Method for biological cracking of spirulina and use in bacterial culture medium

Abstract

The present application relates to a kind of spirulina biological lysis method and application in bacterial culture medium, it relates to the field of microbiological technology, including the following steps: S1: culture Bacillus subtilis to platform, centrifugal, obtain the culture supernatant of the Bacillus subtilis;S2: the culture supernatant of the Bacillus subtilis is added to the solution of sterilized spirulina, lysis, obtain the lysis fluid I of the spirulina;S3: detection, adjust the pH of the lysis fluid I of the spirulina, then sequentially add pH buffer, the culture supernatant of the Bacillus subtilis, lysis again, obtain the lysis fluid II of the spirulina;S4: the lysis fluid II of the spirulina is repeated S3 and lysis again 3-7 times, obtain the supernatant of the lysis fluid II of the spirulina.The present application spirulina biological lysis efficiency is high, bacterial culture effect is good, cost is cheap, can be popularized to large-scale spirulina lysis in industry, for the preparation of bacterial culture medium.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a method for biological lysis of Spirulina and application in bacterial culture medium. BACKGROUND

[0002] Bacteria are widely used in agriculture, industry, medicine, scientific research and other fields due to their short growth cycle, easy large-scale cultivation and other advantages, such as using Escherichia coli prokaryotic expression system to produce Taq enzyme. Using bacteria for basic research and industrial development requires prior separation and large-scale cultivation of related bacteria, and the separation and cultivation of bacteria depend on effective bacterial culture medium. At present, bacterial culture medium is mainly prepared from incomplete lysis of animal (such as beef, fish meat, milk powder, etc.), plant (mainly soybean) and yeast, etc. These raw materials are relatively expensive, and the cost is increasing year by year. In addition, the lysis cost is also increasing, so the culture medium becomes more and more expensive, and it is urgent to find new sources of bacterial culture medium raw materials and low-cost lysis methods.

[0003] Spirulina (scientific name Spirulina) belongs to the genus under the family of Oscillatoriaceae in Cyanophyta, is an ancient lower single-cell or multi-cell aquatic plant, with a length of 200-500 microns and a width of 5-10 microns. Under a microscope, it is in the shape of a spiral filament, hence the name Spirulina. Its cell structure is simple, without nucleus, so it is also called cyanobacteria. Spirulina is rich in nutrients. First, its protein content is very high, which is one of the richest protein sources on earth, with a protein content of about 60%-70%, much higher than traditional raw materials for bacterial culture medium preparation, such as eggs, beer yeast, skimmed milk powder, fish, soybeans and beef, etc., and contains all 20 amino acids. Secondly, Spirulina contains rich vitamins, including vitamins A, E, B1, B7 and B8, etc. Thirdly, Spirulina contains all the minerals and trace elements required for bacterial growth, such as zinc, iron, potassium, calcium, magnesium, phosphorus, selenium, iodine, etc. Finally, the monosaccharides contained in Spirulina are mainly rhamnose (accounting for 53% of total sugar), and glucose is relatively low, which is conducive to preventing / reducing the production of growth inhibitors-acetic acid. Therefore, Spirulina is an ideal raw material for bacterial culture medium preparation. In addition, Spirulina is a photoautotrophic microorganism with low cost, and consumes a large amount of carbon dioxide during photosynthesis, which is environmentally friendly and helps China to achieve the "double carbon" plan.

[0004] However, it is difficult to convert spirulina into a medium raw material available for bacteria. The reasons are as follows: (1) the cell structure of spirulina needs to be destroyed to fully release the cell contents, and the cell wall of spirulina, especially dry powder spirulina, is strong and difficult to be destroyed; (2) for most bacteria, the ideal nitrogen source is free amino acid due to the lack of transport proteins for transporting short peptides. Therefore, the broken spirulina cells need to be further lysed into free amino acids to improve the culture efficiency of bacteria. In view of this, a method for biological lysis of spirulina and application in bacterial culture medium are provided. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for biological lysis of spirulina and application in bacterial culture medium. The purpose is to obtain a medium raw material available for bacteria by biological lysis of spirulina, which is low in cost.

[0006] In order to solve the above technical problem, the first object of the present application is to provide a method for biological lysis of spirulina, comprising the following steps:

[0007] S1: culturing Bacillus subtilis to the platform phase, centrifuging the Bacillus subtilis at the platform phase to obtain the culture supernatant of the Bacillus subtilis, and refrigerating for standby;

[0008] S2: adding the culture supernatant of the Bacillus subtilis into a solution of sterilized spirulina, and lysing for 3-7 hours at 32-42 DEG C to obtain a lysis solution I of the spirulina;

[0009] S3: detecting the pH of the lysis solution I of the spirulina, adjusting the pH to 6.5-7.5, and sequentially adding a pH buffer and the culture supernatant of the Bacillus subtilis into the lysis solution I after adjusting the pH, and again lysing for 3-7 hours at 32-42 DEG C to obtain a lysis solution II of the spirulina;

[0010] S4: repeating the adjustment of pH, the sequential addition of the pH buffer and the culture supernatant of the Bacillus subtilis, and the again lysis for 3-7 times in the S3 for the lysis solution II of the spirulina, and centrifuging to obtain the supernatant of the lysis solution II of the spirulina for standby.

[0011] The Bacillus subtilis is a gram-positive bacillus commonly found in soil. In the case of insufficient or less nutrients in soil, the Bacillus subtilis secretes extracellular proteases to degrade macromolecular proteins or peptides in the soil to maintain its growth and reproduction.

[0012] The present application has the following beneficial effects:

[0013] (1) The present application can more effectively lyse spirulina than other mechanical lysis methods (e.g. water bath, ultrasonic, freeze-thaw), and can lyse spirulina into shorter peptides and release more free amino acids;

[0014] (2) The supernatant of the lysis solution II of spirulina of the present application has better culture effect on bacterial culture medium than the supernatant obtained by mechanical lysis methods (e.g. water bath, ultrasonic, freeze-thaw);

[0015] (3) The present application uses the extracellular secreted protease obtained by culturing bacillus subtilis to lyse spirulina, which is cheaper than specific commercial enzymes, and the spirulina biological lysis has low requirements on environmental conditions and can be performed at about 37℃, without the limitation of other methods required machines, and does not involve repeated processing required by ultrasonic and freeze-thaw, saving labor and being more efficient.

[0016] Therefore, the spirulina biological lysis of the present application has high efficiency, good effect on bacterial culture, low cost, and can be popularized to large-scale spirulina lysis in industry for the preparation of bacterial culture medium.

[0017] On the basis of the above technical solution, the present application can be further improved as follows.

[0018] Further, the supernatant of the culture of the bacillus subtilis comprises extracellular secreted protease.

[0019] Further, the method comprises the following specific steps:

[0020] S1: inoculating the seed liquid of bacillus subtilis with OD600 of 0.5-1.5 into NB culture solution or LB culture solution, the volume ratio of the seed liquid of the bacillus subtilis to the NB culture solution or the LB culture solution being (1-3):(10-30), culturing in a constant-temperature shaking incubator at 150-250 revolutions per minute and 32-42℃ for 60-84 hours until the bacillus subtilis is cultured to the stationary phase, centrifuging to obtain the culture supernatant of the bacillus subtilis, and refrigerating for standby;

[0021] S2: soaking spirulina dry powder in double distilled water, the weight ratio of the spirulina dry powder to the double distilled water being (5-14):(50-70) to obtain a spirulina solution; sterilizing the spirulina solution in high-pressure steam with a temperature of at least 121℃ for at least 15 minutes to obtain a sterilized spirulina solution; then adding the culture supernatant of the bacillus subtilis to the sterilized spirulina solution, the volume ratio of the culture supernatant of the bacillus subtilis to the double distilled water being (0.1-1):(25-35), and placing in a constant-temperature shaking incubator to lyse at 150-250 revolutions per minute and 32-42℃ for 3-7 hours to obtain a spirulina lysis solution I;

[0022] S3: detecting the pH of the spirulina lysate I, adjusting the pH to 6.7-7.3, and then adding a pH buffer and an equal amount of the culture supernatant of the Bacillus subtilis in S2 into the spirulina lysate I after the pH adjustment, and placing the mixture in a constant-temperature shaking incubator at 170-230 rpm and 34-40°C for further lysis for 4-6 hours to obtain the spirulina lysate II;

[0023] S4: repeating the pH adjustment in S3, adding a pH buffer and the culture supernatant of the Bacillus subtilis into the spirulina lysate II, and further lysing 3-7 times, and then centrifuging to obtain the supernatant of the spirulina lysate II for standby use.

[0024] Further, the method comprises the following specific steps:

[0025] S1: inoculating a seed liquid of Bacillus subtilis with OD600 of 0.7-1.3 into NB culture solution or LB culture solution, and the volume ratio of the seed liquid of the Bacillus subtilis to the NB culture solution or the LB culture solution is (0.7-1.3):(7-13), and then placing the mixture in a constant-temperature shaking incubator at 170-230 rpm and 34-40°C for culturing for 66-78 hours until the Bacillus subtilis reaches the stationary phase, and then centrifuging to obtain the culture supernatant of the Bacillus subtilis for standby use in refrigeration;

[0026] S2: soaking spirulina dry powder in double-distilled water, and the weight ratio of the spirulina dry powder to the double-distilled water is (5-12):(54-64) to obtain a spirulina solution, and then sterilizing the spirulina solution in high-pressure steam at a temperature of at least 121°C for at least 15 min to obtain a sterilized spirulina solution, and then adding the culture supernatant of the Bacillus subtilis into the sterilized spirulina solution, and the volume ratio of the culture supernatant of the Bacillus subtilis to the double-distilled water is (0.1-1):(27-32), and then placing the mixture in a constant-temperature shaking incubator at 170-230 rpm and 34-40°C for lysis for 4-6 hours to obtain the spirulina lysate I;

[0027] S3: detecting the pH of the spirulina lysate I, adjusting the pH to 6.5-7.5, and then adding a pH buffer and an equal amount of the culture supernatant of the Bacillus subtilis in S2 into the spirulina lysate I after the pH adjustment, and placing the mixture in a constant-temperature shaking incubator at 150-250 rpm and 32-42°C for further lysis for 3-7 hours to obtain the spirulina lysate II;

[0028] S4: The lysis solution II of the spirulina is repeatedly subjected to the pH adjustment of S3, and pH buffer, the culture supernatant of the Bacillus subtilis, and lysis again for 3-7 times are sequentially added. After lysis, the supernatant of the lysis solution II of the spirulina is obtained by centrifugation and is ready for use.

[0029] Further, the method comprises the following specific steps:

[0030] S1: The seed liquid of the Bacillus subtilis with OD600 of 0.9-1.1 is inoculated into NB culture solution or LB culture solution, and the volume ratio of the seed liquid of the Bacillus subtilis to the NB culture solution or the LB culture solution is (0.9-1.1):(9-11). The culture is incubated in a constant-temperature shaking incubator at 190-210 rpm and 36-38°C for 70-74 hours until the Bacillus subtilis is cultured to the stationary phase. The culture supernatant of the Bacillus subtilis is obtained by centrifugation and is ready for use after refrigeration;

[0031] S2: The dry spirulina powder is soaked in double-distilled water, and the weight ratio of the dry spirulina powder to the double-distilled water is (9-10):(58-60), so as to obtain a spirulina solution. The spirulina solution is sterilized in high-pressure steam at a temperature of at least 121°C for at least 15 min, so as to obtain a sterilized spirulina solution. Then, the culture supernatant of the Bacillus subtilis is added to the sterilized spirulina solution, and the volume ratio of the culture supernatant of the Bacillus subtilis to the double-distilled water is (0.1-1):(29-30). The mixture is placed in a constant-temperature shaking incubator and is lysed at 180-210 rpm and 36-38°C for 5 hours, so as to obtain a lysis solution I of the spirulina;

[0032] S3: The pH of the lysis solution I of the spirulina is detected, and the pH is adjusted to 6.9-7.1. After the pH adjustment, pH buffer and the same amount of the culture supernatant of the Bacillus subtilis as in S2 are sequentially added to the lysis solution I of the spirulina. The mixture is placed in a constant-temperature shaking incubator and is lysed at 190-210 rpm and 36-38°C for 5 hours, so as to obtain a lysis solution II of the spirulina;

[0033] S4: The lysis solution II of the spirulina is repeatedly subjected to the pH adjustment of S3, and pH buffer, the culture supernatant of the Bacillus subtilis, and lysis again for 3-7 times are sequentially added. After lysis, the supernatant of the lysis solution II of the spirulina is obtained by centrifugation and is ready for use.

[0034] Further, the centrifugation step in S1 is specifically centrifugation at 8000-12000 rpm for 15-25 min, so as to obtain the culture supernatant of the Bacillus subtilis;

[0035] The centrifugation step in the S4 is specifically centrifuging at 8000-12000 rpm for 15-25 min to obtain the supernatant of the broken solution II of the spirulina.

[0036] Further, the pH buffer is potassium dihydrogen phosphate and dipotassium hydrogen phosphate, the mass ratio of the potassium dihydrogen phosphate and the dipotassium hydrogen phosphate is (1.577-2.208):(2.336-3.27), and the weight-volume ratio of the pH buffer and the broken solution II of the spirulina is (3.913-5.478):(250-350).

[0037] Further, the pH adjusting step in the S3 is specifically adjusting the pH of the broken solution I of the spirulina to 6.5-7.5 by using 0.5-1.5 mol / L NaOH, and the pH adjusting step in the S4 is specifically adjusting the pH of the broken solution II of the spirulina to 6.5-7.5 by using 0.5-1.5 mol / L NaOH.

[0038] Further, the NB culture solution in the S1 contains 10 g / L of proteose peptone, 3 g / L of beef powder and 5 g / L of sodium chloride.

[0039] The LB culture solution contains 10 g / L of tryptone, 5 g / L of yeast extract and 10 g / L of sodium chloride.

[0040] The second object is to provide a bacterial culture medium comprising the supernatant of the broken solution II of the spirulina prepared by the method according to any one of the above. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The figure is a comparison chart of formaldehyde titration of free amino acids in the supernatant of the broken solution II of the spirulina obtained in the examples 1-6 of the present application, **, P<0.01, *, P<0.05;

[0042] Figure 2 The figure is a comparison chart of formaldehyde titration of the supernatant of the broken solution II of the spirulina obtained in the example 2 of the present application and the supernatant of the broken solution of spirulina obtained in the comparative examples 1-3, **, P<0.01, *, P<0.05;

[0043] Figure 3 The figure is a comparison chart of culture effects of the supernatant of the broken solution II of the spirulina obtained in the examples 1-3 of the present application as a culture medium of Escherichia coli, **, P<0.01, *, P<0.05;

[0044] Figure 4 The figure is a comparison chart of culture effects of the supernatant of the broken solution II of the spirulina obtained in the examples 4-6 of the present application as a culture medium of Escherichia coli, **, P<0.01, *, P<0.05;

[0045] Figure 5 The supernatant of the lysate of the spirulina obtained in Example 2 of the present application and Comparative Examples 1 to 3 was used as a culture medium for E. coli, and the culture effects were compared, **, P<0.01, *, P<0.05;

[0046] Figure 6 The results of the evaluation of the lysing effect of spirulina based on protein gel electrophoresis in the present application are shown in the figure, **, P<0.01, *, P<0.05. DETAILED DESCRIPTION

[0047] The principles and features of the present application are described below, and the examples are only used to explain the present application and not to limit the scope of the present application.

[0048] Explanation of the source of the drug or material:

[0049] Spirulina was purchased from Hunan Zhengdi Biological Resource Development Co., Ltd.

[0050] Bacillus subtilis ATCC6633 was purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd.

[0051] E. coli K-12 was purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd.

[0052] Example 1

[0053] This example relates to a method for biological lysis of spirulina, comprising the following steps:

[0054] Step one, inoculate 1ml of fresh seed liquid of Bacillus subtilis (OD600≈1) into 10ml NB culture solution, and culture the Bacillus subtilis to the stationary phase, about 72 hours;

[0055] Step two, centrifuge at 10000 rpm for 20 min, and centrifuge to obtain the culture supernatant of the Bacillus subtilis, which contains secreted proteases, etc., and store it at 4℃ in a refrigerator for standby;

[0056] Step three, soak 45g of spirulina dry powder in 300ml of double distilled water to obtain a solution of spirulina, sterilize it with high-pressure steam at 121℃ for 15min to eliminate the consumption of microorganisms in the degradation of spirulina, and obtain the sterilized solution of spirulina;

[0057] Step four, after cooling, add 1ml of the culture supernatant of the Bacillus subtilis described above, and use the culture supernatant of the Bacillus subtilis to lyse the spirulina for 5 hours in a constant-temperature shaker (37℃, 200rpm) to obtain the lysate I of the spirulina;

[0058] Step five, measure the pH value of the lysate I of the spirulina, adjust the pH value to 7 using 1 mol / L sodium hydroxide solution, and add 1.893 g of potassium dihydrogen phosphate and 2.803 g of dipotassium hydrogen phosphate and an equal volume of the culture supernatant of the bacillus subtilis in step four;

[0059] Step six, place in a constant temperature oscillation incubator, continue to lyse for 5 hours at 200 rpm and 37°C, to obtain the lysate II of the spirulina;

[0060] Step seven, repeat steps five and six above, a total of 4 times;

[0061] Step eight, centrifuge at 10,000 rpm for 20 min, collect the supernatant of the lysate II of the spirulina, and store in a 4°C refrigerator for standby.

[0062] The NB culture solution in step one is: 10 g / L of proteose peptone, 3 g / L of beef powder, and 5 g / L of sodium chloride.

[0063] Example 2

[0064] Compared with example 1, in step four, after cooling, 3 ml of the culture supernatant of the bacillus subtilis is added, and the other steps are the same as those in example 1.

[0065] Example 3

[0066] Compared with example 1, in step four, after cooling, 9 ml of the culture supernatant of the bacillus subtilis is added, and the other steps are the same as those in example 1.

[0067] Example 4

[0068] Compared with example 1, in step one, the LB culture solution is used, and the other steps are the same as those in example 1. The LB culture solution in step one is: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride.

[0069] Example 5

[0070] Compared with example 2, in step one, the LB culture solution is used, and the other steps are the same as those in example 1. The LB culture solution in step one is: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride.

[0071] Example 6

[0072] Compared with example 3, in step one, the LB culture solution is used, and the other steps are the same as those in example 1. The LB culture solution in step one is: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride.

[0073] Comparative Example 1: Ultrasonic method

[0074] This comparative example relates to a method for ultrasonic lysis of spirulina, comprising the following steps:

[0075] Dry spirulina powder 45 g was soaked in 300 ml of double distilled water, sterilized at 121 °C for 15 min by high pressure steam to eliminate microbial consumption of degradation of spirulina; after cooling, the spirulina solution was ultrasonicated for 8 x 5 min cycles using a sonotrode with a diameter of 2 mm at 100% power input; centrifugation was performed at 10,000 rpm for 20 min to obtain the ultrasonic lysis of spirulina solution, and the supernatant of the ultrasonic lysis of spirulina solution was taken.

[0076] Comparative Example 2: Water bath method

[0077] This comparative example relates to a method for water bath lysis of spirulina, comprising the following steps:

[0078] Dry spirulina powder 45 g was soaked in 300 ml of double distilled water, sterilized at 121 °C for 15 min by high pressure steam to eliminate microbial consumption of degradation of spirulina; after cooling, the spirulina solution was preheated at 60 °C for 15 min to avoid breakage of the flask, and then subjected to water bath at 100 °C for 60 min; centrifugation was performed at 10,000 rpm for 20 min to obtain the water bath lysis of spirulina solution, and the supernatant of the water bath lysis of spirulina solution was taken.

[0079] Comparative Example 3: Freeze-thaw method

[0080] This comparative example relates to a method for freeze-thaw lysis of spirulina, comprising the following steps:

[0081] Dry spirulina powder 45 g was soaked in 300 ml of double distilled water, sterilized at 121 °C for 15 min by high pressure steam to eliminate microbial consumption of degradation of spirulina; after cooling, the spirulina solution was subjected to 4 cycles of freezing (-20 °C for 20 min) and thawing (20 °C for 20 min); centrifugation was performed at 10,000 rpm for 20 min to obtain the freeze-thaw lysis of spirulina solution, and the supernatant of the freeze-thaw lysis of spirulina solution was taken.

[0082] Experimental Example

[0083] 1. Evaluation of lysis effect based on formaldehyde titration

[0084] 1.1 Method for evaluation of lysis effect based on formaldehyde titration

[0085] In this experimental example, the content of free amino acids in the spirulina lysis solution obtained in the examples and comparative examples was evaluated by formaldehyde titration.

[0086] The formaldehyde titration procedure includes: for each of the obtained spirulina lysate solution, three 25 ml conical flasks are prepared, one of which is used as a blank control, 7 ml distilled water is moved into the conical flask with a pipette, and 2 ml of lysate and 5 ml of distilled water are moved into the other two conical flasks with a pipette, one is used for formaldehyde titration preliminary screening, and the other is used for formaldehyde accurate titration. 5 drops of phenolphthalein indicator and 2 ml of neutral formaldehyde solution are added to the three conical flasks, and after being shaken and mixed thoroughly, 0.1 mol / L sodium hydroxide solution is added dropwise until a pink color appears.

[0087] Then the titration amount of the sample is subtracted from the formaldehyde blank titration amount to reflect the free amino acid content of the sample. Each sample of spirulina lysate solution is repeated three times in total.

[0088] Note: (1) The neutral formaldehyde solution is: 50 ml of 36%-37% analytical pure formaldehyde solution is added with 1 ml of 0.1% phenolphthalein ethanol aqueous solution, and titrated to light red with 0.1 mol / L sodium hydroxide solution, and stored in a sealed glass bottle. This reagent is prepared before use.

[0089] (2) The formaldehyde titration is repeated three times for each spirulina lysate solution for statistical analysis.

[0090] (3) The statistical test uses t-test, and the other spirulina lysate products are tested with 3 ml of Bacillus subtilis NB culture supernatant lysed spirulina as reference.

[0091] 1.2 Results

[0092] 1.2.1 Comparison of formaldehyde titration of free amino acids in supernatant of lysate II of spirulina obtained in examples 1 to 6

[0093] The comparison results of formaldehyde titration of free amino acids in supernatant of lysate II of spirulina obtained in examples 1 to 6 are shown in Table 1. Figure 1 As can be seen from Table 1: Figure 1 The supernatant of the lysate II of spirulina obtained by lysing spirulina with 3 ml of supernatant of Bacillus subtilis inoculated in NB culture has the largest volume of 0.1 mol / L sodium hydroxide solution required for formaldehyde titration to the end point, indicating that it has the highest content of free amino acids.

[0094] 1.2.2 Comparison of formaldehyde titration of supernatant of lysate II of spirulina obtained in example 2 with supernatant of lysate of spirulina obtained in comparative examples 1 to 3

[0095] The comparison results of formaldehyde titration of supernatant of lysate II of spirulina obtained in example 2 with supernatant of lysate of spirulina obtained in comparative examples 1 to 3 are shown in Table 2. Figure 2 As can be seen from Table 2: Figure 2It can be seen that the supernatant of the spirulina lysis solution II provided in the embodiment 2 has the largest volume of 0.1 mol / L sodium hydroxide solution required for titration to the end point, indicating that it has the highest free amino acid content, and is significantly higher than that of the other three mechanical methods, proving that the spirulina biological lysis method provided in the present application is significantly better than the three mechanical methods of the comparative examples 1 to 3.

[0096] 2. Evaluation based on the culture effect of E. coli:

[0097] 2.1 Method of evaluation based on the culture effect of E. coli

[0098] In the experimental example, 5 ml of E. coli liquid with OD600 value of about 1 was added into 100 ml of spirulina lysis solution obtained in the embodiment (inoculation amount of 5%), and cultured in a 37℃ constant temperature shaking incubator at 200 revolutions per minute, and the OD600 value was measured every 12 hours, and the culture was performed for a total of 132 hours.

[0099] Note: (1) The spirulina lysis solution for culture was adjusted to pH 7.0 with 1 mol / L sodium hydroxide solution and sterilized at 121℃ for 15 min.

[0100] (2) The strain of E. coli was K-12.

[0101] (3) The culture evaluation of each spirulina lysis solution was repeated three times.

[0102] (4) The OD600 value was obtained by subtracting the OD600 of the corresponding culture medium from the OD600 of the sample, reflecting the growth of E. coli, and was measured by Japan Shimadzu UV-2700 at a wavelength of 600 nm.

[0103] (5) The statistical test used t-test, and the OD600 value in the 3 ml Bacillus subtilis NB culture supernatant lysis spirulina culture medium was used as a reference, and the OD600 values in the other obtained spirulina culture media were tested.

[0104] 2.2 Results

[0105] 2.2.1 Comparison of culture effects of the supernatant of the spirulina lysis solution II obtained in the embodiments 1 to 3 as the culture medium of E. coli

[0106] The comparison of culture effects of the supernatant of the spirulina lysis solution II obtained in the embodiments 1 to 3 as the culture medium of E. coli is shown in the following table. Figure 3 Figure 3 ​It can be seen that: in the NB culture solution, 3 ml of the supernatant of the lysed Spirulina of Bacillus subtilis is taken as the culture medium of Escherichia coli, and the OD600 value of Escherichia coli is up to about 15 after 72 hours of culture. The supernatant of the lysed Spirulina II obtained in Example 1 or Example 3 is taken as the culture medium, and the OD600 value of Escherichia coli is also more than 12 after 72 hours of culture. The culture effect of the supernatant of the lysed Spirulina II obtained in Example 2 as the culture medium of Escherichia coli is significantly higher than that of other culture media at all time points.

[0107] 2.2.2 Comparison of culture effects of the supernatant of the lysed Spirulina II obtained in Examples 4 to 6 as the culture medium of Escherichia coli

[0108] The comparison of culture effects of the supernatant of the lysed Spirulina II obtained in Examples 4 to 6 as the culture medium of Escherichia coli is shown in Table 2.2.2. Figure 4 Figure 4 It can be seen that: in the LB culture solution, 3 ml of the supernatant of the lysed Spirulina of Bacillus subtilis is taken as the culture medium of Escherichia coli, and the OD600 value of Escherichia coli is up to about 15 after 72 hours of culture. The supernatant of the lysed Spirulina II obtained in Example 1 or Example 3 is taken as the culture medium, and the OD600 value of Escherichia coli is also more than 12 after 72 hours of culture. The culture effect of the supernatant of the lysed Spirulina II obtained in Example 2 as the culture medium of Escherichia coli is significantly higher than that of other culture media at all time points.

[0109] 2.2.3 Comparison of culture effects of the supernatant of the lysed Spirulina II obtained in Examples 4 to 6 as the culture medium of Escherichia coli

[0110] The comparison of culture effects of the supernatant of the lysed Spirulina II obtained in Examples 4 to 6 as the culture medium of Escherichia coli is shown in Table 2.2.2. Figure 5 Figure 5 It can be seen that: in the LB culture solution, 3 ml of the supernatant of the lysed Spirulina of Bacillus subtilis is taken as the culture medium of Escherichia coli, and the OD600 value of Escherichia coli is up to about 15 after 72 hours of culture. The supernatant of the lysed Spirulina II obtained in Example 1 or Example 3 is taken as the culture medium, and the OD600 value of Escherichia coli is also more than 12 after 72 hours of culture. The culture effect of the supernatant of the lysed Spirulina II obtained in Example 2 as the culture medium of Escherichia coli is significantly higher than that of other culture media at all time points.

[0111] 3. Evaluation of the lysing effect of Spirulina based on protein gel electrophoresis

[0112] 3.1 Method for evaluating the lysing effect of Spirulina based on protein gel electrophoresis ​​

[0113] The experiment example according to the molecular weight of the spirulina protein lysate sample selects 15% SDS-PAGE, and makes a separation gel and a concentration gel; the lysate of the spirulina obtained above is mixed with a Laoding Buffer dye solution at a volume ratio of 4:1, and is placed in a 95°C water bath for heating for 5 minutes to prepare an electrophoresis sample; 20 microliters of each sample is loaded, and 5 microliters of a protein marker (10-250 kd) sample is also loaded; 60-volt constant voltage electrophoresis is performed for 30 minutes; when the protein marker reaches the separation gel and the bands are separated and clear, the voltage is adjusted to 80 volts for continued constant voltage electrophoresis until the target protein is electrophoresed to a suitable position; after the electrophoresis is completed; the Coomassie blue super-fast staining solution is shaken and dyed for 30 minutes; after the dyeing is completed, the decolorizing solution is used for decolorizing twice, and the bands are recorded.

[0114] 3.2 Results

[0115] The results of evaluating the cracking effect of spirulina based on protein gel electrophoresis are shown in Table 1. Figure 6 As can be seen from Table 1, the relative molecular weight of the spirulina protein or peptide after cracking in Example 2 of the present application is smaller than that of the spirulina protein or peptide after cracking by the mechanical method in Comparative Examples 1 to 3, which proves that the biological cracking method provided by the present application has a higher cracking effect of spirulina. Figure 6

[0116] Therefore, the biological cracking of spirulina has high efficiency, good bacterial culture effect, and low cost, and can be popularized to large-scale cracking of spirulina in industry for the preparation of bacterial culture medium.

[0117] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0118] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.​

Claims

1. A method for biological lysis of Spirulina, characterized by, It comprises the following steps: S1: culture Bacillus subtilis to the plateau, centrifuge the Bacillus subtilis at the plateau, obtain the culture supernatant of the Bacillus subtilis, and store in the refrigerator for standby; the Bacillus subtilis is Bacillus subtilis ATCC6633; S2: add the culture supernatant of the Bacillus subtilis to the solution of sterilized spirulina, lyse at 32-42℃ for 3-7 hours, and obtain the lysate I of the spirulina; S3: detect the pH of the lysate I of the spirulina, adjust the pH to 6.5-7.5, add pH buffer, the culture supernatant of the Bacillus subtilis in sequence in the lysate I after adjusting the pH, and lyse again at 32-42℃ for 3-7 hours, and obtain the lysate II of the spirulina; S4: repeat the adjustment of pH of the S3, add pH buffer, the culture supernatant of the Bacillus subtilis in sequence, and lyse for 3-7 times in the lysate II of the spirulina, and obtain the supernatant of the lysate II of the spirulina after centrifugation, and store for standby.

2. A method of biological lysis of Spirulina according to claim 1, characterized in that, The supernatant of the culture of the Bacillus subtilis comprises extracellular secreted protease.

3. The method of claim 1, wherein the biological lysis of Spirulina is carried out by using a lysis enzyme. It comprises the following specific steps: S1: inoculate the seed liquid of Bacillus subtilis with OD600 of 0.5-1.5 into NB culture solution or LB culture solution, the volume ratio of the seed liquid of the Bacillus subtilis to the NB culture solution or the LB culture solution is (1-3):(10-30), culture in a constant-temperature shaking incubator at 150-250 rpm, 32-42℃ for 60-84 hours until the Bacillus subtilis is cultured to the plateau, centrifuge, obtain the culture supernatant of the Bacillus subtilis, and store in the refrigerator for standby; S2: soak spirulina dry powder in double distilled water, the weight ratio of the spirulina dry powder to the double distilled water is (5-14):(50-70), obtain the solution of the spirulina; then sterilize the solution of the spirulina in high-pressure steam with a temperature of at least 121℃ for at least 15 min, obtain the solution of the spirulina after sterilization; then add the culture supernatant of the Bacillus subtilis in the solution of the spirulina after sterilization, the volume ratio of the culture supernatant of the Bacillus subtilis to the double distilled water is (0.1-1):(25-35), place in a constant-temperature shaking incubator, lyse at 150-250 rpm, 32-42℃ for 3-7 hours, and obtain the lysate I of the spirulina; S3: detect the pH of the lysate I of the spirulina, adjust the pH to 6.7-7.3, add pH buffer, and the culture supernatant of the Bacillus subtilis in sequence in the lysate I after adjusting the pH, place in a constant-temperature shaking incubator, continue to lyse at 170-230 rpm, 34-40℃ for 4-6 hours, and obtain the lysate II of the spirulina; S4: repeat the adjustment of pH of the S3, add pH buffer, the culture supernatant of the Bacillus subtilis in sequence, and lyse for 3-7 times in the lysate II of the spirulina, and obtain the supernatant of the lysate II of the spirulina after centrifugation, and store for standby.

4. The method of claim 1, wherein the Spirulina is subjected to a biological lysis. Comprising the following specific steps: S1: inoculate the seed liquid of Bacillus subtilis with OD600 of 0.7-1.3 into NB culture solution or LB culture solution, the volume ratio of the seed liquid of Bacillus subtilis to the NB culture solution or the LB culture solution (0.7-1.3):(7-13), cultivate in a constant temperature shaking incubator at 170-230 rpm, 34-40℃ for 66-78 hours until the Bacillus subtilis is cultivated to the stationary phase, centrifuge to obtain the culture supernatant of the Bacillus subtilis, and store in the refrigerator for standby; S2: soak the dry spirulina powder in double distilled water, the weight ratio of the dry spirulina powder to the double distilled water is (5-12):(54-64), to obtain the spirulina solution; then sterilize the spirulina solution in high-pressure steam with a temperature of at least 121℃ for at least 15 min to obtain the sterilized spirulina solution; then add the culture supernatant of the Bacillus subtilis to the sterilized spirulina solution, the volume ratio of the culture supernatant of the Bacillus subtilis to the double distilled water is (0.1-1):(27-32), and place it in a constant temperature shaking incubator at 170-230 rpm, 34-40℃ for 4-6 hours to obtain the lysate I of the spirulina; S3: detect the pH of the lysate I of the spirulina, adjust the pH to 6.5-7.5, and then add pH buffer and an equal amount of the culture supernatant of the Bacillus subtilis in S2 to the lysate I of the spirulina after adjusting the pH, and place it in a constant temperature shaking incubator at 150-250 rpm, 32-42℃ for 3-7 hours to obtain the lysate II of the spirulina; S4: repeat the adjustment of pH in S3 for the lysate II of the spirulina, add pH buffer and the culture supernatant of the Bacillus subtilis in sequence, and crackle for 3-7 times again, and then centrifuge to obtain the supernatant of the lysate II of the spirulina for standby.

5. The method for the biolysis of Spirulina according to claim 1, characterized in that, Comprising the following specific steps: S1: inoculate the seed liquid of Bacillus subtilis with OD600 of 0.9-1.1 into NB culture solution or LB culture solution, the volume ratio of the seed liquid of Bacillus subtilis to the NB culture solution or the LB culture solution (0.9-1.1):(9-11), cultivate in a constant temperature shaking incubator at 190-210 rpm, 36-38℃ for 70-74 hours until the Bacillus subtilis is cultivated to the stationary phase, centrifuge to obtain the culture supernatant of the Bacillus subtilis, and store in the refrigerator for standby; S2: dry spirulina powder is soaked in double distilled water, the weight ratio of the dry spirulina powder to the double distilled water is (9-10):(58-60), to obtain a spirulina solution; the spirulina solution is sterilized in high-pressure steam with a temperature of at least 121℃ for at least 15 min, to obtain a sterilized spirulina solution; then the sterilized spirulina solution is added with the culture supernatant of Bacillus subtilis, the volume ratio of the culture supernatant of Bacillus subtilis to double distilled water is (0.1-1):(29-30), and placed in a constant-temperature shaking incubator for lysis at 180-210 rpm, 36-38℃ for 5 h, to obtain a spirulina lysis solution I; S3: the pH of the spirulina lysis solution I is detected, the pH is adjusted to 6.9-7.1, and after the pH adjustment, the spirulina lysis solution I is sequentially added with a pH buffer and an equal amount of the culture supernatant of Bacillus subtilis in S2, and placed in a constant-temperature shaking incubator for lysis at 190-210 rpm, 36-38℃ for 5 h, to obtain a spirulina lysis solution II; S4: the spirulina lysis solution II is repeatedly subjected to the pH adjustment in S3, sequentially added with a pH buffer, the culture supernatant of Bacillus subtilis, and lysed for 3-7 times, and after the lysis, the supernatant of the spirulina lysis solution II is obtained by centrifugation, for standby use.

6. The method of biological lysis of Spirulina according to any one of claims 1 to 5, characterized in that, The centrifugation step in S1 is specifically centrifugation at 8000-12000 rpm for 15-25 min, to obtain the culture supernatant of Bacillus subtilis; and the centrifugation step in S4 is specifically centrifugation at 8000-12000 rpm for 15-25 min, to obtain the supernatant of the spirulina lysis solution II.

7. The method of biological lysis of Spirulina according to any one of claims 1 to 5, characterized in that, The pH buffer is potassium dihydrogen phosphate and dipotassium hydrogen phosphate, the mass ratio of the potassium dihydrogen phosphate to the dipotassium hydrogen phosphate is (1.577-2.208):(2.336-3.27), and the weight-to-volume ratio of the pH buffer to the spirulina lysis solution II is (3.913-5.478):(250-350).

8. The method of biological lysis of Spirulina according to any one of claims 1 to 5, characterized in that, The pH adjustment step in S3 is specifically adjusting the pH of the spirulina lysis solution I to 6.5-7.5 by using 0.5-1.5 mol / L NaOH; and the pH adjustment step in S4 is specifically adjusting the pH of the spirulina lysis solution II to 6.5-7.5 by using 0.5-1.5 mol / L NaOH.

9. The method of biological lysis of Spirulina according to any one of claims 1 to 5, characterized in that, The NB culture solution in S1 contains 10 g / L of proteose peptone, 3 g / L of beef powder, and 5 g / L of sodium chloride; The LB culture solution contains 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride.

10. A bacterial culture medium, characterized in that, The supernatant of the spirulina lysis solution II prepared by the method in any one of claims 1 to 9.

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