阅读说明：本技术 一种凝结芽孢杆菌素的提取方法 (Extraction method of bacillus coagulans ) 是由 樊倩 周盛昌 王丽娜 郑庆礼 张敬学 吴有林 于 2020-12-18 设计创作，主要内容包括：本发明属于细菌培养领域,具体涉及一种凝结芽孢杆菌素的提取方法。本发明提供的培养方法,采用自制培养基进行培养,将成功分离的凝结芽孢杆菌按照1.5～2.5％的接种量接种到培养基中,在36～38℃的温度下发酵培养35～38h,然后经发酵液粗离心、微孔过滤、粗提液浓缩等过程制得。本发明提供的培养方法,可以有效抑制其他细菌生长,制成的凝结芽孢杆菌素的产量大,纯化度高,活性高,适于大规模的生产利用。(The invention belongs to the field of bacterial culture, and particularly relates to a method for extracting coagulans. The culture method provided by the invention adopts a self-made culture medium for culture, inoculates the successfully separated bacillus coagulans into the culture medium according to the inoculation amount of 1.5-2.5%, performs fermentation culture for 35-38 h at the temperature of 36-38 ℃, and then performs the processes of fermentation liquor coarse centrifugation, microfiltration, crude extract concentration and the like to prepare the bacillus coagulans. The culture method provided by the invention can effectively inhibit the growth of other bacteria, and the prepared condensation bacillus element has the advantages of high yield, high purification degree and high activity, and is suitable for large-scale production and utilization.)

1. A method for extracting Bacillus coagulans is characterized by comprising the following steps:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 1.8-2.2% of glucose, 1.2-1.8% of soybean peptone, 0.3-0.7% of yeast extract powder, 0.04-0.08% of magnesium sulfate, 0.02-0.06% of manganese sulfate, 0.1-0.1% of tween-800.06, 0.1-0.2% of a lactein promoter, 94.86-96.48% of water, and adjusting the pH value of the culture medium to 6.8-7.2;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 1.5-2.5%, and performing fermentation culture at the temperature of 36-38 ℃ for 32-40 h to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotating speed of 7000-9000 rpm for 10-20 min, taking the supernatant, performing microporous filtration sterilization on the supernatant, adjusting the pH value to 7.0, precipitating with an ammonium sulfate solution with the mass fraction of 60-70% saturation at 4 ℃, and collecting the lower precipitate;

s4, dissolving the lower-layer precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by Sephadex-G100, drying, and determining the molecular weight by an SDS-PAGE method to obtain the protein.

2. The extraction method according to claim 1, comprising the steps of:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 2.0% of glucose, 1.5% of soybean peptone, 0.5% of yeast extract powder, 0.06% of magnesium sulfate, 0.04% of manganese sulfate, tween-800.08%, 0.15% of a lactein accelerator and 95.67% of water, and adjusting the pH value of the culture medium to 7.0;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 2.0%, and fermenting and culturing at the temperature of 36 ℃ for 36 hours to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotating speed of 8000rpm for 15min, taking the supernatant, carrying out microfiltration sterilization on the supernatant, then adjusting the pH value to 7.0, precipitating with an ammonium sulfate solution with the saturation of 4 ℃ and the mass fraction of 65%, and collecting the lower-layer precipitate;

s4, dissolving the lower-layer precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by Sephadex-G100, drying, and determining the molecular weight by an SDS-PAGE method to obtain the protein.

3. The extraction method according to claim 1, wherein the lactein promoter in step S1 is prepared from arginine and vitamin D in a weight ratio of 10-15: 1 to 4.

4. The extraction method according to claim 3, wherein the lactein promoter of step S1 is prepared from arginine and vitamin D in a weight ratio of 13: 3, and (3).

5. The method according to claim 1, wherein the filtration pore size in the step S3 is 0.45 μm when the microfiltration sterilization is performed.

6. The method according to claim 1, wherein the precipitation in step S3 is performed by mixing the fermentation broth after filtration sterilization with ammonium sulfate solution, and centrifuging at 8000rpm for 20 min.

7. The method of claim 1, wherein the dialysis process in step S4 is dialysis with a dialysis bag with a molecular weight of 1000 Da.

8. The extraction method according to claim 1, wherein the freeze-drying process in step S5 comprises freezing the crude extract at-50 to-40 ℃ under 5 to 10Pa for 20 to 30 min.

Technical Field

The invention belongs to the field of bacterial culture, and particularly relates to a method for extracting coagulans.

Background

Lactein is a polypeptide or precursor polypeptide with bacteriostatic activity produced by certain lactic acid bacteria via the ribosome synthesis mechanism during metabolism. The lactein has no toxicity and side effect, and can be used as antibiotic-free additive in animal production. However, the production of lactein is affected by many factors, and it has been reported that the production of lactein is affected by different strains, and the time of culturing the same strain in fermentation, the temperature of culturing, the pH of the medium, the inoculum size of the bacterial liquid, and even the components of the medium such as carbon source, nitrogen source, phosphate, etc. Wherein the growth of the strain and the synthesis of lactein are influenced by factors such as the composition of the medium, the time of cultivation, the temperature of cultivation, the pH of cultivation, and the like. Therefore, how to obtain the lactein with high yield and high activity by screening strains and setting culture conditions becomes a research hotspot of the lactein.

Bacillus coagulans belongs to the genus Bacillus taxonomically, and has rod-shaped cells, gram-positive bacteria, terminal spores and no flagella. However, it is different from other Bacillus in that it is the only Bacillus that can produce lactic acid (L-lactic acid by decomposing saccharides), and is a homolactic fermentation bacterium, and has an optimum growth temperature of 45 to 50 ℃ and an optimum pH of 6.6 to 7.0. In the prior art, many studies on Bacillus coagulans have been made, but most of them have focused on their application fields, and there is no description in the prior art on how to improve the lactobacilli in Bacillus coagulans.

Disclosure of Invention

Aiming at the defects generally existing in the prior art, the invention creatively provides a method for extracting the coagulation bacillus. The culture method provided by the invention can effectively inhibit the growth of other bacteria, and the prepared condensation bacillus element has the advantages of high yield, high purification degree and high activity, and is suitable for large-scale production and utilization.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for extracting Bacillus coagulans comprises the following steps:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 1.8-2.2% of glucose, 1.2-1.8% of soybean peptone, 0.3-0.7% of yeast extract powder, 0.04-0.08% of magnesium sulfate, 0.02-0.06% of manganese sulfate, 0.1-0.1% of tween-800.06, 0.1-0.2% of a lactein promoter, 94.86-96.48% of water, and adjusting the pH value of the culture medium to 6.8-7.2;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 1.5-2.5% in volume ratio, and performing fermentation culture at the temperature of 36-38 ℃ for 32-40 h to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotating speed of 7000-9000 rpm for 10-20 min, taking the supernatant, performing microporous filtration sterilization on the supernatant, adjusting the pH value to 7.0, precipitating with an ammonium sulfate solution with the mass fraction of 60-70% saturation at 4 ℃, and collecting the lower precipitate;

s4, dissolving the lower-layer precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by Sephadex-G100, drying, and determining the molecular weight by an SDS-PAGE method to obtain the protein.

Preferably, the extraction method comprises the following steps:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 2.0% of glucose, 1.5% of soybean peptone, 0.5% of yeast extract powder, 0.06% of magnesium sulfate, 0.04% of manganese sulfate, tween-800.08%, 0.15% of a lactein accelerator and 95.67% of water, and adjusting the pH value of the culture medium to 7.0;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 2.0%, and fermenting and culturing at the temperature of 36 ℃ for 36 hours to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotating speed of 8000rpm for 15min, taking the supernatant, carrying out microfiltration sterilization on the supernatant, then adjusting the pH value to 7.0, precipitating with an ammonium sulfate solution with the saturation of 4 ℃ and the mass fraction of 65%, and collecting the lower-layer precipitate;

s4, dissolving the lower-layer precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by adopting glucose gel, drying, and determining the molecular weight by using an SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE) method.

Preferably, the lactein promoter in step S1 is prepared from arginine and vitamin D in a weight ratio of 10-15: 1 to 4.

Preferably, the lactein promoter described in step S1 is prepared from arginine and vitamin D in a weight ratio of 13: 3, and (3).

Preferably, the filtration pore size at the time of the microfiltration sterilization in step S3 is 0.45. mu.m.

Preferably, the precipitation in step S3 is performed by mixing the filtered and sterilized fermentation broth with ammonium sulfate solution, and centrifuging at 8000rpm for 20 min.

Preferably, the dialysis process described in step S4 is dialysis with a dialysis bag having a molecular weight of 1000 Da.

Preferably, the freeze-drying process in step S5 is to freeze the crude extract at-50 to-40 ℃ under 5 to 10Pa for 20 to 30 min.

The traditional culture medium for culturing the bacillus coagulans contains components including glucose, beef extract, yeast extract powder, sodium acetate, dipotassium hydrogen phosphate, diamine citrate, magnesium sulfate, manganese sulfate, Tween 80 and the like.

In the actual research and development process, the inventors carry out a large amount of screening on factors such as culture temperature, culture time, inoculum size and the like, and finally screen out an optimal combination range, wherein the optimal combination range can greatly improve the yield of the bacillus coagulans.

Meanwhile, in the further research and development process, the inventor surprisingly finds that the addition of arginine and vitamin D according to a certain proportion can supplement sufficient amino acid and other nutrient components for bacteria, promote the generation of bacteriocin and be beneficial to improving the yield of the bacillus coagulans.

Compared with the prior art, the extraction method of the coagulation bacillus element provided by the invention has the following advantages:

(1) the extraction method of the condensed bacillus element provided by the invention obviously improves the yield, and has high purity and low production cost;

(2) according to the extraction method provided by the invention, the strain is cultured by adopting the self-made culture medium, compared with the traditional culture medium, certain components are removed, the preparation cost of the culture medium is reduced, and the extraction method is suitable for industrial large-scale production of the condensed bacillus;

(3) according to the extraction method provided by the invention, the lactobacillus promoter is added into the culture medium, so that the generation of the condensation bacillus can be effectively promoted, and the yield is improved.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The bacillus coagulans can be purchased from north nano biotechnology limited, and the product number is BNCC 136363; the arginine is L-arginine, which is available from Xianxu Total Biotechnology Co., Ltd, and has CAS number of 74-79-3; the vitamin D can be purchased from Jiangsu Bai Yao biological science and technology limited; the staphylococcus aureus is ATCC6538, the escherichia coli is ATCC 25312, the pasteurella is pasteurella multocida with the number of bio-67665, the salmonella is salmonella paratyphi B with the number of bio-00025, and the salmonella can be purchased from China center for culture of microorganisms; the glucose gel is Sephadex-G100 and can be purchased from Nanjing Senega Biotechnology Ltd.

Example 1A method for extracting Bacillus coagulans

The extraction method of the bacillus coagulans comprises the following steps:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 1.8% of glucose, 1.2% of soybean peptone, 0.3% of yeast extract powder, 0.04% of magnesium sulfate, 0.02% of manganese sulfate, tween-800.06%, 0.1% of a lactein accelerator and 96.48% of water, and adjusting the pH value of the culture medium to 6.8; the lactein accelerant is prepared from arginine and vitamin D in a weight ratio of 10-15: 1-4;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 1.5%, and fermenting and culturing for 32h at the temperature of 36 ℃ to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotating speed of 7000rpm for 10min, taking the supernatant, performing microfiltration sterilization on the supernatant (the size of the filter pore diameter is 0.45 mu m), then adjusting the pH value to 7.0, mixing the supernatant with ammonium sulfate solution with the mass fraction of 60% saturation at the temperature of 4 ℃, centrifuging the mixture at the rotating speed of 8000rpm for 20min, and collecting the lower-layer precipitate;

s4, dissolving the lower precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing by using a dialysis bag with the molecular weight of 1000Da to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by Sephadex-G100, drying, and determining the molecular weight by an SDS-PAGE method to obtain the protein; the freeze drying process comprises freezing the crude extract at-50 deg.C under 5Pa for 20 min.

Example 2 extraction of Bacillus coagulans

The extraction method of the bacillus coagulans comprises the following steps:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 2.2% of glucose, 1.8% of soybean peptone, 0.7% of yeast extract powder, 0.08% of magnesium sulfate, 0.06% of manganese sulfate, 800.1% of tween-2, 0.2% of a lactein accelerator and 94.86% of water, and adjusting the pH value of the culture medium to 7.2; the lactein accelerator is prepared from arginine and vitamin D according to the weight ratio of 15: 4, preparing a composition;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 2.5%, and fermenting and culturing at the temperature of 38 ℃ for 40h to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotating speed of 9000rpm for 20min, taking the supernatant, performing microfiltration sterilization on the supernatant (the size of the filter pore diameter is 0.45 mu m), adjusting the pH value to 7.0, mixing the supernatant with an ammonium sulfate solution with the mass fraction of 70% saturation at the temperature of 4 ℃, centrifuging at the rotating speed of 8000rpm for 20min, precipitating, and collecting the lower precipitate;

s4, dissolving the lower-layer precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing by a dialysis bag with the molecular weight of 1000Da to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by Sephadex-G100, drying, and determining the molecular weight by an SDS-PAGE method to obtain the protein; the freeze drying process comprises freezing the crude extract at-40 deg.C under 10Pa for 30 min.

Example 3A method for extracting Bacillus coagulans

The extraction method of the bacillus coagulans comprises the following steps:

s1, preparation of a culture medium: adding the following components in percentage by weight, and uniformly mixing: 2.0% of glucose, 1.5% of soybean peptone, 0.5% of yeast extract powder, 0.06% of magnesium sulfate, 0.04% of manganese sulfate, tween-800.08%, 0.15% of a lactein accelerator and 95.67% of water, and adjusting the pH value of the culture medium to 7.0; the lactein accelerator described in step S1 is prepared from arginine and vitamin D in a weight ratio of 13: 3, preparing a composition;

s2, inoculation: taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 2.0%, and fermenting and culturing at the temperature of 36 ℃ for 36 hours to obtain fermentation liquor;

s3, centrifuging the fermentation liquor prepared in the step S2 at the rotation speed of 8000rpm for 15min, taking the supernatant, performing microfiltration sterilization on the supernatant (the size of the filter pore diameter is 0.45 mu m), then adjusting the pH value to 7.0, mixing the supernatant with ammonium sulfate solution with the saturation of 4 ℃ and the mass fraction of 65%, centrifuging the mixture at the rotation speed of 8000rpm for 20min, precipitating, and collecting the lower precipitate;

s4, dissolving the lower-layer precipitate collected in the step S3 in a sodium phosphate buffer solution with the pH value of 6.5, and dialyzing by a dialysis bag with the molecular weight of 1000Da to obtain a crude extract containing bacteriocin;

s5, freeze-drying and concentrating the crude extract prepared in the step S4, purifying by Sephadex-G100, drying, and determining the molecular weight by an SDS-PAGE method to obtain the protein; the freeze drying process comprises freezing the crude extract at-45 deg.C under 8Pa for 25 min.

Comparative example 1 extraction method of Bacillus coagulans

The extraction method of the bacillus coagulans is similar to the example 3;

the difference from example 3 is that in comparative example 1, in preparing the culture medium, the lactein promoter is prepared from arginine and vitamin D in a weight ratio of 1: 1.

Comparative example 2 extraction method of Bacillus coagulans

The extraction method of the bacillus coagulans is similar to the example 3;

the difference from example 3 is that in comparative example 2, in preparing the medium, the lactein promoter was arginine only.

Comparative example 3 extraction method of Bacillus coagulans

The extraction method of the bacillus coagulans is similar to the example 3;

the difference from example 3 is that in comparative example 2, the lactein promoter was vitamin D only when preparing the culture medium.

Test example 1 screening of inoculum size

1. Test samples: bacillus coagulans; staphylococcus aureus bacteria; e.coli; salmonella; pasteurella; 2. the test method comprises the following steps: according to the extraction method disclosed by the embodiment 3 of the invention, the inoculation amounts are respectively modified to be 1%, 1.5%, 2.0%, 2.5% and 3.0%, and the purity of the prepared bacteriocin is detected by an Oxford cup bacteriostasis test.

And (3) an oxford cup bacteriostasis test:

the specific operation steps are as follows:

1. taking out the bacillus coagulans separated in a laboratory, inoculating the bacillus coagulans into 100mL of culture medium according to the inoculation amount of 1%, 1.5%, 2.0%, 2.5% and 3.0%, culturing for 36h in an environment with the temperature of 37 ℃ to obtain fermentation liquor, finally performing dialysis through ammonium sulfate precipitation and a dialysis bag, purifying by Sephadex-G100, and drying to obtain a purified dry product.

2. And (3) recovering the indicator bacteria: staphylococcus aureus, Escherichia coli, Salmonella and Pasteurella were added to the high-pressure LB Broth at a ratio of 1: 100. Wherein, 5% serum is added into the pasteurella culture medium.

3. LB Broth Agar medium was heated, then about 20mL of solid medium was poured into a sterile plate, and 200. mu.L of indicator medium was added after the plate had solidified.

4. And after the indication bacterium liquid is dried, placing the sterile oxford cup into a flat plate containing the indication bacterium, and adding 50 mu L of sample into each oxford cup. The experimental design was as follows: group 1 is the 1% inoculum group; group 2 was 1.5% inoculum group; group 3 was the 2.0% inoculum group; group 4 was the 2.5% inoculum group; group 5 was the 3.0% inoculum group.

(4) And (3) putting the solid plate into a constant-temperature incubator at 37 ℃, culturing for 24h, and measuring the size of the bacteriostatic zone by using a vernier caliper and making a record.

3. And (3) test results: the specific test results are shown in table 1.

TABLE 1 size of different inoculum sizes to the diameter of the bacteria inhibition zone of the indicator bacteria

As is clear from Table 1, when the inoculum size was 2%, the diameters of the zones of inhibition were the largest for all four bacteria, and therefore, the inoculum size of 2% was the optimum inoculum size.

Test example 2 cultivation time screening

1. Test samples: bacillus coagulans; staphylococcus aureus bacteria; e.coli; salmonella; pasteurella; 2. the test method comprises the following steps: according to the extraction method disclosed by the embodiment 3 of the invention, the culture time is respectively changed into 24h, 32h, 36h, 40h and 48h, and the purity of the prepared bacteriocin is detected by utilizing an Oxford cup bacteriostasis test. 3. And (3) test results: the specific test results are shown in Table 2.

TABLE 2 measurement of different culture times for the diameter of the zone of inhibition of the indicator bacteria

As is clear from Table 2, the maximum diameter of the zone of inhibition for various bacteria was observed when the culture time was 36 hours, and therefore, the culture time of 36 hours was the optimum culture time.

Test example 3 screening of culture temperature

1. Test samples: bacillus coagulans; staphylococcus aureus bacteria; e.coli; salmonella; pasteurella; 2. the test method comprises the following steps: according to the extraction method disclosed by the embodiment 3 of the invention, the culture temperature is respectively changed into 25 ℃, 36 ℃, 37 ℃, 38 ℃ and 40 ℃, and the purity of the prepared bacteriocin is detected by utilizing an Oxford cup bacteriostasis test.

3. And (3) test results: the specific test results are shown in Table 3.

TABLE 3 size of inhibition zone of indicator bacteria for different cultivation time

As is clear from Table 3, the diameter of the zone of inhibition for each strain reached the maximum value at the culture temperature of 37 ℃ and thus the culture temperature of 37 ℃ was the optimum culture temperature.

Test example 4 Bacillus coagulans production assay (ca)

1. Test samples: the extracted coagulans of examples 1-3 and comparative examples 1-3;

2. the test method comprises the following steps: the method comprises the steps of detecting the diameter of a bacteriostatic zone of the bacillus coagulans by adopting an oxford cup method and taking escherichia coli and pasteurella as indicator bacteria, wherein the larger the diameter of the bacteriostatic zone is, the more the yield of the bacillus coagulans extracted by a corresponding test method is.

3. And (3) test results: the specific test results are shown in Table 4.

TABLE 4 comparison of the diameter of zone of inhibition for different test samples

Test sample	Escherichia coli (mm)	Pasteurella (mm)
			Example 1	26.56	27.35
Example 2	25.88	28.52
			Example 3	28.52	30.38
Comparative example 1	21.06	22.32
			Comparative example 2	19.96	20.56
Comparative example 3	18.75	19.46

As can be seen from Table 4, the content of Bacillus coagulans extracted by the method of examples 1-3 of the present invention is significantly higher than that of the groups of comparative examples 1, 2 and 3, especially the group of example 3, the diameters of inhibition zones for Escherichia coli and Pasteurella bacillus are 28.52 and 30.38, respectively, and the yield is extremely high, therefore, example 3 is the best example of the present invention, while the mutual synergy among the components of the lactic acid promoter is destroyed in the groups of comparative examples 1 and 2, which leads to a significant decrease in yield.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.