Bacillus amyloliquefaciens and application thereof in improving lipase activity

Abstract

The invention provides bacillus amyloliquefaciens and application thereof in improving lipase activity, and belongs to the technical field of microorganisms, the bacillus amyloliquefaciens is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20232293. Fermentation liquid of the bacillus amyloliquefaciens is centrifuged, fermentation supernate is taken, the fermentation supernate is subjected to high-temperature inactivation and then is mixed with lipase, the activity of the lipase can be improved, and therefore the fermentation supernate can be used for preparing the lipase synergist. The method disclosed by the invention is low in cost and high in safety, can obviously enhance the activity of lipase, is particularly suitable for preparing health-care products and medicines for helping people with gastrointestinal discomfort to better digest, and has a good market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Bacillus amyloliquefaciens and application thereof in improving lipase activity. Background Art

[0002] Lipase, or triacylglycerol acyl hydrolase, is an ester bond hydrolase that is widely present in nature, especially in various tissues of plants and animals and a variety of microorganisms. Lipase can catalyze triglycerides to form diglycerides, monoglycerides or glycerol and free fatty acids at the oil-water interface. Lipase has multiple catalytic abilities. In addition to catalyzing hydrolysis and esterification reactions, it can also catalyze a variety of chemical reactions such as transesterification, transesterification, and acidolysis. Due to the unique properties of lipase, such as high catalytic efficiency, simple reaction conditions, few by-products, and no need for auxiliary factors, lipase is widely used in industrial production such as food, medicine, biofuels, diagnosis, bioremediation, chemicals, and cosmetics, becoming one of the three major industrial enzymes after proteases and amylases. As people's awareness of health, eating habits, and the upcoming high-tech food and beverage industry continues to increase, the global lipase market will grow at a faster rate. For example, cheap, low-nutritional oils and fats can be upgraded to expensive, high-nutritional oils and fats through lipase-catalyzed modification.

[0003] Lipase has shown attractive application prospects in many fields. However, the "natural" activity of this catalyst cannot meet the requirements of its catalytic reaction in industry in most cases. Therefore, improving the activity of lipase to enhance the market competitiveness of products has always been one of the focuses of lipase research. The reported methods include using protein engineering technology to improve the activity of lipase; using some chemical reagents to react with -NH2, -OH or -COOH groups on the surface of lipase, covalently binding these chemical reagents to the side chain of lipase molecules, changing the side chain structure, and thus improving the activity of lipase. For example, Ding Hui et al. modified the lipase from Candida rugosa with synthetic glutamic acid dodecyl ester ribitol and used it to hydrolyze olive oil in oil / water two-phase system. In this reaction system, the maximum activity of the surfactant-modified enzyme reached 48.8mmol / min / g, which is 1.8 times the activity of the natural enzyme. However, there are currently no reports on microbial lipase enhancers at home and abroad.

[0004] Lipase in animals is mainly found in the stomach and pancreatic tissues, such as gastric lipase and pancreatic lipase in humans, which can mainly help the human body digest and absorb oils. Lipase in plants is mainly found in plant seeds. The lipase content in oil-bearing crop seeds is relatively high, and its main function is to catalyze the hydrolysis of oil components in seeds to provide material basis and energy for seed development. Compared with the previous animal lipase and plant lipase, microbial lipase has better reaction conditions, such as a wide range of temperature and pH applications, higher substrate specificity, and higher stability. At the same time, it is now found that there are a wide range of microorganisms that can secrete lipase, and microorganisms have the characteristics of fast reproduction, short growth cycle, convenient mass cultivation, and good control of growth conditions, which are suitable for modern industrial production. Therefore, today, microbial lipase has replaced animal lipase and plant lipase and become the main source of industrial lipase. The present invention isolates a strain of Bacillus amyloliquefaciens X7 that produces a lipase enhancer from high-sugar persimmon enzymes. The supernatant of the strain is incubated with pancreatic lipase and microbial lipase for 5 minutes, which can significantly improve the activities of the two lipases from animals and microorganisms, respectively. In addition, experiments show that heat treatment at 100°C for 20 minutes has little effect on the activity of the lipase enhancer produced by Bacillus amyloliquefaciens X7, which is also an obvious advantage for the industrial application of the lipase enhancer. Summary of the invention

[0005] The invention firstly discovers a strain of Bacillus amyloliquefaciens X7 (Bacillus amyloliquefaciens X7) CCTCC NO: M 20232293, and the fermentation supernatant after inactivation can significantly enhance the activity of lipase. The method is safer and more reliable than the method of promoting gastrointestinal digestion by exogenous intake of lipase, and is beneficial to human health.

[0006] Persimmon enzyme contains a variety of biologically active ingredients. The inventors found that some persimmon enzyme supernatants prepared in the laboratory have effective ingredients that improve the activity of porcine pancreatic lipase. In order to obtain the key strains, the inventors inoculated the persimmon enzyme samples into high-sugar YEPD medium for cultivation. In each separation and purification process, the improvement of porcine pancreatic lipase activity was used as an indicator, so that the key strain X7 in persimmon enzyme was gradually separated and purified from the flora. The colony of strain X7 is light yellow, with ridges on the surface and irregular edges. Under a microscope, strain X7 is shaped like a curved rod, Gram-positive, and evenly colored. Endogenous spores can be formed, which are oval, blunt at both ends, and the spore capsules do not swell.

[0007] The research team deposited it in the China Center for Type Culture Collection on November 21, 2023. The preservation number of Bacillus amyloliquefaciens X7 is: CCTCC No: M 20232293, Latin name: Bacillus amyloliquefaciens X7, address: Wuhan University, Wuhan, China, postal code: 430072, telephone: 027-68754052.

[0008] The Bacillus amyloliquefaciens is inoculated into a liquid culture medium containing a carbon source and a nitrogen source, and after shaking culture, the solid and liquid are separated, and the supernatant is taken to obtain the fermentation supernatant of the Bacillus amyloliquefaciens.

[0009] The fermentation supernatant of Bacillus amyloliquefaciens is used to improve the activity of lipase.

[0010] The fermentation supernatant of Bacillus amyloliquefaciens is used for preparing a lipase enhancer.

[0011] Wherein, the lipase includes porcine pancreatic lipase and microbial lipase.

[0012] The present invention also provides a method for improving the activity of lipase, specifically, mixing the fermentation supernatant of the preserved Bacillus amyloliquefaciens X7 with lipase.

[0013] Wherein, the mixing time of the fermentation supernatant of Bacillus amyloliquefaciens and lipase is 0 to 10 minutes.

[0014] The method for preparing the fermentation supernatant of Bacillus amyloliquefaciens is as follows: inoculating Bacillus amyloliquefaciens into a liquid culture medium containing a carbon source and a nitrogen source, shaking and culturing, separating the solid and the liquid, and taking the supernatant to obtain the fermentation supernatant of Bacillus amyloliquefaciens.

[0015] Wherein, the solid-liquid separation method is: centrifugation at 2-6°C and 5000-15000 r / min for 1-15 min.

[0016] Beneficial Effects

[0017] There are many reports on using live probiotics to feed animals to improve the activity of digestive enzymes in their intestines. The mechanism is that these live probiotics colonize, grow and multiply after entering the intestines, producing a variety of extracellular digestive enzymes that enter the "enzyme pool" of the intestines, thereby improving the activity of animal digestive enzymes. So far, there are no reports at home and abroad on using extracellular metabolites of microorganisms to directly improve the activity of digestive enzymes.

[0018] The present invention uses the isolated metabolites of Bacillus amyloliquefaciens to enhance the activity of lipase, or in other words, the metabolites of Bacillus amyloliquefaciens are lipase synergists, and the synergist has strong thermal stability. Experiments show that the synergist can withstand heat treatment at 121° C., but its synergistic effect is reduced. The specific substance produced and the mechanism of synergistic effect with lipase need further in-depth research.

[0019] The present invention utilizes a strain of Bacillus amyloliquefaciens, and the fermentation supernatant of the strain can significantly increase the activity of lipase. However, due to too many substances in the fermentation broth, it is temporarily impossible to compare what kind of substance or combination of substances it is, and further in-depth research is needed. As for the Bacillus amyloliquefaciens strain of the present invention, its method has stable effect, good high temperature resistance and high safety. This method is more beneficial to human health than the method of promoting gastrointestinal digestion by exogenous intake of lipase.

[0020] The present invention activates lipase through the metabolites of microorganisms, has a simple preparation method, and the metabolites are highly stable, easy to preserve, and have certain thermal stability. Therefore, it is expected that it can be applied in a wide range of fields in the future. In addition to being used for producing digestion-promoting health products and high-activity pancreatic enzymes, it can also be used for producing meat products, beverages and other foods with digestion-promoting functions, and animal feed that can improve breeding efficiency. The present invention has low cost for enhancing lipase activity and good market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The colony morphology and microscopic morphology photos of Bacillus amyloliquefaciens of the present invention are shown.

[0022] Figure 2 The phylogenetic tree of the deposited strains of the present invention based on ITS sequence homology.

[0023] Figure 3 These are the growth curve of Bacillus amyloliquefaciens X7 and the fermentation curve of lipase enhancer.

[0024] Figure 4 The effect of heat treatment on the activity of Bacillus amyloliquefaciens X7 lipase enhancer. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to examples. All methods and techniques are conventional methods and techniques.

[0026] The porcine pancreatic lipase used in the present invention is from Nanjing Dulai Biotechnology Co., Ltd., the commercial microbial lipase is from Hefei BASF Biotechnology Co., Ltd., and the lipase is from Aspergillus niger. Staphylococcus xylosus M10 and M11 strains, Lactobacillus plantarum R2 strain, and Lactobacillus pentosus R5 strain are all isolated from commercial low-temperature meat products.

[0027] The method for determining pancreatic lipase activity in the present invention is as follows:

[0028] (1) Establishment of standard curve

[0029] Weigh 0.07 g of PNP and dissolve it in phosphate buffer (0.20 mol / L, pH 7.4) and dilute to 100 mL to prepare a mother solution with a concentration of 5 mmol / L. Pipette 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 mL into a 10 mL stoppered colorimetric tube, respectively, and add phosphate buffer to dilute to 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, and 0.07 mmol / L PNP solution. Measure the absorbance at a wavelength of 405 nm, and draw a standard curve with PNP concentration as the abscissa and the corresponding absorbance as the ordinate to obtain the correlation coefficient and regression equation Y=10.69X-0.0139(R 2 =0.9991).

[0030] (2) Establishment of enzyme activity determination method

[0031] Weigh 0.10g pancreatic lipase, dilute to 50mL with phosphate buffer, centrifuge at 8000r / min for 10min, take the supernatant, and obtain 2mg / mL pancreatic lipase stock solution; take 5mL of the test bacterial solution, centrifuge at 8000r / min for 5min, take 2mL of the supernatant and inactivate in boiling water for 20min, cool and set aside. Take 131.09μL PNPP and dissolve it in 0.25mL N,N-dimethylformamide, dilute to 100mL with phosphate buffer, add 25 drops of Triton X-100, and obtain 11.2mmol / L PNPP solution (substrate solution).

[0032] Experimental group: Take 2mL of pancreatic lipase solution in a 10mL stoppered colorimetric tube, add 2mL of the inactivated bacterial solution supernatant, react at 37℃ for 5min, and obtain a mixture of bacterial solution and pancreatic lipase solution. Add 4mL of phosphate buffer, keep at 37℃ for 10min, add 2mL of substrate solution, react in a water bath at 37℃ in the dark for 20min, and measure the absorbance at 405nm. Substitute into the PNP standard curve to obtain the PNP concentration produced by the pancreatic lipase-catalyzed substrate.

[0033] Control group: Take 2 mL of pancreatic lipase solution in a 10 mL stoppered colorimetric tube, add 2 mL of phosphate buffer and react at 37°C for 5 min, add 4 mL of phosphate buffer and keep warm at 37°C for 10 min, then add 2 mL of substrate solution, react in a water bath at 37°C in the dark for 20 min, and measure the absorbance at a wavelength of 405 nm.

[0034] The calculation formula of pancreatic lipase activity is as follows: the mixture is incubated at 37°C for 10 minutes, and the activity of pancreatic lipase is defined as 1 μmol PNP produced by catalyzing the substrate per minute, unit (U).

[0035]

[0036] Wherein, n is the dilution factor, V is the volume of the sample solution to be tested / mL, c is the PNP concentration calculated by substituting into the PNP standard curve / (mmol / L), t is the reaction time / min, and 0.004 is the mass of pancreatic lipase in the reaction system / g.

[0037] Microsoft Excel software was used to organize the data, and SPSS22.0 software was used for statistical analysis. The experimental data were expressed as mean ± standard deviation (`x±s), and P<0.05 indicated a significant difference.

[0038] Example 1

[0039] This embodiment provides a Bacillus amyloliquefaciens isolated from a high-sugar persimmon enzyme stock solution prepared in a laboratory.

[0040] Persimmon is a temperate fruit, rich in vitamin C, pectin, carotenoids, etc., and can be used to prepare enzymes. It is reported that persimmon enzyme has a high antioxidant capacity, thus having the effect of anti-aging and enhancing human resistance.

[0041] Persimmon enzyme contains a variety of biologically active ingredients. The inventors found that some persimmon enzyme supernatants prepared in the laboratory contain effective ingredients that increase the activity of porcine pancreatic lipase. In order to obtain the key strains, the inventors cultured the persimmon enzyme samples in high-sugar YEPD culture medium. In each separation and purification process, the increase in porcine pancreatic lipase activity was used as an indicator, thereby gradually separating and purifying the key strain X7 in persimmon enzyme from the bacterial community.

[0042] The plate streak method was used to separate and purify the enzyme stock solution made in the laboratory. The single colonies on the plate were picked out one by one, inoculated into the YEPD liquid medium containing 8% NaCl, and cultured at 28°C to determine the multiple of the fermentation supernatant on the pancreatic lipase activity. The high-yield bacterial solution was selected and inoculated into the YEPD liquid medium containing 8% NaCl at a 2% inoculation amount for further culture. After the bacterial solution became turbid, an appropriate amount of the bacterial solution was taken for gradient dilution, and the dilution was spread on the YEPD plate and cultured at 28°C. The operation was repeated to finally obtain the strain X7 with high-yield lipase enhancer.

[0043] Figure 1 The colony morphology (left) and microscopic morphology (right) of strain X7 are shown in Figure 1. Figure 1It can be seen that the colony of strain X7 is light yellow, with ridges on the surface and irregular edges. Under the microscope, strain X7 is in the shape of a curved rod, Gram-positive, and evenly colored. Endospores can be formed, which are oval in shape, with blunt ends, and the spore capsule does not swell.

[0044] After the strain X7 was sent to a biological company for identification, its 16S rDNA gene sequence was obtained. It was compared with the gene sequences in the BLAST and databases in NCBI. The results showed that the 16S rDNA gene sequence of strain X7 was 99.65% similar to the 16S rDNA gene of the known Bacillus amyloliquefaciens in the database. The phylogenetic tree was constructed using MEGA11.0 software. The results showed that it was clustered with the strain NR117946.1-Bacillus amyloliquefaciens in the phylogenetic tree. The phylogenetic tree is as follows: Figure 2 Therefore, combined with the observation of morphological characteristics, strain X7 was determined to be Bacillus amyloliquefaciens.

[0045] The research team deposited it in the China Center for Type Culture Collection on November 21, 2023. The preservation number of Bacillus amyloliquefaciens X7 is: CCTCC No: M 20232293, Latin name: Bacillus amyloliquefaciens X7, address: Wuhan University, Wuhan, China, postal code: 430072, telephone: 027-68754052.

[0046] Example 2

[0047] This example provides the effect of the mixing and incubation time of the synergist and porcine pancreatic lipase on the synergistic effect of the synergist. According to literature reports, the incubation time of commonly used enzymes and effectors such as inhibitors is generally 5-10 minutes, so the mixing and incubation time of the fermentation supernatant of Bacillus amyloliquefaciens X7 and porcine pancreatic lipase was set to 5 minutes in the aforementioned experiment. If the lipase synergist produced by Bacillus amyloliquefaciens X7 can immediately play a synergistic role after contacting the lipase, it will be beneficial to its subsequent application. Therefore, this study determined the synergistic multiples when the mixing and incubation time of the fermentation supernatant of Bacillus amyloliquefaciens X7 and porcine pancreatic lipase was 0, 5, and 10 minutes at a temperature of 37°C.

[0048] The experimental method is as follows: the Bacillus amyloliquefaciens X7 strain preserved in Example 1 is inoculated into a liquid culture medium, the culture medium formula is 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume is 50 mL / 100 mL, and the culture is carried out at a constant temperature of 30° C. and 150 rpm until the turbidity (OD600) is 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution. The seed liquid of Bacillus amyloliquefaciens X7 was inoculated into a liquid culture medium at a volume ratio of 5%, the culture medium formula was 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract powder, 20 g / L NaCl, the liquid volume was 50 mL / 100 mL, and the culture was shaken at 30° C. and 150 rpm for 6 h. After the culture of Bacillus amyloliquefaciens was completed, the fermentation liquid of Bacillus amyloliquefaciens was taken, centrifuged at 4° C. and 8000 r / min for 5 min, and the supernatant was taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7.

[0049] The experiment showed that when the mixed incubation time of the fermentation supernatant of Bacillus amyloliquefaciens X7 and porcine pancreatic lipase was 0, 5, and 10 minutes, the enzymatic activity of porcine pancreatic lipase was increased by 0.99±0.03 times, 1.0±0.03 times, and 1.0±0.01 times, respectively. There was no significant difference among the three, and the mixed incubation temperature could achieve synergism at the temperature at which porcine pancreatic lipase was active. It can be seen that the lipase enhancer produced by Bacillus amyloliquefaciens X7 has strong activity and can immediately increase the enzymatic activity of lipase without mixed incubation with lipase.

[0050] Example 3

[0051] The above experiments show that the fermentation supernatant of Bacillus amyloliquefaciens X7 can improve the activity of porcine pancreatic lipase. Since the main source of industrial lipase is microorganisms, this example studies the synergistic effect of the supernatant of Bacillus amyloliquefaciens X7 on microbial lipase. The specific method is as follows.

[0052] (1) Establishment of standard curve for determination of microbial lipase activity

[0053] Prepare 100 mL of 6 mmol / L p-nitrophenol solution by first dissolving it with a small amount of 95% ethanol and then making up to volume with water. The substrate buffer is 50 mmol / L Tris-HCl (pH=8.0).

[0054] Table 1 Drawing of standard curve for determination of microbial lipase activity

[0055]

[0056] Each reagent was added according to Table 1, mixed in a test tube, and the absorbance value was measured at 410 nm. The measurement results were plotted into a standard curve to obtain the relevant linear equation Y = 45.516X-1.1323, R2 =0.9993 (X: absorbance value, Y: P-NP concentration μmol / L).

[0057] (2) Synergistic effect of Bacillus amyloliquefaciens X7 on microbial lipase

[0058] Preparation of microbial lipase solution: Commercially available microbial lipase was prepared into microbial lipase solution at 40 mg / mL, and the enzyme activity was determined.

[0059] Solution A: 10 mol / L 4-nitrophenyl palmitate

[0060] Solution B: 50mmol / L Tris-HCl (pH=8.0) buffer solution

[0061] Solution C: Mix solution A and solution B evenly in a volume ratio of 1:9, add 1% Triton X-100 and 0.1% gum arabic powder, and prepare it before use.

[0062] Enzyme activity assay group: Take two test tubes, a control tube and a sample tube, with a total reaction system of 2 mL. Add 0.9 mL of solution C (keep warm at 37°C for 5 min) to each test tube, add 0.1 mL of enzyme solution to the sample tube, and add 0.1 mL of enzyme solution inactivated in a 100°C water bath to the control tube. Mix immediately and start timing. React in a 37°C constant temperature water bath for 10 min. Immediately add 95% ethanol to terminate the reaction, and then measure the absorbance at 410 nm.

[0063] Synergistic experimental group: Take two test tubes, one for the control tube and one for the sample tube, and the total reaction system is 2mL. Add 0.9mL of C solution (37℃ constant temperature for 5min) to each test tube, add 0.05mL of enzyme solution and 0.05mL of fermentation supernatant of strain X7 inactivated in a 100℃ water bath for 20min to the sample tube, and add 0.05mL of enzyme solution inactivated in a 100℃ water bath for 20min and 0.05mL of fermentation supernatant of strain X7 inactivated in a 100℃ water bath for 20min to the control tube. Mix well immediately and start timing, react in a 37℃ constant temperature water bath for 10min, immediately add 95% ethanol to terminate the reaction, and then measure the absorbance value at 410nm.

[0064] Enzyme activity calculation formula: A = ([A1-A0]*K+C0)*V1*n / (V2*t)

[0065] In the formula, A is the sample enzyme activity / (U / mL), A1 is the absorbance OD value of the sample enzyme solution, A0 is the blank absorbance value of the corresponding enzyme solution, K is the slope, C0 is the intercept, n is the dilution factor, V l is the volume of the reaction solution / mL, V2 is the volume of the enzyme solution / mL, and t is the reaction time / min.

[0066] Example 4

[0067] This embodiment provides a method for increasing the activity of commercially available Aspergillus niger lipase, and the specific method is as follows.

[0068] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was carried out at a constant temperature of 30° C. and 150 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0069] Step 2: Take the seed liquid of Bacillus amyloliquefaciens X7 and inoculate it into the liquid culture medium at an inoculum volume ratio of 1%. The culture medium formula is 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract powder, 20 g / L NaCl, and the liquid volume is 50 mL / 100 mL. Shake and culture at a constant temperature of 35°C and 50 rpm for 12 hours.

[0070] Step 3: Prepare a microbial lipase solution with a commercially available microbial lipase concentration of 40 mg / mL, and perform enzyme activity determination.

[0071] Step 4: After the cultivation of the Bacillus amyloliquefaciens obtained in step 2 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 2°C and 5000r / min for 15min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the microbial lipase solution obtained in step 2 at a volume ratio of 1:3, and the activity of the lipase is determined.

[0072] Experiments show that the fermentation supernatant of Bacillus amyloliquefaciens X7 has a synergistic effect on commercially available microbial lipase, which can increase the enzyme activity by 0.86 times.

[0073] Example 5

[0074] This embodiment provides a method for increasing the activity of commercially available Aspergillus niger lipase, and the specific method is as follows.

[0075] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was carried out at a constant temperature of 30° C. and 150 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0076] Step 2: Take the seed liquid of Bacillus amyloliquefaciens X7 and inoculate it into the liquid culture medium at an inoculum volume ratio of 10%. The culture medium formula is 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract powder, 20 g / L NaCl, and the liquid volume is 50 mL / 100 mL. Shake and culture at a constant temperature of 25°C and 200 rpm for 16 hours.

[0077] Step 3: Prepare a microbial lipase solution with a commercially available microbial lipase concentration of 40 mg / mL, and perform enzyme activity determination.

[0078] Step 4: After the cultivation of the Bacillus amyloliquefaciens obtained in step 2 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 6°C and 15000r / min for 1min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the microbial lipase solution obtained in step 2 at a volume ratio of 3:1, and the activity of the lipase is measured.

[0079] Experiments show that the fermentation supernatant of Bacillus amyloliquefaciens X7 has a synergistic effect on commercially available microbial lipase, which can increase the enzyme activity by 0.79 times.

[0080] Example 6

[0081] This embodiment provides a method for improving the lipase activity of Staphylococcus xylosus M10, and the specific method is as follows.

[0082] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was carried out at a constant temperature of 30° C. and 150 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0083] Step 2: Take the seed liquid of Bacillus amyloliquefaciens X7 and inoculate it into the liquid culture medium at a mass volume ratio of 5%. The culture medium formula is 20g / L peptone, 20g / L glucose, 10g / L yeast extract powder, 20g / L NaCl, the liquid volume is 50mL / 100mL, and the culture is shaken at 30°C and 150rpm for 6h.

[0084] Step 3: Preparation of crude enzyme solution of Staphylococcus xylosus M10 lipase: The Staphylococcus xylosus M10 strain was inoculated into a liquid culture medium at a 4% inoculation rate. The culture medium formula was 10 g / L mannitol, 10 g / L peptone, 1 g / L beef extract, 75 g / L sodium chloride, 2.5 mL / L 1% phenol red reagent, pH 7.2-7.4, and the liquid volume was 50 mL / 100 mL. After static cultivation in a 37°C incubator for 18 hours, the fermentation broth was centrifuged at 4°C and 10,000 r / min for 5 minutes, and the supernatant was taken as the crude enzyme solution of M10 lipase for enzyme activity determination.

[0085] Step 4: After the cultivation of the Bacillus amyloliquefaciens obtained in step 2 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 4°C and 8000r / min for 5min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the crude M10 lipase solution obtained in step 2 at a volume ratio of 1:1, and the activity of the lipase is determined.

[0086] The experiment showed that the lipase activity in the supernatant of Staphylococcus xylosus M10 was 2.22U / mL, and the fermentation supernatant of Bacillus amyloliquefaciens X7 had a synergistic effect on the lipase of Staphylococcus xylosus M10, which could increase the enzyme activity by 1.17 times.

[0087] Example 7

[0088] Bacillus amyloliquefaciens is an aerobic bacterium. In order to explore the correlation between the growth of Bacillus amyloliquefaciens X7 and the production of synergists, the growth curve and synergist fermentation curve of strain X7 under oscillation conditions (150r / min) were studied in this experiment.

[0089] from Figure 3 It can be seen that the enhancer is produced synchronously with the growth of Bacillus amyloliquefaciens X7, so the lipase enhancer produced by Bacillus amyloliquefaciens X7 is a primary metabolite. However, the fermentation curves of porcine pancreatic lipase and Staphylococcus xylosus M10 lipase enhancers are not exactly the same. The production of porcine pancreatic lipase enhancer reaches a peak at 8h, and the activation of Staphylococcus xylosus M10 lipase reaches a peak at 6h, indicating that the porcine pancreatic lipase enhancer produced by Bacillus amyloliquefaciens X7 and the Staphylococcus xylosus M10 lipase enhancer may not be the same component. However, both lipase enhancers showed a downward trend after reaching the peak.

[0090] Example 8

[0091] This example studies the effect of heat treatment on the synergistic effect of Bacillus amyloliquefaciens X7 on lipase.

[0092] If the lipase enhancer produced by Bacillus amyloliquefaciens X7 is used in the industrial field, it is necessary to understand the heat resistance of the enhancer. If the heat resistance is strong, the processing technology can be simplified and the production cost can be reduced. Therefore, this experiment studied the effect of high temperature treatment on the activity of the enhancer in the fermentation supernatant of Bacillus amyloliquefaciens X7. Figure 4 As shown in the results, the fermentation supernatant of Bacillus amyloliquefaciens X7 without high temperature treatment can increase the activity of porcine pancreatic lipase and Staphylococcus xylosus M10 lipase by 1.03 times and 1.32 times, respectively, while the fermentation supernatant of strain X7 treated at 100℃ and 121℃ for 20 minutes can increase the activity of porcine pancreatic lipase by 1.03 times and 0.95 times, and Staphylococcus xylosus M10 lipase by 1.17 and 0.91 times, respectively. Therefore, high temperature treatment at 100℃ for 20 minutes has no effect on the activity of porcine pancreatic lipase enhancer, but it will significantly reduce the activity of Staphylococcus xylosus M10 lipase enhancer, with a decrease of 11.36%; treatment at 121℃ for 20 minutes will significantly reduce the activity of porcine pancreatic lipase and Staphylococcus xylosus M10 lipase enhancer, with a decrease of 7.77% and 31.10%, respectively. It can be seen that the lipase enhancer in the fermentation supernatant of Bacillus amyloliquefaciens X7 may be a mixed component, which can enhance the activities of porcine pancreatic lipase and Staphylococcus xylosus M10 lipase, respectively, among which the component that enhances the activity of porcine pancreatic lipase is more heat-resistant.

[0093] Example 9

[0094] This example provides a method for an acute oral toxicity test on mice of a Bacillus amyloliquefaciens X7 bacterial liquid and its concentrated fermentation supernatant. The specific method is as follows.

[0095] (1) Preparation of bacterial solution: Under sterile operation conditions, strain X7 was inoculated into YEPD liquid medium and activated for three generations. The bacterial solution was centrifuged and the supernatant was discarded. The bacterial slurry was washed at least three times with sterilized saline. The concentration of the bacterial solution was adjusted to 1×10 10 CFU / mL was used to prepare X7 bacterial solution for subsequent experiments.

[0096] (2) Preparation of fermentation supernatant of Bacillus amyloliquefaciens X7 with different concentration ratios: After centrifuging the fermentation broth of strain X7 (8000r / min, 5min, 4℃), the supernatant was taken and concentrated by rotary evaporation (120r / min, 60℃). The concentrate was transferred into a separatory funnel and extracted with ethyl acetate at a volume ratio of 1:1. After standing for the solution to separate, the fermentation supernatant and organic phase were taken out and stored separately. The fermentation supernatant was mixed with ethyl acetate at a volume ratio of 1:1 and continued to shake for 1min and stand for equal separation. After separation, the previous experimental operation was repeated until the organic phase became transparent and the extraction experiment was completed (about 3 to 4 times). After the extraction was completed, the obtained organic phase was evaporated to dryness by rotary evaporation (10r / min, 60℃), and a certain volume of pure water was added to re-dissolve to obtain fermentation supernatant of strain X7 with different concentration ratios.

[0097] (3) Mouse grouping: 40 healthy SPF Kunming mice, weighing 18-22 g, half male and half female. After 7 days of adaptive feeding, they were randomly divided into 4 groups according to body weight, with 10 mice in each group, namely the control group, 60-fold concentrated group, 100-fold concentrated group, and bacterial solution group.

[0098] The control group was gavaged with normal saline, and the experimental group was gavaged with the fermentation liquid of the strain (the concentration of the bacterial liquid was 10 10 CFU / mL) and the treated supernatant.

[0099] The mice were fasted for 16 hours before the experimental treatment. They were kept for 0.5 hours on the day of gavage for continuous observation. The weight changes of the mice were recorded after gavage, and the observation was continued for 14 days. The clinical signs of the mice were observed once in the morning and afternoon every day, and the abnormal conditions were recorded in detail. At the end of the experiment, orbital blood was collected from all surviving animals and hematological indicators were measured; finally, the animals were killed by cervical dislocation, and the tissues and organs of the animals were collected and observed.

[0100] After intragastric administration, the general state of the mice showed no obvious abnormalities. The mice grew well within 14 days, and there were no abnormalities in drinking water, eating, defecation, daily activities, etc. No obvious abnormalities were found in the normal state of the experimental mice and the various organ tissues, no obvious symptoms of poisoning were found, and no deaths were found. The organ indexes and weight growth of the mice are shown in Table 2.

[0101] Table 2 Organ index and final weight of experimental mice

[0102]

[0103] Note: Data in the same column with different lowercase letters indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences.

[0104] After 14 days of acute toxicity test, the mice were dissected and no visible changes were observed. The weight of the heart, liver, spleen, lung and kidney was weighed, and the organ index of the mice was calculated (as shown in Table 2). The results showed that the relative weight of each organ was not significantly different between the experimental groups and the control group after administration of the bacterial solution / concentrated supernatant of the strain.

[0105] The hematopoietic system is one of the most sensitive targets of toxic substances and is a good indicator of the physiological status of animals and humans. Changes in these indicators usually precede clinical symptoms. Therefore, the blood condition and the occurrence of diseases can be judged by observing changes in the number of blood cells, thereby reflecting the side effects of the test strain in the blood. The results of mouse hematological indicators are summarized in Table 3.

[0106] Table 3 Hematological parameters of experimental mice

[0107]

[0108] Note: Data in the same column with different lowercase letters indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences.

[0109] After 14 days of acute toxicity test, whole blood of experimental animals was collected, and the results are shown in Table 3. There was no statistically significant difference in PLT, RBC, WBC, HGB, MCV, and MCH between the experimental group and the control group. The above experimental results show that Bacillus amyloliquefaciens X7 bacterial liquid and its fermentation supernatant are safe and non-toxic.

[0110] Example 10

[0111] This embodiment provides a method for improving the lipase activity of Staphylococcus xylosus M11, and the specific method is as follows.

[0112] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was carried out at a constant temperature of 30° C. and 150 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0113] Step 2: Take the seed liquid of Bacillus amyloliquefaciens X7 and inoculate it into the liquid culture medium at an inoculum volume ratio of 5%. The culture medium formula is 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract powder, 20 g / L NaCl, the liquid volume is 50 mL / 100 mL, and the culture is shaken at a constant temperature of 30°C and 150 rpm for 6 hours.

[0114] Step 3: Preparation of crude enzyme solution of Staphylococcus xylosus M11 lipase: The Staphylococcus xylosus M11 strain was inoculated into a liquid culture medium at a 4% inoculation rate. The culture medium formula was 10 g / L mannitol, 10 g / L peptone, 1 g / L beef extract, 75 g / L NaCl, 2.5 mL / L 1% phenol red reagent, pH 7.2-7.4, and the liquid volume was 50 mL / 100 mL. After static culture in a 37°C incubator for 18 hours, the fermentation broth was centrifuged at 4°C and 10,000 r / min for 5 minutes, and the supernatant was taken as the crude enzyme solution of M11 lipase for enzyme activity determination.

[0115] Step 4: After the cultivation of the Bacillus amyloliquefaciens obtained in step 2 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 4°C and 8000r / min for 5min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant of Bacillus amyloliquefaciens X7 is mixed with the crude M11 lipase solution obtained in step 2 at a volume ratio of 1:1, and the activity of the lipase is determined.

[0116] The experiment showed that the lipase activity in the supernatant of Staphylococcus xylosus M11 was 1.68U / mL, and the fermentation supernatant of Bacillus amyloliquefaciens X7 had a synergistic effect on the lipase of Staphylococcus xylosus M11, which could increase the enzyme activity by 1.09 times.

[0117] Embodiment 11

[0118] This embodiment provides a method for improving the lipase activity of Lactobacillus plantarum R2, and the specific method is as follows.

[0119] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was cultured at a constant temperature of 30° C. and 100 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0120] Step 2: Take the seed liquid of Bacillus amyloliquefaciens X7 and inoculate it into the liquid culture medium at a volume ratio of 5%. The culture medium formula is 20% peptone, 20% glucose, 10% yeast extract powder, and 20g / L NaCl. The liquid volume is 50mL / 100mL. Shake and culture at a constant temperature of 30°C and 150rpm for 6h.

[0121] Step 3: the preparation of the crude enzyme liquid of plant lactobacillus R2 lipase: the plant lactobacillus R2 bacterial strain is accessed in the liquid nutrient medium with 2% inoculum size, culture medium recipe is tryptone 10g / L, beef extract 5g / L, yeast extract powder 4g / L, glucose 20g / L, sodium acetate (three waters) 5g / L, diammonium citrate 2g / L, tween 1mL, K2HPO4.7H2O 2g / L, MnSO4﹒4H2O 0.05g / L, calcium carbonate 20g / L, pH 6.2, liquid capacity is 50mL / 100mL, after leaving standstill to cultivate 14h in 37 ℃ of incubators, with fermented liquid at 4 ℃, centrifugal 5min of 10000r / min, getting supernatant is the crude enzyme liquid of plant lactobacillus R2 lipase, carries out the enzyme activity assay.

[0122] Step 4: After the cultivation of Bacillus amyloliquefaciens X7 obtained in step 1 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 4°C and 8000r / min for 5min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the crude enzyme liquid of Lactobacillus plantarum R2 obtained in step 2 at a volume ratio of 1:1, and the activity of lipase is determined.

[0123] The experiment showed that the lipase activity in the fermentation supernatant of Lactobacillus plantarum R2 was 1.62U / mL, and the fermentation supernatant of Bacillus amyloliquefaciens X7 had a synergistic effect on the lipase of Lactobacillus plantarum R2, which could increase the enzyme activity by 0.90 times.

[0124] Example 12

[0125] This embodiment provides a method for improving the lipase activity of Lactobacillus pentosus R5, and the specific method is as follows.

[0126] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was shaken at a constant temperature of 30° C. and 150 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0127] Step 2: The seed liquid of Bacillus amyloliquefaciens X7 was inoculated into the liquid culture medium at a volume ratio of 5%. The culture medium formula was 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract powder, and 20 g / L NaCl. The liquid volume was 50 mL / 100 mL. The culture was shaken at a constant temperature of 30°C and 150 rpm for 6 hours.

[0128] Step 3: the preparation of the crude enzyme liquid of plant lactobacillus R5 lipase: the plant lactobacillus R5 bacterial strain is accessed in the liquid nutrient medium with 2% inoculum size, culture medium recipe is tryptone 10g / L, beef extract 5g / L, yeast extract powder 4g / L, glucose 20g / L, sodium acetate (three waters) 5g / L, diammonium citrate 2g / L, tween 1mL, K2HPO4.7H2O 2g / L, MnSO4﹒4H2O 0.05g / L, calcium carbonate 20g / L, pH 6.2, liquid capacity is 50mL / 100mL, after leaving standstill to cultivate 14h in 37 ℃ of incubators, with fermented liquid at 4 ℃, centrifugal 5min of 10000r / min, getting supernatant is the crude enzyme liquid of plant lactobacillus R5 lipase, carries out the enzyme activity assay.

[0129] Step 4: After the cultivation of Bacillus amyloliquefaciens X7 obtained in step 1 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 4°C and 8000r / min for 5min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the crude enzyme liquid of Lactobacillus pentosus R5 obtained in step 2 at a volume ratio of 1:1, and the activity of lipase is determined.

[0130] The experiment showed that the lipase activity in the fermentation supernatant of Lactobacillus pentosus R5 was 1.54U / mL, and the fermentation supernatant of Bacillus amyloliquefaciens X7 had a synergistic effect on the lipase of Lactobacillus pentosus R5, which could increase the enzyme activity by 0.82 times.

[0131] Embodiment 13

[0132] This embodiment provides a method for improving the activity of microbial lipase, and the specific method is as follows.

[0133] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 is inoculated into a liquid culture medium, the culture medium formula is 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume is 50 mL / 100 mL, and cultured at a constant temperature of 30° C. until the turbidity (OD600) is 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0134] Step 2: The seed liquid of Bacillus amyloliquefaciens X7 was inoculated into a liquid culture medium at a volume ratio of 5%. The culture medium formula was 20 g / L glucose, 15 g / L tryptone, and 20 g / L NaCl. The liquid volume was 50 mL / 100 mL. The culture was shaken at a constant temperature of 30°C and 150 rpm for 6 hours.

[0135] Step: 3: Preparation of crude enzyme solution of Staphylococcus xylosus M10 lipase: The Staphylococcus xylosus M10 strain was inoculated into a liquid culture medium at a 4% inoculation rate. The culture medium formula was 10 g / L mannitol, 10 g / L peptone, 1 g / L beef extract, 75 g / L NaCl, 2.5 mL / L 1% phenol red reagent, pH 7.2-7.4, and the liquid volume was 50 mL / 100 mL. After static culture in a 37°C incubator for 18 hours, the fermentation broth was centrifuged at 4°C and 10,000 r / min for 5 minutes, and the supernatant was taken as the crude enzyme solution of M10 lipase for enzyme activity determination.

[0136] Step 4: After the cultivation of the Bacillus amyloliquefaciens obtained in step 2 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 4°C and 8000r / min for 5min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the crude M10 lipase solution obtained in step 2 at a volume ratio of 1:1, and the activity of the lipase is determined.

[0137] The experiment showed that the lipase activity in the fermentation supernatant of Staphylococcus xylosus M10 was 2.13U / mL, and the fermentation supernatant of Bacillus amyloliquefaciens X7 had a synergistic effect on the lipase of Staphylococcus xylosus M10, which could increase the enzyme activity by 1.26 times.

[0138] Embodiment 14

[0139] This embodiment provides a method for improving the activity of microbial lipase, and the specific method is as follows.

[0140] Step 1: The Bacillus amyloliquefaciens X7 strain preserved in Example 1 was inoculated into a liquid culture medium, the culture medium formula was 20 g / L glucose, 10 g / L peptone, 5 g / L yeast powder, the liquid volume was 50 mL / 100 mL, and the culture was carried out at a constant temperature of 30° C. and 200 rpm until the turbidity (OD600) was 1.40±0.15 to obtain a Bacillus amyloliquefaciens X7 seed solution.

[0141] Step 2: Take the seed liquid of Bacillus amyloliquefaciens X7 and inoculate it into the liquid culture medium at a volume ratio of 5%. The culture medium formula is 20g / L glucose, 10g / L ammonium sulfate, and 20g / L NaCl. The liquid volume is 50mL / 100mL. Shake and culture at a constant temperature of 30°C and 150rpm for 6h.

[0142] Step 3: Preparation of crude enzyme solution of Staphylococcus xylosus M10 lipase: The Staphylococcus xylosus M10 strain was inoculated into a liquid culture medium at a 4% inoculation rate. The culture medium formula was 10 g / L mannitol, 10 g / L peptone, 1 g / L beef extract, 75 g / L NaCl, 2.5 mL / L 1% phenol red reagent, pH 7.2-7.4, and the liquid volume was 50 mL / 100 mL. After static culture in a 37°C incubator for 18 hours, the fermentation broth was centrifuged at 4°C and 10,000 r / min for 5 minutes, and the supernatant was taken as the crude enzyme solution of M10 lipase for enzyme activity determination.

[0143] Step 4: After the cultivation of the Bacillus amyloliquefaciens obtained in step 2 is completed, the fermentation liquid of Bacillus amyloliquefaciens is taken, centrifuged at 4°C and 8000r / min for 5min, the supernatant is taken to obtain the fermentation supernatant of Bacillus amyloliquefaciens X7, inactivated at high temperature, and the fermentation supernatant is mixed with the crude M10 lipase solution obtained in step 2 at a volume ratio of 1:1, and the activity of the lipase is determined.

[0144] The experiment showed that the lipase activity in the fermentation supernatant of Staphylococcus xylosus M10 was 2.17U / mL, and the fermentation supernatant of Bacillus amyloliquefaciens X7 had a synergistic effect on the lipase of Staphylococcus xylosus M10, which could increase the enzyme activity by 0.84 times.

Claims

1. A Bacillus amyloliquefaciens, characterized in that: The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with the accession number: CCTCC M 20232293.

2. An application of the Bacillus amyloliquefaciens according to claim 1, characterized in that: The fermentation supernatant of Bacillus amyloliquefaciens is used to improve the activity of lipase.

3. An application of the Bacillus amyloliquefaciens according to claim 1, characterized in that: The fermentation supernatant of Bacillus amyloliquefaciens is used for preparing a lipase enhancer.

4. An application of the Bacillus amyloliquefaciens according to claim 1, characterized in that: The lipase includes porcine pancreatic lipase and microbial lipase.

5. The use of Bacillus amyloliquefaciens according to claims 2, 3, and 4, characterized in that: The method for preparing the fermentation supernatant of Bacillus amyloliquefaciens is as follows: inoculating Bacillus amyloliquefaciens into a liquid culture medium containing a carbon source and a nitrogen source, performing oscillating culture, performing solid-liquid separation, and taking the supernatant to obtain the fermentation supernatant of Bacillus amyloliquefaciens.

6. A method for improving lipase activity, characterized in that: The fermentation supernatant of the Bacillus amyloliquefaciens according to claim 1 is mixed with lipase.

7. The method for improving lipase activity according to claim 6, characterized in that: The mixing time of the fermentation supernatant of Bacillus amyloliquefaciens and lipase is 0 to 10 min.

8. The method for improving lipase activity according to claim 6, characterized in that: The method for preparing the fermentation supernatant of Bacillus amyloliquefaciens is as follows: inoculating Bacillus amyloliquefaciens into a liquid culture medium containing a carbon source and a nitrogen source, performing oscillating culture, performing solid-liquid separation, and taking the supernatant to obtain the fermentation supernatant of Bacillus amyloliquefaciens.

9. The method for improving lipase activity according to claim 6, characterized in that: The solid-liquid separation method is: centrifugation for 1 to 15 minutes at 2 to 6°C and 5000 to 15000 r / min.

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