIG. 17, and a liquid milk hydrolysis graph of the protease produced by the Bacillus cereus C58 strain at 10℃shown in FIG. 18 were obtained, and it was found that a protein precipitation phenomenon was observed from FIGS. 15 and 16 at 16h at 28℃and that a clear whey precipitation was observed at 24h, and a small amount of whey precipitation phenomenon was observed at 6d at 10 ℃. From FIGS. 17 and 18, it can be seen that the protease produced by Bacillus cereus C58 strain first hydrolyzes kappa-CN in liquid milk, the kappa-CN is partially hydrolyzed when hydrolyzed at 28℃for 8 hours, almost completely hydrolyzed when hydrolyzed for 12 hours, while the band of beta-CN becomes gradually weaker, and at 20 hours, beta-CN and alpha S CN is extensively hydrolyzed. When this occurs at 10℃and at 3d, a progressive hydrolysis of kappa-CN by the protease, and beta-CN and alpha at 5d and 6d can be observed S -CN, whereas proteases have almost no hydrolysis on β -LG and α -LA. The hydrolysis sequence of casein by protease produced by bacillus cereus C58 strain is kappa-CN>β-CN>α S -CN。
This means that even if the bacillus cereus cells die, the protease will be active and once the thermostable bacillus cereus and its protease are present in the liquid milk, it will be difficult to ensure quality safety during the shelf life of the liquid milk.
Claims (6)
1. A strain producing a high temperature protease, characterized in that the strain is Bacillus cereus C58.
2. A process for preparing high-temp. resistant proteinase, which is characterized by that the strain of claim 1 is inoculated in primary LB seed culture medium, shake-cultured at 37 deg.C for 12 hr, the rotation speed is 2 Xg, then inoculated in 50mL secondary LB seed culture medium according to 2% inoculum size, and the bacterial suspension after culturing for 12 hr is diluted to 10 6 CFU/mL, inoculating to protease and lipase agar culture medium, culturing at 37deg.C for 24 hr to obtain bacterial suspension containing protease, centrifuging to obtain supernatant, and purifying to obtain the high temperature resistant protease.
3. The preparation method of claim 2, wherein the composition of the LB seed medium comprises 8% of skim milk powder, 1.5% of agar powder and the balance of water in percentage by weight.
4. A method of preparation according to claim 3, wherein the purification steps are ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography in that order.
5. The method of claim 2, wherein the thermostable protease is a disulfide-containing metal-dependent alkaline serine protease.
6. The method according to claim 2, wherein the protease has an optimum pH of 9 and an optimum temperature of 60 ℃.
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