CN106636299B - Method for detecting bacterial contamination of edible fungus liquid strain - Google Patents
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Abstract
The invention provides a method for detecting bacterial contamination of liquid strains of edible fungi, which comprises the following steps: 1) respectively carrying out NB plate culture counting and fluorescent staining determination on the representative diluted bacterial suspension of the pollution bacteria of each dilution degree, wherein the representative pollution bacteria are common pollution bacterial strains of edible fungus liquid strains; establishing a standard curve according to the plate counting result (Bq) of each dilution bacterial suspension and the corresponding fluorescent staining determination result (Fg); 2) filtering the liquid strain culture solution of the edible fungi by using a filter membrane suitable for filtering the edible fungi hyphae; then, centrifuging the filtrate, removing the culture solution, washing the thallus precipitate by using sterile physiological saline, re-precipitating, taking sterile water to re-suspend the precipitated bacterial cells, and performing gradient dilution; 3) dyeing the gradient bacterial suspension of the series of the polluted bacteria obtained in the step 2), measuring the fluorescence value, and calculating the concentration of the polluted bacteria in the liquid strains of the edible fungi according to a standard curve. The method can rapidly and accurately determine the number of bacteria polluted by the liquid strains of the edible fungi.
Description
Technical Field
The invention relates to a method for detecting the quality of a liquid strain, in particular to a method for detecting the quality of the liquid strain in the industrial cultivation process of edible fungi, belonging to the field of microorganisms.
Background
The edible fungus industrial production in China has undergone the development process of more than 20 years, and the cultivation facility hardware and the technology level have great progress and innovation. The strain preparation is an important link in the industrial cultivation process of edible fungi, and is gradually developed from the traditional two-stage and three-stage strain production process based on solid culture base materials into a liquid strain production process with the advantages of high purity, strong activity, short period, low cost and the like. The application of the liquid spawn greatly shortens the cultivation period of the edible fungi, lays an important foundation for the industrial high-efficiency standardized production of the edible fungi, but the production process of the liquid spawn is easy to generate bacterial pollution, and the traditional solid spawn quality inspection method is difficult to be used for quality control of the liquid spawn.
The detection method related to the bacterial contamination of the liquid strains of the edible fungi reported in the literature at present mainly comprises sensory judgment, microscopic observation, pH value determination, culture detection and the like. Through sensory judgment of indexes such as color, smell and form, although the accuracy is high for early pollution of strain fermentation, errors are easily caused for pollution starting at the later stage of fermentation, meanwhile, not all bacterial pollution can form obvious sensory characteristics, and subjective sensory judgment lacks a unified standard; the detection precision of the microscopic observation and the measurement based on the change of the pH value on the pollution of a small amount of bacteria in the liquid strains is not enough, so that the methods can form misjudgment, thereby possibly causing serious loss on production. Compared with the detection method, the culture method can accurately judge the pollution condition of the liquid strain, but the culture time generally needs 24 to 48 hours, the detection timeliness is poor, the optimal inoculation time of the liquid strain can be delayed, and the utilization efficiency of liquid strain fermentation equipment is reduced. Therefore, the establishment of a rapid and accurate bacterial contamination detection technology is the key for the safe and efficient application of the liquid strain technology.
The patent technical application related to the quality detection of liquid strains of edible fungi only has 1 piece (CN 105087750A, "liquid strain detector for edible fungi"), which is a device for detecting the concentration of liquid strains. The patent application information for the detection of bacterial contamination by liquid strains is hardly available.
Disclosure of Invention
The invention aims to provide a detection method for rapidly and accurately measuring the number of bacteria polluted by liquid strains of edible fungi. The basic idea is as follows: the method is characterized in that the BAClight staining method is used for carrying out fluorescent staining on the polluted bacteria in the edible fungus liquid strain subjected to specific separation, and the quantity of the polluted bacteria in the edible fungus liquid strain is rapidly and accurately determined by establishing a mathematical relation model of the bacteria concentration and the fluorescent quantitative index.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a method for detecting bacterial contamination of liquid strains of edible fungi, which comprises the following steps:
1) preparation of a standard curve:
respectively carrying out NB plate culture counting and fluorescence staining determination on the obtained representative diluted bacterial suspension of the polluted bacteria with each dilution degreeBacteria G for common pollution of edible fungus liquid strains-And G+At least one of the contaminant-like bacteria, such as: pseudomonas (G)-) And Bacillus subtilis (G)+) (ii) a A standard curve was then established based on the plate count (Bq) and the corresponding fluorescent staining assay (Fg) for each dilution of bacterial suspension to define the range of linear relationship between the number of each representative contaminating bacteria and the fluorescence value.
Further, the representative contaminating bacteria may be two or more representative contaminating bacterial strains; preferably, the standard curve may be a fitted standard curve representing a standard curve of the contaminating bacterial strain used.
2) According to the difference of individual size and shape of edible fungus hypha and bacterial cell, a filter membrane with proper aperture is adopted, namely: suitable filters for filtering the hyphae of edible fungi are, for example: the nylon net with the aperture of 5 mu m is used for filtering the liquid strains, and the edible mycelium pellets are filtered from the liquid strain culture solution to avoid the interference on the quantitative detection of the polluted bacteria, and preferably, the filtering is repeated for 3 times; and then, centrifuging the filtrate, removing the culture solution, washing the thallus precipitate by using sterile physiological saline, reprecipitating, taking sterile water to resuspend the precipitated bacterial cells, and performing gradient dilution to form a series of gradient bacterial suspensions.
3) Dyeing the gradient bacterial suspension of the contaminated bacteria series obtained in the step 2) and determining the fluorescence value, preparing the standard curve in the step 1) by the method, and calculating the concentration of the contaminated bacteria in the liquid strains of the edible fungi according to the standard curve.
4) Calculation of correction coefficients and correction equations: quantitatively adding typical pollution bacteria into a strain to be detected to form a strain simulating bacteria pollution liquid, detecting and calculating the concentration (measured value) of the pollution bacteria by adopting the steps 2) and 3), and comparing the concentration with the concentration of the pollution bacteria actually doped into the liquid strain to obtain a correction coefficient; the correction formula is as follows: the actual value is the measured value × the correction coefficient. Wherein, the correction coefficient is to consider that part of the polluted bacteria thallus is still adhered to the mycelium pellet of the edible fungus in the suction filtration process, so that the concentration of the polluted bacteria obtained by the method is lower than the actual concentration, and a correction formula is established.
Through the technical scheme, the invention achieves the following beneficial effects:
the liquid strain bacterial contamination detection method provided by the invention can control the detection time within 90 minutes, and the error between the detection result and the detection result based on culture is small; compared with the sensory judgment and microscopic examination detection method in the prior art, the method has the obvious accuracy advantage, and has the advantages of convenience and rapidness compared with the traditional culture detection method, so that the method is a rapid and accurate detection method for the bacterial contamination of the liquid strain of the edible fungi; the method of the invention carries out accurate quantitative determination for the living cells of the polluted bacteria in the liquid strains, thereby providing a rapid, accurate and convenient detection means for the industrial application of the liquid strains of the edible fungi and having wider application and popularization values.
Drawings
FIG. 1 is a flow chart for establishing a standard curve for quantitative fluorescent staining measurement of the number of bacteria contaminated by liquid strains of edible fungi.
FIG. 2 is a flow chart of the separation and detection of contaminating bacteria in liquid strains of edible fungi of the present invention.
FIG. 3 is a flow chart of the determination of the number of bacteria in the liquid strain of edible fungi simulating bacterial contamination and the determination of the correction factor according to the present invention.
FIG. 4 is a graph of fluorescence staining intensity versus plate count results (standard curve) for two typical contaminating bacteria of the present invention. The figure respectively lists the mathematical relationship between the concentration of viable pseudomonas fluorescens and bacillus subtilis and the intensity of BAClight staining green fluorescence and the mathematical relationship between the number of viable pseudomonas fluorescens and bacillus subtilis and the intensity of green fluorescence after fitting.
Fig. 5 is a schematic view of the suction filtration apparatus. Wherein, the sterile environment refers to an ultra-clean workbench environment, and ensures that the foreign bacteria in the air pollute the suction filtration process. In the figure, 1 is a filter bowl, 2 is a clamp, 3 is a filter head, 4 is a conical flask, and 5 is a vacuum pump.
Detailed Description
In order to clarify the technical solution and technical object of the present invention, the present invention will be further described with reference to the accompanying drawings and the detailed description.
Example 1:
1. preparation of Standard Curve
FIG. 1 is a flow chart for establishing a standard curve for quantitative fluorescent staining measurement of the number of bacteria contaminated by liquid strains of edible fungi, wherein the concentration values of the bacteria are obtained by using two methods, namely plate counting and fluorescent staining respectively, and a mathematical relationship between the two methods is established to form the standard curve. The invention takes two typical contamination bacteria pseudomonas and bacillus subtilis pure strains as samples, and the process is also suitable for constructing the fluorescence quantitative determination standard curve of other common contamination bacteria in the production process of specific edible fungus liquid strains.
The specific operation steps are as follows:
1.1 contamination of the bacterium Pseudomonas with common liquid strains of edible fungi (G)-) And Bacillus subtilis (G)+) As representative strains of the contaminating bacteria, the strains are respectively inoculated into a sterilized NB liquid medium for shake flask culture under the following culture conditions: constant temperature of 28 ℃, shaking table rotating speed of 180r/min, and bacterial concentration (OD) of culture solution after 24 hours of culture600) In the range of about 1.8 to about 2.0;
1.2 taking 10ml to 50ml of culture solution of pseudomonas and bacillus respectively, centrifuging for 5min at 10000r/min, and discarding the supernatant;
1.3 the thalli sediment is fully washed by 30ml of sterilized normal saline for 3 times, each time is centrifuged for 5min at 10000r/min, and the supernatant is discarded;
1.4 taking 10ml of sterile water to carry out resuspension treatment on the precipitated bacterial cells, uniformly dispersing the bacterial precipitates in liquid by using a vortex instrument, and carrying out gradient dilution by 10 times to form a series of gradient bacterial suspensions; respectively taking 200 mu l of each gradient diluted bacterium liquid, coating the gradient diluted bacterium liquid on a sterilized NB plate culture medium, culturing at the constant temperature of 28 ℃ for 24 hours, counting, and calculating the viable bacteria concentration (Bq) in each gradient diluted bacterium liquid;
1.5 gradient dilution bacterial suspension (dilution factor is 10) for the pseudomonas and the bacillus subtilis prepared above respectively0～10-10) Performing fluorescent staining, and separating with staining agent2.5mL of sterilized deionized water is respectively used for dissolving, and the sterilized deionized water is mixed in equal proportion during the experiment, and the specific method comprises the following steps: adding 25 μ l SYTO 9+ PI (1:1 mixing) into 1ml serial diluted bacterial suspension, mixing, and dyeing at room temperature in dark for 15 min;
1.6 the fluorescence intensity of the bacterial dye solution with each dilution is measured by utilizing a Shanghai prism light F97Xp fluorescence spectrophotometer, and the instrument measurement conditions are as follows: the excitation wavelength is 470nm, the emission wavelength is 510nm, the bandwidth of the excitation and emission spectrums is 5nm, and the peak value of the absorption wavelength (510nm) of the green light is measured and is counted as Fg;
1.7 establishing a standard curve according to the plate counting result (Bq) of each dilution bacterial suspension and the corresponding fluorescent staining determination result (Fg), wherein the standard curve of the pseudomonas is as follows: fg1＝250.7lg(Bq1)+249.2，R20.997, detection range 5.2 × 100～5.2×107cfu/ml; bacillus standard curve: fg2＝248.5lg(Bq2)+216.7，R20.995, detection range 8.5 × 100～8.5×107cfu/ml; fitting a standard curve: fg 249.6lg (bq) +232.9, R20.996. As shown in fig. 4. Considering the complexity possibility of the bacterial contamination species in the fermentation process of the edible fungus liquid strain, the invention takes a fitting standard curve mathematical formula as the following calculation formula of the number of the contaminating bacteria.
2. Separating and detecting the polluted bacteria in the edible fungus liquid strain:
the separation and detection process of the contaminated bacteria in the liquid strains of the edible fungi can be seen in figure 2.
The method specifically comprises the following steps:
2.1, using a Jinteng GM-0.33II diaphragm vacuum pump to pump and filter 250ml of edible fungus liquid strains, and collecting the polluted bacterial bodies in the strains. The filter membrane is made of nylon net with the aperture of 5 mu m, the pressure is 1500Pa, and the suction filtration process is carried out on a superclean bench;
2.2 taking 30ml of sterile physiological saline to fully wash the hypha on the filter membrane, pumping and filtering the hypha into a bacteria collection bottle (shown in figure 5), and repeating the steps for 3 times to wash out the bacterial cells adsorbed on the hypha and the surface of the filter membrane;
2.3, filling the filtrate (about 330ml) in the bacteria collecting bottle into 2 sterile centrifuge tubes (centrifuge tube 1 and centrifuge tube 2) with the volume of 50ml in batches, and centrifuging for 5min at the speed of 10000r/min to finally obtain the thalli precipitate of the filtrate;
2.4 taking 30ml of sterilized normal saline, fully washing the thallus precipitate for 2 times, centrifuging for 5min at 10000r/min each time, and removing the supernatant; adding 10ml of sterilized normal saline into the centrifuge tube 1, merging the suspension into the centrifuge tube 2 after suspension treatment, washing the centrifuge tube 1 by using 10ml of sterilized normal saline, merging the washed centrifuge tube 2 into the centrifuge tube, repeating the washing for 1 time, and merging the washing liquid into the centrifuge tube 2. And (4) centrifuging the centrifugal tube 2 by the same method, and finally obtaining the polluted bacterial cell sediment in the edible fungus liquid strain.
2.5 taking 10ml of sterile water to carry out resuspension treatment on the precipitated bacterial cells, uniformly suspending the thalli by using a vortex instrument, and carrying out gradient dilution by 10 times to form serial dilution bacterial suspension;
2.6 taking the dilution of 10-1、10-3、10-5、10-7Adding 25 μ l SYTO 9+ PI (1:1 mixing) into 1ml of the bacterial suspension, mixing, and dyeing at room temperature in dark for 15 min;
2.7 measurement of the fluorescence intensity of the dye solution by means of Shanghai prism F97Xp fluorescence spectrophotometer (same as step 1.6 in example 1), selection of samples whose fluorescence intensity values lie in the linear range of the standard curve, calculation of the viable count (Bq) on the basis of the fitted standard curve (see step 1.7 in example 1), and calculation of the average value of the viable count of the samples.
Example 2:
in this example 2, the bacterial count of the liquid strain of Flammulina velutipes which simulates bacterial contamination was determined by the method of example 1, and the flow chart is shown in FIG. 3.
The method specifically comprises the following steps:
1) preparation of a culture medium: plate culture medium: PDA culture medium; liquid culture medium: corn flour 5%, wheat bran 3%, yeast extract 1%, sucrose 4%, KH2PO4 0.1％，MgSO4 0.15％,VB1 10mg/L，pH＝5.5。
2) Preparing flat strains of needle mushrooms: sterilizing the prepared PDA culture medium in a high-pressure steam sterilization pot (121 ℃, 30min), subpackaging in a sterilization culture dish, cooling, solidifying, inversely placing in a constant-temperature incubator at 28 ℃ for overnight culture, and removing a polluted plate; inoculating needle mushroom strain on PDA plate culture medium, and culturing at 28 deg.C for 7 d;
3) preparing a needle mushroom liquid strain: placing 200ml liquid culture medium into 500ml conical flask, sterilizing with high pressure steam autoclave (121 deg.C, 30min), cooling, and inoculating 3 pieces of 1cm per flask2Culturing Flammulina velutipes (Fr.) Sing strain at 28 deg.C for 8 days at 180 r/min; a sterile wet towel is placed in the shaking table to ensure that the strain is shaken and cultured in a constant humidity environment.
4) Preparing a needle mushroom liquid strain simulating bacterial pollution: the pseudomonas suspensions were aspirated separately (dilution plate count concentration 2.9X 107cfu/ml), bacillus suspension (8.7X 10 dilution plate count concentration)7cfu/ml) of the culture medium is respectively added into the liquid strains of the needle mushrooms cultured for 8 days and shaken up;
5) separating the polluted bacteria of the flammulina velutipes liquid strain: respectively carrying out suction filtration on the mixed solution by using a Jinteng GM-0.33II diaphragm vacuum pump (see figure 5), selecting a nylon net with the aperture of 5 mu m for a filter membrane, fully washing hyphae on the filter membrane for 3 times by using sterilized normal saline, respectively carrying out suction filtration, carrying out centrifugal precipitation on collected thalli, resuspending, and carrying out gradient dilution to obtain a series of bacterial suspensions with dilution degrees (the specific operation is the same as the steps 2.1-2.4 in the example 1);
6) respectively taking the dilution of 10 in serial dilution bacterial suspensions-1、10-3、10-5、10-7Adding 25 μ l SYTO 9+ PI (1:1 mixed) into 1ml of the bacterial suspension respectively, mixing uniformly, and dyeing for 15min at room temperature in a dark place;
7) measuring the fluorescence intensity of the dye solution by using a Shanghai prism light F97Xp fluorescence spectrophotometer, wherein the excitation wavelength is 470nm, the emission wavelength is 510nm, the bandwidth of the excitation and emission spectrums is 5nm, and the peak value Fg of the green light absorption wavelength (510nm) is measured; and calculating viable bacteria concentration according to fitting calculation formula, wherein the calculated values of the pseudomonas are respectively 1.37 multiplied by 106cfu/ml、1.48×106cfu/ml、1.54×106cfu/ml、1.33×106cfu/ml, compared with the theoretical value, the error range is-8.28% - + 6.21%; the number of bacillus is 4.01 multiplied by 10 respectively6cfu/ml、4.78×106cfu/ml、3.86×106cfu/ml、4.41×106cfu/ml, compared with the theoretical value, the error range is-11.26% + 9.89%.
Example 3:
in this example 3, the bacterial count of the liquid strain of apricot spore mushroom simulating bacterial contamination was measured by the method of example 1, and the flowchart is shown in FIG. 3.
1) Preparation of a culture medium: plate culture medium: PDA culture medium; liquid culture medium: 3% of glucose, 0.2% of peptone, 0.5% of yeast extract and KH2PO4 0.05％，MgSO4 0.05％，VB1 10mg/L，pH＝7.5。
2) Preparing flat strains of pleurotus eryngii: sterilizing PDA culture medium in high pressure steam sterilizing pot (121 deg.C, 30min), subpackaging the culture medium into sterilized flat plates, cooling, solidifying, placing in constant temperature incubator at 28 deg.C, culturing overnight, and removing contaminated flat plates; inoculating Pleurotus eryngii strain on PDA culture medium, and culturing at 28 deg.C for 7 d;
3) preparing pleurotus eryngii liquid strains: subpackaging liquid culture medium into 500ml conical flasks (200 ml per flask), sterilizing in high pressure steam sterilizer, cooling, inoculating 3 pieces of 1cm culture medium into each flask2Culturing Pleurotus eryngii strain at 28 deg.C for 8 days at 180 r/min; a sterile wet towel is placed in the shaking table to ensure that the strain is shaken and cultured in a constant humidity environment.
4) Preparation of simulated bacterial-contaminated pleurotus eryngii liquid strain and respective absorption of pseudomonas suspension (the counting concentration of a dilution plate is 2.9 multiplied by 10)7cfu/ml), bacillus suspension (8.7X 10 dilution plate count concentration)7cfu/ml) of the culture medium is respectively added into the pleurotus eryngii liquid strains cultured for 8d and mixed evenly;
5) separating the polluted bacteria of the pleurotus eryngii liquid strain: performing suction filtration on the above mixed solution with a Jinteng GM-0.33II diaphragm vacuum pump (see FIG. 5), washing mycelia on the filter membrane with sterilized normal saline for 3 times, and performing suction filtration; the filter membrane is made of a 5-micron nylon net with the pressure of 1500 Pa; fully washing hypha on a filter membrane for 3 times by using sterilized normal saline, respectively carrying out suction filtration, carrying out centrifugal precipitation on collected thalli, then suspending, and carrying out gradient dilution to obtain a series of diluted bacterial suspensions (the specific operation is the same as the steps 2.1-2.4 in the example 1);
6) respectively taking the dilution of the serial gradient bacterial suspensions as 10-1、10-3、10-5、10-7Adding 25 μ l SYTO 9+ PI (1:1 mixed) into 1ml of the bacterial suspension respectively, mixing uniformly, and dyeing for 15min at room temperature in a dark place;
7) measuring the fluorescence intensity of the dye solution by using a Shanghai prism light F97Xp fluorescence spectrophotometer, wherein the excitation wavelength is 470nm, the emission wavelength is 510nm, the bandwidth of the excitation and emission spectrums is 5nm, and the peak value Fg of the green light absorption wavelength (510nm) is measured; and calculating viable bacteria concentration according to fitting calculation formula, wherein the number of the pseudomonads is 1.34 multiplied by 106cfu/ml、1.42×106cfu/ml、1.58×106cfu/ml、1.30×106cfu/ml, compared with the theoretical value, the error range is-10.34% + 8.97%; the number of bacillus is 3.93X 106cfu/ml、4.16×106cfu/ml、4.56×106cfu/ml、4.81×106cfu/ml, compared with the theoretical value, the error range is-9.66% + 6.89%.
Example 4:
in this example 4, the bacterial count of different batches of liquid strains of needle mushroom of a certain edible mushroom factory production enterprise was measured. The method specifically comprises the following steps:
1) performing gradient dilution on a liquid strain sample of needle mushroom in an edible mushroom factory, and respectively taking 200 mul each gradient (10 mul)-1、10-3、10-5、10-7) The diluted bacteria liquid is spread on a sterile NB plate and cultured for 24 hours at a constant temperature of 28 ℃ for counting, each gradient is repeated for 3 times, and samples with the plate colony number between 30 and 300 are used for calculating the average value of the viable bacteria number.
2) 250ml of the liquid strain of the needle mushroom is taken and filtered and centrifuged according to the method (the steps 2.1 to 2.4 in the example 1) to prepare the gradient diluted bacterial suspension of the polluted bacteria in the liquid strain.
3) 1mL of gradient bacterial suspension (10) was taken separately-1、10-3、10-5、10-7) Adding 25 μ l SYTO 9+ PI (1:1 mixture), mixing, and dyeing at room temperature in dark for 15 min; by using shanghai arrisThe fluorescence intensity of the dye liquor is measured by a light F97Xp fluorescence spectrophotometer, the excitation wavelength is 470nm, the emission wavelength is 510nm, the bandwidth of both the excitation spectrum and the emission spectrum is 5nm, and the peak value Fg of the green light absorption wavelength (510nm) is measured.
4) According to the actual value of the number of bacteria in the liquid strains of the needle mushrooms and the pleurotus eryngii with simulated bacterial contamination and the calculated value of the fitting formula in the embodiments 2 and 3, the correction coefficient between the actual value and the measured value is calculated to be 1.869, namely the actual value is multiplied by 1.896; and calculating the viable count according to the fitting curve and the correction coefficient. The results of detecting the number of the polluted bacteria in different batches of the flammulina velutipes liquid strains are shown in table 1.
TABLE 1 detection results of the amount of bacteria contaminated by different batches of flammulina velutipes liquid strains
Example 5:
in example 5, the number of bacteria in different liquid strains of Pleurotus eryngii in a certain industrial production facility of edible fungi was measured.
The method specifically comprises the following steps:
1) performing gradient dilution on a pleurotus eryngii liquid strain sample in a certain edible fungus factory, and respectively taking 200 mu l of each gradient diluted bacterial liquid (10)-1、10-3、10-5、10-7) Inoculating to sterilized NB plate medium at 28 deg.C, culturing at constant temperature for 24 hr, counting, and repeating for 3 dilutions by the same method as 5.3.1;
2) taking 250ml of pleurotus eryngii liquid strains, carrying out suction filtration, centrifugation, washing and precipitation according to the method (step 2.1-2.4 in the example 1), and then preparing a gradient diluted bacterial suspension of polluted bacteria in the liquid strains;
3) respectively taking the dilution of the serial gradient bacterial suspensions as 10-1、10-3、10-5、10-7Adding 25 μ l SYTO 9+ PI (1:1 mixing) into 1ml of the bacterial suspension, mixing uniformly, and dyeing for 15min at room temperature in a dark place; measuring the fluorescence intensity of the dye solution by using a Shanghai prism light F97Xp fluorescence spectrophotometer with the excitation wavelength of 470nm, the emission wavelength of 510nm and the excitationMeasuring the peak value Fg of green light absorption wavelength (510nm) when the emission spectrum bandwidth and the emission spectrum bandwidth are both 5 nm; and calculating the viable count according to the fitted curve and the correction coefficient in the step 4) of the embodiment 4. The results of detecting the number of bacteria polluted by different batches of pleurotus eryngii liquid strains are shown in table 2.
TABLE 2 detection results of the number of bacteria contaminated by different batches of Pleurotus eryngii liquid strains
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Claims (2)
1. A method for detecting bacterial contamination of liquid strains of edible fungi is characterized by comprising the following steps:
1) preparation of a standard curve:
respectively carrying out NB plate culture counting and fluorescence staining determination on the obtained representative diluted bacteria suspension of each dilution, wherein the representative diluted bacteria is edible fungus liquid strain G-And G+At least one of the contaminating bacteria; then establishing a standard curve according to the plate counting result (Bq) of each dilution bacterial suspension and the corresponding fluorescent staining determination result (Fg); wherein, represents contaminating bacteria G-And G+Pseudomonas and bacillus subtilis respectively;
the specific operation steps are as follows:
1.1 using common edible fungus liquid strain polluted bacteria pseudomonas and bacillus subtilis as representative strains of polluted bacteria, respectively inoculating the representative strains in a sterilized NB liquid culture medium for shake flask culture, wherein the culture conditions are as follows: constant temperature of 28 ℃, table rotation speed: 180r/min, and the bacterial concentration range of the culture solution is 1.8-2.0 after 24 hours of culture;
1.2 taking 10mL to 50mL of culture solution of pseudomonas and bacillus respectively, centrifuging for 5min at 10000r/min, and discarding the supernatant;
1.3, fully washing the thalli precipitate for 3 times by using 30mL of sterilized normal saline, centrifuging for 5min at 10000r/min each time, and removing supernatant;
1.4 taking 10mL of sterile water to carry out resuspension treatment on the precipitated bacterial cells, uniformly dispersing the bacterial precipitates in liquid by using a vortex instrument, and carrying out gradient dilution by 10 times to form a series of gradient bacterial suspensions; respectively taking 200 mu L of each gradient diluted bacterium liquid, coating the gradient diluted bacterium liquid on a sterilized NB plate culture medium, culturing at the constant temperature of 28 ℃ for 24 hours, counting, and calculating the viable bacterium concentration Bq in each gradient diluted bacterium liquid;
1.5 respectively carrying out fluorescent staining on the pseudomonas and bacillus subtilis gradient diluted bacterial suspensions, respectively dissolving coloring agents by using 2.5mL of sterilized deionized water, and mixing in equal proportion during experiments, wherein the specific method comprises the following steps: adding 25 μ L SYTO 9+ PI into 1mL serial diluted bacterial suspension respectively, mixing at a ratio of 1:1, mixing uniformly, and dyeing for 15min at room temperature in a dark place;
1.6 the fluorescence intensity of the bacterial dye solution with each dilution is measured by utilizing a Shanghai prism light F97Xp fluorescence spectrophotometer, and the instrument measurement conditions are as follows: the excitation wavelength is 470nm, the emission wavelength is 510nm, the bandwidth of an excitation spectrum and the emission spectrum is 5nm, and the peak value of the absorption wavelength of the green light is measured and is counted as Fg;
1.7 establishing a standard curve according to the plate counting result Bq of each dilution bacterial suspension and the corresponding fluorescent staining determination result Fg, wherein the pseudomonas standard curve is as follows: fg1＝250.7lg(Bq1)+249.2，R20.997, detection range 5.2 × 100～5.2×107cfu/mL; bacillus standard curve: fg2＝248.5lg(Bq2)+216.7，R20.995, detection range 8.5 × 100～8.5×107cfu/mL; fitting a standard curve: fg 249.6lg (bq) +232.9, R2＝0.996；
2) Quantitatively adding the bacteria to be detected into the typical polluted bacteria used in the step 1) to form a liquid strain simulating bacterial pollution, and filtering a liquid strain culture solution by using a filter membrane suitable for filtering edible fungus hyphae; then, centrifuging the filtrate, removing the culture solution, washing the thallus precipitate by using sterile physiological saline, re-precipitating, taking quantitative sterile water to perform resuspension treatment on the precipitated bacterial cells, and performing gradient dilution to form a series of gradient bacterial suspensions; dyeing the obtained gradient bacterial suspension of the series of the polluted bacteria, measuring a fluorescence value, calculating the concentration of the polluted bacteria in the liquid strain of the edible fungi according to a standard curve, and comparing the concentration of the polluted bacteria with the concentration of the polluted bacteria actually participating in the liquid strain to obtain a correction coefficient; the correction formula is as follows: actual value is measured value x correction coefficient;
3) calculating the bacterial number of the liquid strain: filtering the liquid strain culture solution by using a filter membrane suitable for filtering edible fungus hyphae; then, centrifuging the filtrate, removing the culture solution, washing the thallus precipitate by using sterile physiological saline, re-precipitating, taking quantitative sterile water to perform resuspension treatment on the precipitated bacterial cells, and performing gradient dilution to form a series of gradient bacterial suspensions; and dyeing the obtained gradient bacterial suspension of the contaminated bacteria series, measuring the fluorescence value, fitting a standard curve to calculate the concentration of the contaminated bacteria in the liquid strain of the edible fungi, and calculating the actual value of the bacteria in the liquid strain of the edible fungi according to a correction formula.
2. The method for detecting bacterial contamination of liquid spawn of edible fungi according to claim 1, wherein in the step 2), the filter membrane suitable for filtering the hypha of the edible fungi is a nylon mesh with a pore size of 5 μm.
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