阅读说明：本技术 一种具有芥子苷高降解能力的菌种筛选鉴定方法 (Screening and identifying method for strain with high sinapiside degradation capability ) 是由 杨吉霞 于 2019-07-18 设计创作，主要内容包括：本发明公开了一种具有芥子苷高降解能力的菌种筛选鉴定方法,包括以下步骤：s1.进行芥子苷的提取和含量的测定,并设定芥子苷标准品的浓度；s2.利用MRS和PDA为培养基纯化菌种并进行形态学观察,将单个菌落进行液体培养之后做菌种的保藏和革兰氏染色；s3.对菌株进行芥子苷降解率的测定并进行第一次筛选,将筛选出的菌株进行耐受性实验并进行第二次筛选；s4.将筛选出来的菌株以16S rDNA基因序列分析法进行分子生物学快速鉴定。(The invention discloses a method for screening and identifying strains with high sinapiside degradation capability, which comprises the following steps: s1. extracting sinapine and measuring the content of sinapine, and setting the concentration of standard sinapine; s2, purifying strains by using MRS and PDA as culture media, observing morphology, and preserving and gram-staining the strains after carrying out liquid culture on a single bacterial colony; s3. measuring sinapin degradation rate of the strain and screening for the first time, and subjecting the screened strain to tolerance test and screening for the second time; s4. the selected strain is rapidly identified by 16S rDNA gene sequence analysis.)

1. A method for screening and identifying strains with high sinapiside degradation capability is characterized by comprising the following steps:

s1. extracting sinapine and measuring the content of sinapine, and setting the concentration of standard sinapine;

s2, purifying strains by using MRS and PDA as culture media, observing morphology, and preserving and gram-staining the strains after carrying out liquid culture on a single bacterial colony;

s3. measuring sinapin degradation rate of the strain and screening for the first time, and subjecting the screened strain to tolerance test and screening for the second time;

s4. the selected strain is rapidly identified by 16S rDNA gene sequence analysis.

2. The method for screening and identifying strains with high sinapiside degradation capability according to claim 1, wherein the method comprises the following steps: in step s1, the steps of extracting sinapiside and measuring the content of sinapiside comprise:

s11, weighing a certain mass of raw material containing sinapiside and preheating in a water bath;

s12, stirring and smashing the raw materials, and adding a methanol solution to carry out water bath extraction;

s13, adding barium acetate, mixing, subpackaging the mixture by using a centrifuge tube and centrifuging;

s14, carrying out water bath on the centrifuged supernatant until methanol is completely removed, and dissolving and filtering the obtained dry matter by using ultrapure water;

s15, adding a palladium chloride color developing solution and sodium carboxymethylcellulose into a set amount of sample solution, standing for color development, and setting a standard solution control group and a blank control group as a series a;

replacing the palladium chloride color developing solution in the a series with distilled water to be used as a b series;

oscillating and mixing the solutions of the a series and the b series, standing for color development, performing spectrophotometry on the solutions, and calculating the sinapiside content by using the following formula:

wherein C is sinaposide content (mg/g); e₁Subtracting the OD value of the blank control from the developing solution of the a series of samples; e₂Subtracting the OD value of the blank control from the developing solution of the b series samples; e₁Marked as the OD value of the a series standard color developing solution after blank deduction; e₂Marked as OD value of b series standard color development liquid after blank deduction; c is labeled as concentration of sinapioside standard solution (mg/mL); v is the volumetric volume (mL) of sinaposide sample; m is the mass (g) of the raw material used for extracting sinapiside.

3. The method for screening and identifying strains with high sinapiside degradation capability according to claim 1, wherein the method comprises the following steps: step s2 specifically includes:

s21, preparing a plurality of aseptic plates of PDA and MRS, and dripping a set amount of bacterial liquid into the corresponding aseptic plates after cooling and solidification;

s22, marking by using the inoculating loop after heating sterilization, marking the type time and the number of the marked culture dish, taking out the culture dish after the marking is finished, and putting the culture dish into an incubator for culture;

s23, after the first culture is finished, picking and placing a single bacterial colony into a corresponding liquid culture medium by using an inoculating loop subjected to heat sterilization, recording the type, the number and the date, and placing the bacterial colony into an incubator for culture after all the bacterial colonies are finished;

s24, sucking the bacterial liquid after the second culture, placing the bacterial liquid on a glass slide, heating the glass slide on flame of an alcohol lamp to fix the thalli, observing the shape of the thalli under a microscope after gram staining, and taking a picture;

and S25, adding the secondarily cultured bacterial liquid into glycerol, shaking and uniformly mixing, and freezing and storing.

4. The method for screening and identifying strains with high sinapiside degradation capability according to claim 1, wherein the method comprises the following steps: in step s3, the first screening of the strains comprises:

s31, taking a set amount of sinapiside solution, adding the sinapiside solution into the sterilized liquid culture medium by using a filter membrane, and then subpackaging the liquid culture medium into test tubes;

s32, adding the bacterial liquid after the set second culture into a corresponding test tube containing a PDA or MRS culture medium, setting a blank control, and marking date and number;

and S33, placing the bacteria into an incubator for culture, measuring the sinapioside concentration in each test tube after the culture is finished, calculating the degradation rate, screening out the bacteria liquid with higher degradation rate, and recording the serial number.

5. The method for screening and identifying strains with high sinapiside degradation capability according to claim 1, wherein the method comprises the following steps: in step s3, the second screening of the strains comprises:

s34, adding sinapine glycoside solution into an acid-resistant experiment culture medium to enable the sinapine glycoside solution to reach a set value, subpackaging the solution into test tubes, inoculating a set amount of bacteria solution, and ensuring that each pH value leaves an uninoculated test tube as a blank to measure the initial concentration of the sinapine glycoside; measuring the sinapine concentration after culturing, and calculating the degradation rate by using the initial value after culturing and the amount of degraded sinapine;

s35, adding sinapine glycoside solution into a salt tolerance experiment culture medium to enable the sinapine glycoside solution to reach a set value, subpackaging the solution into test tubes, inoculating a set amount of bacterial liquid, and ensuring that each test tube without inoculation is reserved at each salinity to serve as a blank test tube for measuring the initial concentration of the sinapine glycoside; measuring the sinapine concentration after culturing, and calculating the degradation rate by using the initial value after culturing and the amount of degraded sinapine;

and s36, integrating the degradation rate of each strain in the acid resistance experiment and the salinity resistance experiment, and screening out the strain with relatively good tolerance for recording.

6. The method for screening and identifying strains with high sinapiside degradation capability according to claim 1, wherein the method comprises the following steps: in step s4, the specific steps of strain identification are as follows:

s41. extraction of genomic DNA:

s42, performing gel electrophoresis on the extracted genome DNA solution;

s43, performing 16SrDNAPCR amplification;

s44, after all amplification is successful, carrying out 16S rDNA sequence determination on the PCR amplification product;

s45, strain 16S rDNA sequence alignment is carried out by using Blast of NCBI official network to identify strains.

Technical Field

The invention belongs to the technical field of degradation determination of sinaposide by microorganisms, and particularly relates to a method for screening and identifying strains with high sinaposide degradation capability.

Background

1. Brief introduction to sinapine

Sinapioside is also called glucosinolate, is glycoside formed by combining a sulfhydryl (-SH) -containing aglycone and glucose, and is mainly present in cruciferous plants. The structural formula is shown in figure 1, wherein R is various aryl and alkane. Sinapioside is an anti-nutritional factor, and is degraded to form isothiocyanates under the conditions of myrosinase or heat.

2. Characteristic research on glucosinolate degradation product isothiocyanate

Isothiocyanates are a family of compounds characterized by sulfur containing N ═ C ═ S functional groups. It is an important and unique plant product of two types. Isothiocyanates have a wide range of applications in the food, pharmaceutical and agricultural fields. Allyl isothiocyanate induces the death of colorectal and prostate cancer cells and inhibits the proliferation of various types of human cancer cells. Other pharmacological properties include antibacterial activity against a range of gram-negative and gram-positive pathogenic bacteria, antifungal activity, thyroid-inducing properties, and they have also been shown to be potent phase II enzyme inducers that can protect cells from the toxic and tumorigenic effects of carcinogens, inhibit nitric oxide production and inhibit the expression of inducible nitric oxide synthase enzymes involved in inflammation and cancer.

The degradation of sinaponin is very important for the formation of the flavor of tuber mustard, but more important isothiocyanate has a health-care effect, such as propenyl isothiocyanate compounds, 2-phenethyl isothiocyanic acid compounds, 3-butenenitrile, 3-phenylpropylcyanogen and other substances generated by the hydrolysis of the Fuling brassica oleracea glucosinolate have unique medicinal and health-care effects, can inhibit the growth of tumors such as breast cancer, gastric cancer, intestinal cancer, lung cancer and the like, has various pharmacological activities such as cough relieving, asthma relieving, slow pressure reducing and the like, has the function of regulating endocrine, can effectively prevent myocardial infarction, and has antioxidant, sexual improvement function and antibacterial effects. Isothiocyanates also do enhance immunity, enhance anti-mutation, anti-oxidant and anti-cancer properties; in addition, microorganisms in the intestinal tract of a human body can degrade the sulforaphane, the product sulforaphane is a common antioxidant and is a plant active substance with the best anti-cancer effect in vegetables, and the enterobacter coli is confirmed to have a gene for degrading sinaposide and can degrade the sinaposide. However, the degradation rate of the sinapioside cannot be guaranteed by human bodies, but most researches only study the properties of the sinapioside, and relatively few researches are carried out on the degradation rate of the sinapioside. Therefore, if the degradation rate of sinapioside can be improved in the process of pickling the preserved szechuan pickle, the flavor of the preserved szechuan pickle can be improved, the efficacy of the preserved szechuan pickle can be improved, and the healthy life of people is promoted.

3. Degradation of sinaposide

As early as 1983, it was experimentally determined that Bacillus cereus can degrade sinapioside; since the factors (such as temperature and glucose concentration) influencing the degradation of sinapioside by the myrosinase produced by Salmonella and Listeria monocytogenes were studied, the ability of bacteria to degrade sinapioside was long established.

In 1992, Aspergillus was found to produce sinapinase and was subsequently isolated from soil and screened for an Aspergillus strain that degrades sinapine; not only Aspergillus, but also lactic acid bacteria were found to be effective in converting sinaposide to nitrile, and fermentation time also affected the degradation of sinaposide by lactic acid bacteria, and it was observed that fermentation of lactic acid bacteria promoted complete degradation of sinaposide and the production of isothiocyanates. Thus, recent studies on sinaposide degradation have biased the fungal aspect.

4. Conventional microorganism identification method

The traditional microorganism classification and identification method is to carry out pure culture and separation on bacteria, and the used culture medium is artificially prepared and is suitable for the growth and the propagation of microorganisms. The conventional methods are a dilution coating flat plate method and a flat plate scribing method. The conventional identification technology is culture characteristic observation, morphological structure, biological biochemical test and the like. However, many microorganisms have been shown to be ineffective using current culture techniques. The conventional methods therefore have their limitations, which are described laterally by the diversity of the microorganisms.

Disclosure of Invention

The purpose of the invention is: the method comprises the steps of taking strains purified and preserved from a preserved pickle preserving solution as raw materials, culturing and purifying, then determining the degradation rate of each strain for degrading sinaposide by a palladium chloride method, primarily screening out the strains with strong degradation capability, then carrying out tolerance (acid resistance and salt resistance) experiments, carrying out secondary screening, identifying the strains screened out secondarily by using 16S rDNA sequence homology analysis, and constructing a phylogenetic tree.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

a method for screening and identifying strains with high sinapiside degradation capability comprises the following steps:

s1. extracting sinapine and measuring the content of sinapine, and setting the concentration of standard sinapine;

s2, purifying strains by using MRS and PDA as culture media, observing morphology, and preserving and gram-staining the strains after carrying out liquid culture on a single colony;

s3. measuring sinapin degradation rate of the strain and screening for the first time, and subjecting the screened strain to tolerance test and screening for the second time;

s4. the selected strain is rapidly identified by 16S rDNA gene sequence analysis.

Further, in step s1, the steps of extracting sinapiside and measuring the content of sinapiside comprise:

s11, weighing a set weight of sinapiside-containing raw material (white radish or tuber mustard) and preheating in a water bath;

s12, stirring and smashing the raw materials, and adding a methanol solution for water bath extraction;

s13, adding barium acetate, mixing, subpackaging the mixture by using a centrifuge tube and centrifuging;

s14, carrying out water bath on the centrifuged supernatant until the methanol is completely removed, and dissolving the obtained dry matter by using ultrapure water

Filtering;

s15, adding a palladium chloride color developing solution and sodium carboxymethylcellulose into a set amount of sample solution, standing for color development, and setting a standard solution control group and a blank control group as a series a;

replacing the palladium chloride color developing solution in the a series with distilled water to be used as a b series;

oscillating and mixing the solutions of the a series and the b series, standing for color development, performing spectrophotometry on the solutions, and calculating the sinapiside content by using the following formula:

wherein C is sinaposide content (mg/g); e1 is the OD value of the a series sample developing solution minus the blank control; e2 is the OD value of b series sample color development liquid minus the blank control; e1 is marked as the OD value of the a series standard developing solution minus the blank; e2 is marked by the OD value of b series standard developing solution minus the blank.

Further, step s2 specifically includes:

s21, preparing a plurality of aseptic plates of PDA and MRS, and dripping a set amount of bacterial liquid into the corresponding plates after cooling and solidification;

s22, marking by using the inoculating loop after heating sterilization, marking the type time and the number of the marked culture dish, taking out the culture dish after the marking is finished, and putting the culture dish into an incubator for culture;

s23, after the first culture is finished, picking and placing a single bacterial colony into a corresponding liquid culture medium by using an inoculating loop subjected to heat sterilization, recording the type, the number and the date, and placing the bacterial colony into an incubator for culture after all the bacterial colonies are finished;

s24, sucking the bacterial liquid after the second culture, placing the bacterial liquid on a glass slide, heating the glass slide on flame of an alcohol lamp to fix the thalli, observing the shape of the thalli under a microscope after gram staining, and taking a picture;

and S25, adding the secondarily cultured bacterial liquid into glycerol, shaking and uniformly mixing, and freezing and storing.

Further, in step s3, the first screening of the strains comprises:

s31, taking a set amount of sinapiside solution, adding the sinapiside solution into the sterilized liquid culture medium by using a filter membrane, and then subpackaging the liquid culture medium into test tubes;

s32, adding the bacterial liquid after the set second culture into a corresponding test tube containing a PDA or MRS culture medium, setting a blank control, and marking date/number;

and S33, placing the bacteria into an incubator for culture, measuring the sinapine concentration in each test tube after the culture is finished, calculating the degradation rate, screening out the bacteria liquid with higher degradation rate, and recording the serial number.

Further, in step s3, the second screening of the strains comprises:

s34, adding sinapine glycoside solution into an acid-resistant experiment culture medium to enable the sinapine glycoside solution to reach a set value, subpackaging the solution into test tubes, inoculating a set amount of bacteria solution, and ensuring that each pH value leaves an uninoculated test tube as a blank test tube for testing the initial concentration of the sinapine glycoside; measuring the sinapine concentration after culturing, and calculating the degradation rate by using the initial value after culturing and the amount of degraded sinapine;

s35, adding sinapine glycoside solution into a salt tolerance experiment culture medium to enable the sinapine glycoside solution to reach a set value, subpackaging the sinapine glycoside solution into test tubes, inoculating a set amount of bacterial liquid, and ensuring that each test tube without inoculation is reserved at each salinity to serve as a blank test tube for measuring the initial concentration of the sinapine glycoside; measuring the sinapioside concentration after culture, and calculating the degradation rate by using the initial value after culture and the degradation amount of the sinapioside;

and s36, integrating the degradation rate of each strain in the acid resistance experiment and the salinity resistance experiment, and screening out the strain with relatively good tolerance for recording.

Further, in step s4, the specific steps of strain identification are as follows:

s41. extraction of genomic DNA:

s42, performing gel electrophoresis on the extracted genome DNA solution;

s43, performing 16S rDNAPCR amplification;

and S44, after all amplification is successful, carrying out 16S rDNA sequence determination on the PCR amplification product.

Drawings

FIG. 1 shows a simplified structural formula of sinapioside;

FIG. 2 shows sinapiside concentration and E_{1 Label}-E_{2 label}A relationship graph;

FIG. 3 is a DNA gel electrophoresis diagram;

FIG. 4 is a diagram showing PCR amplification of a 16S rDNA fragment;

FIG. 5 is a 16S rDNA-based phylogenetic tree of strains.

Detailed Description

The experimental materials required by the screening and identification method of the strain with high sinapiside degradation capability are as follows:

experimental samples: the strain culture solution is obtained by culturing, separating, purifying and preserving the strain culture solution from the preserved szechuan pickle.

The main reagents are as follows: PDA culture medium: 200g of potato, 20g of glucose and 15g of agar. Peeling 200g potato, cutting into small pieces, boiling in a beaker for 30min, stirring with a glass rod to prevent bottom sticking, and filtering with double-layer gauze. Adding 20g glucose and 15g agar into the filtrate, heating to dissolve completely, adding distilled water to make the volume of the solution to 1000mL, and autoclaving at 121 deg.C for 15 min.

MRS culture medium: weighing 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 1mL of Tween reagent, 5g of sodium acetate, 2g of disodium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and 18g of agar, adding the materials into 1000mL of distilled water, heating to dissolve, adjusting the pH value to 6.2-6.6 after constant volume, and sterilizing at 121 ℃ for 15min under high pressure.

MRS liquid medium: the MRS culture medium is prepared by adding no agar, and sterilizing at 121 deg.C for 15 min.

PDA liquid culture medium: the PDA culture medium is prepared by dissolving agar completely in the same manner, and autoclaving at 121 deg.C for 15 min.

Acid-resistant experimental culture medium: preparing a mixture containing 5.5mmol glucose, 7g/L yeast extract, and 10g/L NaH₂PO₄、10g/LNa₂HPO₄The solution of (2) was mixed by a vortex shaker, and the mixture was divided into 6 flasks, and the pH was adjusted to 3.0, 3.5, 4.0, 4.5, 5.0, and 6.5 with lactic acid (control). Packaging the prepared liquid into high temperature sterilizing bottles, and autoclaving at 121 deg.C for 15 min.

Salt tolerance experiment culture medium: preparing a mixture containing 5.5mmol glucose, 7g/L yeast extract, and 10g/L NaH₂PO₄、10g/LNa₂HPO₄The solution is mixed evenly by a vortex oscillator, and the mixture is divided into 7 conical flasks, and the salinity is adjusted to 4%, 6%, 8%, 10%, 12%, 14% and 16% by sodium chloride respectively. Packaging the prepared liquid into high temperature sterilizing bottles, and autoclaving at 121 deg.C for 15 min.

The main drugs used in this experiment are shown in table 1.

TABLE 1 major drugs

(1)50 × TAE: 121g Tris and 18.6g Na were weighed out separately₂EDTA·2H₂Adding 400mL of deionized water and 28.55mL of glacial acetic acid into a beaker, stirring to dissolve the deionized water and the glacial acetic acid, adjusting the pH value to 8.3 by using NaOH, adding the deionized water to a constant volume of 500mL, and storing at room temperature.

(2)8mmol/L palladium chloride color developing solution: 354mg of palladium chloride powder is accurately weighed in a 150mL beaker, 4.0mL and 20mL of distilled water are respectively added into 2mol/L hydrochloric acid solution, after heating and dissolving (stirring continuously by a glass rod in the heating process), the distilled water is added to a constant volume of 250mL, and then the solution is transferred into a brown bottle for storage and standby.

(3) 1.5% sodium carboxymethyl cellulose solution (CMC): accurately weighing 1.5g of sodium carboxymethylcellulose in a 250mL beaker, adding a small amount of distilled water, heating, continuously stirring by using a glass rod until the sodium carboxymethylcellulose is completely dissolved, adding the distilled water for dilution, fixing the volume to 100mL by using a volumetric flask, standing overnight, and taking supernatant liquid to a brown reagent bottle for storage for later use the next day.

Experimental instrument equipment

The main instrumentation used in this experiment is shown in table 2.

TABLE 2 Main instrumentation

The method for screening and identifying the strain with sinapine glycoside high degradation capability comprises the following steps:

s1. extracting sinapine and measuring the content of sinapine, and setting the concentration of standard sinapine;

(1) extraction of sinapioside from raw material

The radish was refrigerated, cut into pieces and weighed 400 g. Placing into 80 deg.C water bath to preheat for 10 min. Breaking radix Raphani and juice in a blender, taking out, adding 200mL 70% methanol solution, and extracting in 75 deg.C water bath for 20 min. After cooling, 1mL of barium acetate was added and mixed thoroughly with a vortexer. And subpackaging the obtained mixture by using a 50mL centrifuge tube, wherein the height of the centrifuge tube does not exceed 2/3, centrifuging for 10min at 4000r/min, and preserving the supernatant. And subpackaging the supernatant into conical flasks, performing water bath until the methanol is completely removed, and dissolving the obtained dry matter with a small amount of ultrapure water for filtering for later use.

(2) Determination of sinapiside content

Sucking 1mL of each sample solution, placing the sample solution in a small graduated test tube, adding 1mL of 8mmol/L palladium chloride color development solution and 2mL of 0.15% sodium carboxymethylcellulose (CMC) solution, and standing for color development; taking 1mL of sinapiside standard solution, adding 1mL of 8mmol/L palladium chloride color developing solution and 2mL of 0.15% sodium carboxymethylcellulose (CMC) solution as standard control; meanwhile, 1mL of 8mmol/L palladium chloride color developing solution and 2mL of 0.15% CMC solution are added into 1mL of distilled water to serve as blank control (a series). In order to correct errors possibly caused by the light absorption value of the sample liquid, a b series without adding a color developing solution is prepared at the same time, namely 1mL of distilled water is used for replacing 1mL of 8mmol/L palladium chloride color developing solution and is added into a small graduated test tube mixed with 1mL of sample liquid (standard solution) and 2mL of 0.15% CMC solution for color development, and meanwhile, blank control is carried out.

Namely a series: sample solution 1mL + PdCl₂Color development liquid 1mL + CMC 2mL

Standard solution 1mL + PdCl₂Color development liquid 1mL + CMC 2mL

Distilled water 1mL + PdCl₂Color development liquid 1mL + CMC 2mL

b series: sample solution 1mL + distilled water 1mL + CMC 2mL

Standard solution 1mL, distilled water 1mL and CMC 2mL

1mL of distilled water, 1mL of distilled water and 2mL of CMC

Transferring the solutions of the a series and the b series into a centrifuge tube respectively, placing the centrifuge tube on a vortex oscillator for oscillation for a few seconds, after uniformly mixing, developing for 6 hours at room temperature, carrying out spectrophotometry on the solutions at 540nm, repeating the steps for 4 times for each sample, and taking an average value.

Sinapiside content calculation formula:

c: the content (mg/g) of sinapioside in the raw material

E₁: OD value of a series of samples after blank contrast is deducted from developing solution

E₂: OD value of b series sample developing solution minus blank control

E_{1 Label}: OD value of a series standard color developing solution after blank deduction

E_{2 label}: OD value of b series standard color developing liquid after blank deduction

C_{Sign board}: concentration of sinapioside Standard solution (mg/mL)

V: constant volume (mL) of sinapioside sample

M: quality (g) of raw material for extracting sinapioside

(3) Determination of sinapin Standard concentration

Preparing small amount of sinapiside solution into 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2.0mg/mL, respectively, and determining E as standard solution_{1 Label}And E_{2 label}Making a variance line, and determining the concentration of the standard substance used in the later experiment.

S2, purifying strains by using MRS and PDA as culture media, observing morphology, and preserving and gram-staining the strains after carrying out liquid culture on a single colony;

(1) separation and purification of bacterial liquid

After alcohol sterilization is carried out on the sterile operating table, a culture medium, a gun head, a pipette gun, a culture dish and the like are placed on the sterile operating table for ultraviolet sterilization for 30min, then a plurality of sterile plates of PDA and MRS are prepared, after cooling and solidification, 0.1mL of bacterial liquid is dripped into the corresponding plates by the pipette gun, and then lineation is carried out by an inoculating loop after heating sterilization. Marking the type time and numbering of the marked culture dish, taking out the culture dish after all the types are marked, and putting the culture dish into a constant temperature incubator at 28 ℃ for 3 days.

After the first culture is completed, picking and placing a single colony into a corresponding liquid culture medium by using an inoculating loop after heat sterilization in an aseptic operation table, recording the type, the number and the date, and putting the single colony into a constant-temperature incubator at 28 ℃ for culture for 3 days after all the single colonies are completed.

(2) Morphological observation

The colonies after plate culture were observed for color, edge, transparency, and shape.

And sucking a small amount of bacteria liquid obtained after secondary culture in a sterile operating platform by using a liquid transfer gun, placing the bacteria liquid on a glass slide, heating the glass slide on flame of an alcohol lamp to fix the bacteria, observing the shape of the bacteria under a microscope after gram staining, and taking a picture.

(3) Preservation of strains

And adding 500 mu L of the liquid culture bacterial liquid after the secondary culture into 500 mu L of 30% glycerol (the final concentration of the glycerol is 15%) by using a pipette gun, uniformly shaking, and placing in a refrigerator at the temperature of-20 ℃ for freezing and storing.

s3. measuring sinapin degradation rate of the strain and screening for the first time, and subjecting the screened strain to tolerance test and screening for the second time;

(1) screening of strains

mu.L of sinapioside solution was taken in a sterile operating station, added to the autoclaved liquid medium using a 20 μm filter and then dispensed into test tubes. And (3) taking 25 mu L of the bacterial liquid after the second culture by using a pipette, adding the bacterial liquid into a corresponding test tube containing a PDA or MRS culture medium, simultaneously making a blank control, and marking the date and the number. After all the components are finished, the mixture is put into a constant temperature incubator at 28 ℃ for 3 days. And measuring the sinapine concentration in each test tube after the culture is finished and calculating the degradation rate. And screening out the bacterial liquid with higher degradation rate and recording the number.

(2) Acid resistance test

After the culture medium of the acid resistance experiment is sterilized, sinapiside solution is added after cooling to ensure that the concentration of the sinapiside solution is 1.5 g/L. And (3.5 mL of bacteria liquid is inoculated into the sterilized test tube by a pipette gun to inoculate 100-200 mL of bacteria liquid, and an uninoculated test tube is left at each pH value to be used as a blank for measuring the initial concentration of sinapiside. Sinapine concentration was measured after 3 days of culture. And calculating the degradation rate by using the initial value after culture and the amount of degraded sinapiside.

(3) Salinity tolerance test

After the culture medium for the salt tolerance experiment is sterilized, sinapiside solution is added after the liquid is cooled to ensure that the concentration of the sinapiside solution is 1.5 g/L. And (3.5 mL of bacteria liquid is inoculated into the sterilized test tube by a pipette gun to inoculate 100-200 mL of bacteria liquid, and an uninoculated test tube is left at each salinity as a blank for measuring the initial concentration of sinapine. The concentration of glucosinolates was measured after 3 days of culture. And calculating the degradation rate by using the initial value after culture and the amount of degraded sinapiside.

s4. the selected strain is rapidly identified by 16S rDNA gene sequence analysis.

(1) Extraction of genomic DNA

Bacterial genomic DNA was extracted using the Tiangen "bacterial genomic DNA extraction kit (DP 302)" following the instructions.

1. Centrifuging 1mL of the bacterial culture solution at 1000rpm for 1min, and completely sucking the supernatant as much as possible;

2. adding 180mL of buffer solution GA into the thallus precipitate, oscillating until the thallus is completely suspended, adding 10 mu L of lysozyme, and carrying out water bath at 37 ℃ for 15 min;

3. adding 10 mu LProteinase K solution into the tube, uniformly mixing, and placing in a water bath at 37 ℃ for 30 min;

4. adding 220 μ L buffer solution GB, shaking for 15s, standing in 70 deg.C water bath for 10min, cleaning the solution, and centrifuging to remove water drop on the inner wall of the tube cover;

5. adding 220 μ L of anhydrous ethanol, fully shaking and mixing uniformly for 15s, wherein flocculent precipitate may appear, and centrifuging to remove water drops on the inner wall of the tube cover;

6. the solution and flocculent precipitate obtained in the previous step were added to an adsorption column CB3 (adsorption column added to adsorption tube) and centrifuged at 12000rpm for 30 s. Pouring out waste liquid, and placing adsorption column CB3 into the collecting pipe

7. Adding 500mL GD into an adsorption column CB3, centrifuging at 12000rpm for 30s, pouring waste liquid, and putting the adsorption column GB3 into a collecting pipe;

8. adding 600 μ L of rinsing liquid PW into adsorption column GB3, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column GB3 into a collecting tube;

9. repeating operation 8;

10. putting the adsorption column GB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, pouring off the waste liquid, placing the adsorption column GB3 at room temperature for several minutes to completely dry the rinsing liquid remained in the adsorption column material;

11. transferring the adsorption column GB3 into a clean centrifugal tube, suspending and dripping 50-200 mu L of elution buffer TE into the central part of the adsorption membrane, standing at room temperature for 2-5 min, and centrifuging at 12000rpm for 2min to collect the solution into the centrifugal tube.

(2) DNA gel electrophoresis

The extracted genomic DNA solution was subjected to 1.5% agarose gel electrophoresis at a voltage of 90V for 30min, and subjected to image analysis using a gel imager.

(3)16S rDNAPCR amplification

Using the extracted genome DNA as a template for PCR amplification, selecting the most common general primers for bacteria, namely a forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided. The reaction system and the reaction procedure are shown in table 3 and table 4.

TABLE 3 PCR reaction System

Table 3 Reaction system of PCR amplification

TABLE 4 PCR reaction procedure

Table 4 Procedure of PCR amplification

And (3) carrying out 1.5% agarose gel electrophoresis on the product obtained by the PCR for 30-40 min under the voltage of 90V. If the PCR amplification was successful, a band of about 1500bp could be seen.

(4)16S rNDA sequencing

Re-amplifying the amplification failure until all the amplification failures are successful, and sending the PCR amplification product to the company of Biotechnology engineering (Shanghai) to carry out sequencing.

s5. results and analysis

(1) Selection of sinapiside concentration

It is evident from FIG. 2 that the most satisfactory concentration of sinapiside is used at 1 mg/mL.

(2) Determination of sinaposide content and degradation rate of each strain in primary screening

The sinapiside content of the cultured strain was measured by the palladium chloride method. The sinapioside content and degradation rate in each medium are shown in Table 5.

TABLE 5 bacterial Strain sinapin concentration and degradation Rate

Table 5 Concentration and degradation rate of glucosides

As can be seen from the results in the table above, the degradation rates of the strains are different and have obvious difference values, the extreme value reaches 77.65%, and only eleven strains with degradation rates of more than 50%, namely 13-1, 13-2, 13-6, 13-9, 13-11, 13-14, 13-18, 13-19, 13-20, 13-23 and 13-26, are selected for carrying out tolerance experiments.

(3) Determination of sinaposide content and degradation rate of each strain in secondary screening

The sinapiside content of the cultured strain was measured by the palladium chloride method. The sinapioside content and degradation rate in each medium are shown in the table below.

Results at various pH values:

TABLE 6 sinapioside concentration (mg/mL)

Table 6 Concentration of glucosides

TABLE 7 degradation rates of sinapioside

Table 7 Degradation rate of glucosinolates

Results at various salinity:

TABLE 8 sinapioside concentration (mg/mL)

Table 8 Concentration of glucosides

TABLE 9 degradation rates of sinapioside

Table 9 Degradation rate of glucosinolates

The degradation rate of each strain at each acidity and salinity is shown in the table above, and strains with stronger capacity, namely strains with the degradation rate higher than 45% at each pH and salinity, are screened out.

(4) Morphological characteristics of bacterial colony and thallus

The individual colonies of the screened strains were morphologically observed, and the color, edge, transparency, shape, etc. of the colonies were recorded, and their shapes and colors under a microscope were recorded, see table 10.

TABLE 10 form of the strain and microscopic examination results

Table 10 Strain morphology and microscopic examination resuLts

(5) Genomic DNA gel electrophoresis

The agarose gel is used for separating DNA fragments with the size of 0.2-50 kb, and completely accords with the DNA detection of the experiment. The extracted genomic DNA was subjected to 1.5% agarose gel electrophoresis, photographed and observed by a gel imager, and the result is shown in FIG. 3.

As can be seen from FIG. 3, a fluorescence band appeared, indicating that DNA was extracted, and that amplification and sequencing of 16SrDNA could be performed although the brightness was different.

(6) PCR amplification results for 16S rDNA

The PCR-amplified products of each strain were subjected to 1.5% agarose gel electrophoresis using 16S rDNA, and photographed and observed by a gel imager, and the results are shown in FIG. 4.

(7) Results of sequencing and similarity analysis

The DNA database of the NCBI official website was registered, and the sequence of the strain thus determined was subjected to similarity analysis using Blast, the results of which are shown in Table 11.

TABLE 1116S rDNA sequence homology comparison results

Table 11 Homology comparison of 16S rDNA Sequence

As can be seen from Table 11, the similarity was greater than 97.5%, and the sequencing was successful.

And (3) constructing a phylogenetic tree by utilizing the sequence of the identification result and the sequence of the measured strain by a Neighbor-Joining method through MEGA 6 software, and deleting the base groups which are not aligned at two ends after comparing all the sequences by using actinomycetes (Actinobacteria strain PB90-5) of the sequence outside the phylogenetic tree. The evolutionary tree is displayed by adopting a method of 'computer Linear zero tree', and the evolutionary tree is converted into a picture file through a function of 'Image → Save as Enhanced Metafile (EMF)', so that the result is clearer and more intuitive as shown in figure 5.

Conclusion

(1) In the research, 26 strains are selected from the strains separated, purified and preserved from the preserved liquid of the tuber mustard, the sinapiside content of the strains is determined by a palladium chloride method through primary culture, and 11 strains with stronger degradation capability are screened out; these 11 strains were cultivated at 6 pH (3, 3.5, 4, 4.5, 5, 6.5) and 7 salinity (4%, 6%, 8%, 10%, 12%, 14%, 16%) and 5 more tolerant strains were selected: 13-9, 13-14, 13-18, 13-19 and 13-23. Wherein 13-9, 13-19, 13-23 are identified as Bacillus amyloliquefaciens; 13-14 identified as corynebacterium mutans; 13-18 are marine bacteria.

(2) The experiment measures the sinapiside degradation capability of each strain under a plurality of pH and salinity, and provides reference for production strains in the preserved szechuan pickle process.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, 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 may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.