CN106636299A - Bacterial pollution detection method for edible fungus liquid strain - Google Patents

Bacterial pollution detection method for edible fungus liquid strain Download PDF

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CN106636299A
CN106636299A CN201611223693.6A CN201611223693A CN106636299A CN 106636299 A CN106636299 A CN 106636299A CN 201611223693 A CN201611223693 A CN 201611223693A CN 106636299 A CN106636299 A CN 106636299A
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bacteria
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liquid strain
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edible fungi
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CN106636299B (en
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胡建
梅丽娟
张洋
李青
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Yangzhou University
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Abstract

The invention provides a bacterial pollution detection method for an edible fungus liquid strain. The bacterial pollution detection method comprises the following steps: 1) performing NB plate cultivation counting and fluorescent staining determination on representative polluted bacteria diluted bacterial suspensions of various dilution degrees, and building a standard curve according to plate counting results (Bq) and corresponding fluorescent staining determination results (Fg) of the bacterial suspensions of various dilution degrees, wherein the representative polluted bacteria are common polluted bacterial strains in an edible fungus liquid strain; 2) filtering an edible fungus liquid strain nutrient solution by using a filtering membrane which is suitable for filtering edible fungi mycelia, centrifuging filtrate, removing the nutrient solution, washing thallus precipitate by using aseptic normal saline, precipitating, re-suspending precipitated bacterial cells with sterile water, and performing gradient dilution; 3) straining a series of gradient bacterial suspensions of the polluted bacteria obtained in the step 2), determining fluorescence values, and calculating the concentration of the polluted bacteria in the edible fungus liquid strain according to the standard curve. By adopting the method, the number of the polluted bacteria in the edible fungus liquid strain can be measured rapidly and accurately.

Description

A kind of edible fungi liquid strain germ contamination detection method
Technical field
The present invention relates to a kind of method of liquid spawn quality testing, more particularly to a kind of edible fungus industrial cultivation process The quality determining method of middle liquid spawn, belongs to microorganism field.
Background technology
China's edible fungus industrial industry experienced the development course of more than 20 years, and cultivating facility hardware and technical merit have Huge progress and innovation.Bacterial classification prepares the important step as edible fungus industrial cultivation process, gradually by traditional base Two grades in solid culture base-material, three-level kind production technology develop into purity is high, energetic, cycle is short, cost it is low excellent The liquid spawn production technology of point.The application of liquid spawn greatly shortens the edible fungus culturing cycle, is that edible fungus industrial is efficient Standardized production has established important foundation, but liquid spawn production process easily produces germ contamination, and conventional solid bacterial classification Detection method for quality is difficult for carrying out Quality Control to liquid spawn.
The detection method of the relevant edible fungi liquid strain germ contamination of current document report, mainly judge including sense organ, Micro- sem observation, acid-base value determine and cultivate detection etc..Judged by the sense organ of the indexs such as color, smell and form, though So for strain fermentation early stage pollution has high accuracy, but the pollution to starting from the fermentation later stage easily causes error, while not All germ contaminations can form obvious organoleptic feature, and the sense organ of subjectivity judges to lack unified standard；Microscope is seen The measure examined and changed based on acid-base value to liquid spawn in a small amount of germ contamination accuracy of detection not enough, therefore these methods Erroneous judgement is likely to form, consequently, it is possible to causing the heavy losses for producing.Compared with above-mentioned detection method, cultural method can be more Judge liquid spawn pollution situation exactly, but because incubation time is generally required 24 hours to 48 hours, detect it is ageing compared with Difference, may be delayed the optimal inoculation time of liquid spawn, reduce the utilization ratio of liquid fungus seed equipment.Hence set up it is quick, Accurate germ contamination detection technique, is the key of liquid spawn technical security, efficient application.
Patented technology application about edible fungi liquid strain quality testing only has 1 (CN 105087750A, " edible mushroom Liquid spawn detector "), it is a kind of device for liquid spawn Concentration Testing.For liquid spawn germ contamination detection APPLICATION INFORMATIONThe is nearly no.
The content of the invention
It is an object of the invention to provide a kind of quick, accurate detection for determining edible fungi liquid strain polluted bacteria quantity Method.Its basic concept is：With BAClight decoration methods to the dirt in the edible fungi liquid strain that have passed through specific isolation Dye bacterium carries out fluorescent staining, quick, accurate to determine by the numerical relationship model for setting up bacterial concentration and fluorescent quantitation index Edible fungi liquid strain polluted bacteria quantity.
In order to realize object above, technical scheme is as follows：
The invention provides a kind of edible fungi liquid strain germ contamination detection method, comprises the steps：
1) prepared by calibration curve：
Respectively each dilution polluted bacteria dilution bacteria suspension that represents to obtaining carries out NB flat board culture countings and fluorescence Dyeing is determined, and described represents polluted bacteria as the common polluted bacteria polluted bacteria G of edible fungi liquid strain-And G+Class pollution is thin It is at least one respectively in bacterium, such as：Pseudomonad (G-) and bacillus subtilis (G+)；Subsequently according to each dilution factor bacteria suspension flat board Count results (Bq) and corresponding fluorescent dyeing determination result (Fg) set up calibration curve, and with clearly every kind of polluted bacteria quantity is represented The linear relationship scope between fluorescent value.
Further, described representative polluted bacteria can two kinds or more of represent polluted bacteria strain；Preferably, institute The calibration curve stated can be the fit standard curve of used representative polluted bacteria strain calibration curve.
2) according to hypha of edible fungus and the otherness of bacterial cell Individual Size and form, using the suitable filter membrane in aperture, I.e.：It is adapted to filter the filter membrane of hypha of edible fungus, such as：5 μm of aperture nylon wire, filters, by edible mycelium pellet to liquid spawn Filter from strain cultivation liquid, it is to avoid it produces interference to polluted bacteria quantitative determination, it is preferable that repeat filter 23；With Afterwards, filtrate is centrifuged, discards nutrient solution, and bacterial sediment thing is washed using SPSS, reprecipitation takes Sterilized water carries out settling flux process to precipitum cell, and carries out gradient dilution formation graded series bacteria suspension.
3) to step 2) in obtain polluted bacteria graded series bacteria suspension dyeed and determined fluorescent value, method synchronization It is rapid 1) in calibration curve prepare, and establishing criteria curve calculate edible fungi liquid strain in polluted bacteria concentration.
4) calculating of correction coefficient and updating formula：Typical contaminated bacteria is quantitatively adding by bacterial classification to be measured, simulation is formed thin Bacterium pollution liquid bacterial classification, and using step 2) and 3) detection calculate its polluted bacteria concentration (measured value), and with liquid spawn in It is actual to mix polluted bacteria concentration ratio pair, obtain correction coefficient；Its updating formula is：Actual value=measured value × correction coefficient. Wherein, correction coefficient is to consider suction filtration process contamination microorganism some is remained adhered in edible fungi pompon, so that The polluted bacteria concentration that must be obtained by said method is less than actual concentrations, has hence set up a updating formula.
By above-mentioned technical proposal, following beneficial effect is invention achieves：
The liquid spawn germ contamination detection method that the present invention is provided can control detection time in 90 minutes, to determine As a result it is little with the testing result error based on culture；Have relative to sense organ of the prior art judgement and microscopy detection method bright Aobvious degree of accuracy advantage, and there is convenient quickly advantage relative to tradition culture detection method, therefore be a kind of quick, accurate Edible fungi liquid strain germ contamination detection method；The inventive method is carried out accurately for polluted bacteria living cells in liquid spawn Quantitative determination, so as to for edible fungi liquid strain batch production application provide quickly, accurately, easily detection means, have Wide application and popularization value.
Description of the drawings
Fig. 1 is edible fungi liquid strain polluted bacteria quantity fluorescent staining quantitative determination calibration curve Establishing process of the present invention Figure.
Fig. 2 is that polluted bacteria is separated and overhaul flow chart in a kind of edible fungi liquid strain of the invention.
Fig. 3 is that bacterial number is determined and correction coefficient determines stream in the edible fungi liquid strain for simulating germ contamination of the invention Cheng Tu.
Fig. 4 is the relation curve of the fluorescent staining intensity of two kinds of typical polluted bacterias and plate count result in the present invention (calibration curve).List Pseudomonas fluorescens and bacillus subtilis viable bacteria concentration in figure respectively green glimmering with BAClight dyeing The number of viable after mathematical relationship and both fittings and the mathematical relationship between green fluorescence intensity between luminous intensity.
Fig. 5 is Suction filtration device schematic diagram.Wherein, gnotobasis refers to superclean bench environment, it is ensured that miscellaneous bacteria is to taking out in air Filtration journey produces pollution.In figure, 1. filter bowl, 2. clip, 3. filter, 4. conical flask, 5. vavuum pump.
Specific embodiment
In order to illustrate technical scheme and technical purpose, below in conjunction with the accompanying drawings and specific embodiment is to the present invention It is described further.
Embodiment 1：
Embodiment 1 is a kind of specific embodiment party of edible fungi liquid strain germ contamination detection method provided by the present invention Formula, comprises the following steps：
1. prepared by calibration curve
Fig. 1 is that edible fungi liquid strain polluted bacteria quantity fluorescent staining quantitative determination calibration curve of the present invention sets up stream Journey, is respectively adopted plate count and obtains bacterial concentration numerical value with two methods of fluorescent staining, and builds both mathematical relationships, is formed Calibration curve.The present invention is using two kinds of typical polluted bacteria pseudomonads and the pure bacterial strain of bacillus subtilis as sample, sheet It is bent that flow process is equally applicable to other common polluted bacteria fluorescent quantitative measurement standards in concrete edible fungi liquid strain production process The structure of line.
Its concrete operation step is as follows：
1.1 with common edible fungi liquid strain polluted bacteria pseudomonad (G-) and bacillus subtilis (G+) as pollution Bacterium representative strain, is inoculated in respectively Shaking culture in sterilizing NB fluid nutrient mediums, condition of culture：28 DEG C of constant temperature, shaking speed: 180r/min, nutrient solution bacterial concentration (OD after cultivating 24 hours600) scope is about 1.8-2.0；
1.2 take respectively in pseudomonad, bacillus nutrient solution 10ml to 50ml sterile centrifugation tubes, 10000r/min centrifugations 5min, abandons supernatant；
1.3 are precipitated 3 times with the abundant washing thallines of sterile saline 30ml, and every time 10000r/min centrifugations 5min, abandons Supernatant；
1.4 take 10ml sterilized waters carries out settling flux process to precipitum cell, and bacterial precipitation is made in liquid using vortex instrument It is uniformly dispersed in body, and carries out 10 times of gradient dilutions and forms graded series bacteria suspension；Take each gradient dilution bacterium solutions of 200 μ l respectively to apply Sterilizing NB plating mediums are distributed in, 28 DEG C count after incubated 24 hours, calculate viable bacteria concentration (Bq) in each gradient dilution liquid；
1.5 pseudomonads respectively to above-mentioned preparation, (extension rate is 10 to bacillus subtilis gradient dilution bacteria suspension0 ～10-10) fluorescent staining is carried out, coloring agent is dissolved respectively with 2.5mL sterile deionized waters, equal proportion mixing during experiment, concrete side Method：Bacteria suspension is serially diluted in 1ml be separately added into 25 μ l SYTO 9+PI (1:1 mixing), uniform mixing, the dyeing of room temperature lucifuge 15min；
1.6 utilize the above-mentioned each dilution factor bacterium dye liquor fluorescence intensity of Shanghai rib light F97Xp fluorescent spectrophotometer assay, instrument Device condition determination：Excitation wavelength 470nm, launch wavelength 510nm is excited, emission spectrum bandwidth is 5nm, is determined green glow and is absorbed Wavelength (510nm) peak value is calculated as Fg；
1.7 set up mark according to each dilution factor bacteria suspension plate count result (Bq) and corresponding fluorescent dyeing determination result (Fg) Directrix curve, pseudomonad calibration curve is：Fg1=250.7lg (Bq1)+249.2, R2=0.997, detection range 5.2 × 100~ 5.2×107cfu/ml；Bacillus calibration curve：Fg2=248.5lg (Bq2)+216.7, R2=0.995, detection range 8.5 ×100～8.5 × 107cfu/ml；Fit standard curve：Fg=249.6lg (Bq)+232.9, R2=0.996.As shown in Figure 4. Possible in view of the complexity of edible fungi liquid strain sweat germ contamination species, the present invention is with fit standard curve math Computing formula of the formula as following polluted bacteria quantity.
2. the separation of polluted bacteria and detection in edible fungi liquid strain：
Polluted bacteria is separated and the visible Fig. 2 of testing process in edible fungi liquid strain.
Specifically include following steps：
2.1 rise GM-0.33II vacuum diaphragm pumps using Tianjin carries out suction filtration to 250ml edible fungi liquid strains, collects bacterial classification Middle polluted bacteria body.Filter membrane adopts 5 μm of aperture nylon wire, pressure 1500Pa, suction filtration process to carry out in superclean bench；
2.2 take the mycelia that SPSS 30ml is fully washed on filter membrane, and suction filtration enters bacterium receiving flask (see Fig. 5), It is repeated 3 times, to wash out absorption in mycelia and the bacterial cell of filter membrane surface；
2.3 filtrate (about 330ml) in above-mentioned bacterium receiving flask is loaded in batches 2 50ml sterile centrifugation tubes (centrifuge tube 1, Centrifuge tube 2) in, 10000r/min centrifugation 5min finally obtain above-mentioned filtrate bacterial sediment；
2.4 take the abundant washing thallines of sterile saline 30ml precipitates 2 times, and every time 10000r/min centrifugations 5min, abandons Supernatant；Take sterile saline 10ml to add in centrifuge tube 1, suspension is integrated with centrifuge tube 2 by suspension after processing, and takes go out again Centrifuge tube 2 is incorporated to after bacterium physiological saline 10ml washing centrifuge tubes 1, cleaning solution is simultaneously incorporated to centrifuge tube 2 by repeated washing 1 time.To from The centrifugal treating of heart pipe 2, method is ibid, final to obtain polluted bacteria cell precipitation in edible fungi liquid strain.
2.5 take 10ml sterilized waters carries out settling flux process to precipitum cell, and using vortex instrument thalline even suspension is made, And carry out 10 times of gradient dilutions and form dilution series bacteria suspensions；
2.6 take dilution factor for 10-1、10-3、10-5、10-7Bacteria suspension 1ml, is separately added into 25 μ l SYTO 9+PI (1:1 mixes Close), uniform mixing, room temperature lucifuge dyeing 15min；
2.7 utilize Shanghai rib light F97Xp fluorescent spectrophotometer assay dye liquor fluorescence intensities, and (method is walked with embodiment 1 Rapid 1.6) to select fluorescence intensity level to be located at the sample of the calibration curve range of linearity, the calibration curve of foundation fitting is (see embodiment 1 Middle step 1.7) viable count (Bq) calculating is carried out, and calculate sample viable count average measurement.
Embodiment 2：
In the present embodiment 2, the golden mushroom liquid spawn for simulating germ contamination is carried out using the method in embodiment 1 The measure of bacterial number, its flow chart is shown in Fig. 3.
Specifically include following steps：
1) preparation of culture medium：Plating medium：PDA culture medium；Fluid nutrient medium：Corn flour 5%, wheat bran 3%, yeast Cream 1%, sucrose 4%, KH2PO40.1%, MgSO40.15%, VB1 10mg/L, pH=5.5.
2) prepared by asparagus flat board bacterial classification：The PDA culture medium for preparing is sterilized in high-pressure steam sterilizing pan (121 DEG C, During sterilizing culture dish is sub-packed in after 30min) processing, 28 DEG C of overnight incubations of constant incubator are inverted in after cooled and solidified, reject dirty Dye flat board；Needle mushroom strain is connected on PDA plate culture medium, in 28 DEG C of constant temperature and humidity culture 7d；
3) prepared by flammulina velutipes liquid strains：By conical flask of the 200ml fluid nutrient mediums loaded on 500ml, and steamed by high pressure Vapour autoclave sterilizes (121 DEG C, 30min), 3 pieces of 1cm of per bottle of access after cooling2Needle mushroom strain, 28 DEG C of constant temperature, 180r/min shakes Training 8d；Aseptic wet towel is placed in shaking table, to ensure that bacterial classification shakes training in constant humidity environment.
4) simulate germ contamination flammulina velutipes liquid strains to prepare：(dilution plate is counted to draw pseudomonad bacteria suspension respectively Concentration is 2.9 × 107Cfu/ml), (dilution plate number concentration is 8.7 × 10 to bacillus bacteria suspension7Cfu/ml) each 10ml point Do not add in the flammulina velutipes liquid strains of above-mentioned culture 8d and shake up；
5) flammulina velutipes liquid strains polluted bacteria is separated：Using Tianjin GM-0.33II vacuum diaphragm pumps are risen respectively to above-mentioned mixed Closing liquid carries out suction filtration (see Fig. 5), and filter membrane is selected 5 μm of aperture nylon wire, the bacterium on filter membrane is fully washed using sterile saline Silk 3 times and respectively suction filtration, the thalline centrifugation to collecting, settling flux, and it is a series of dilution to carry out gradient dilution acquisition Bacteria suspension (concrete operations are with step 2.1-2.4 in embodiment 1)；
6) it is 10 to take dilution factor in dilution series bacteria suspension respectively-1、10-3、10-5、10-7Bacteria suspension 1ml, is separately added into 25μl SYTO 9+PI(1:1 mixing), and be well mixed, room temperature lucifuge dyeing 15min；
7) using Shanghai rib light F97Xp fluorescent spectrophotometer assay dye liquor fluorescence intensities, excitation wavelength 470nm, transmitting Wavelength 510nm, excites, emission spectrum bandwidth is 5nm, determines peak F g of green glow absorbing wavelength (510nm)；And according to fitting Computing formula calculates viable bacteria concentration, and pseudomonad calculated value is respectively 1.37 × 106cfu/ml、1.48×106cfu/ml、1.54 ×106cfu/ml、1.33×106Cfu/ml, compared with theoretical value, error range is -8.28%～+6.21%；Bacillus number Amount is respectively 4.01 × 106cfu/ml、4.78×106cfu/ml、3.86×106cfu/ml、4.41×106Cfu/ml, with theory Value is compared, and error range is -11.26%～+9.89%.
Embodiment 3：
In the present embodiment 3, the apricot spore mushroom bacteria liquid bacterial classification for simulating germ contamination is carried out using the method in embodiment 1 The measure of bacterial number, its flow chart is shown in Fig. 3.
1) preparation of culture medium：Plating medium：PDA culture medium；Fluid nutrient medium：Glucose 3%, peptone 0.2%, Yeast extract 0.5%, KH2PO40.05%, MgSO40.05%, VB1 10mg/L, pH=7.5.
2) prepared by pleurotus eryngii flat board bacterial classification：PDA culture medium is sterilized into (121 DEG C, 30min) in high-pressure steam sterilizing pan, After sterilizing, culture medium is distributed in sterilizing flat board, 28 DEG C of overnight incubations of constant incubator are inverted in after cooled and solidified, reject dirty Dye flat board；Pleurotus eryngii quel strains are connected in PDA culture medium, in 28 DEG C of constant temperature and humidity culture 7d；
3) prepared by pleurotus eryngii liquid strain：Fluid nutrient medium is sub-packed in the conical flask of 500ml, every bottle of 200ml, juxtaposition Sterilized in high-pressure steam sterilizing pan, after cooling, every bottle of culture medium accesses 3 pieces of 1cm2Pleurotus eryngii quel strains, 28 DEG C of constant temperature, 180r/min shakes training 8d；Aseptic wet towel is placed in shaking table, to ensure that bacterial classification shakes training in constant humidity environment.
4) simulate germ contamination pleurotus eryngii liquid strain and prepare and draw respectively pseudomonad bacteria suspension (dilution plate counts dense Spend for 2.9 × 107Cfu/ml), (dilution plate number concentration is 8.7 × 10 to bacillus bacteria suspension7Cfu/ml) each 10ml difference In adding the pleurotus eryngii liquid strain of above-mentioned culture 8d, it is well mixed；
5) pleurotus eryngii liquid strain polluted bacteria is separated：Using Tianjin GM-0.33II vacuum diaphragm pumps are risen respectively to above-mentioned mixed Closing liquid carries out suction filtration (see Fig. 5), and mycelia 3 times and the difference suction filtration on filter membrane is fully washed using sterile saline；Filter membrane From 5 μm of nylon wires, pressure 1500Pa；Mycelia 3 times and the difference suction filtration on filter membrane is fully washed using sterile saline, it is right The thalline centrifugation of collection, settling flux, and (concrete operations are with real to carry out a series of dilution bacteria suspensions of gradient dilution acquisition Apply step 2.1 in example 1-2.4)；
6) it is 10 to take dilution factor in graded series bacteria suspension respectively-1、10-3、10-5、10-7Bacteria suspension 1ml, is separately added into 25 μ l SYTO 9+PI(1:1 mixing), and be well mixed, room temperature lucifuge dyeing 15min；
7) using Shanghai rib light F97Xp fluorescent spectrophotometer assay dye liquor fluorescence intensities, excitation wavelength 470nm, transmitting Wavelength 510nm, excites, emission spectrum bandwidth is 5nm, determines peak F g of green glow absorbing wavelength (510nm)；And according to fitting Computing formula calculates viable bacteria concentration, and pseudomonad quantity is respectively 1.34 × 106cfu/ml、1.42×106cfu/ml、1.58× 106cfu/ml、1.30×106Cfu/ml, compared with theoretical value, error range is -10.34%～+8.97%；Bacillus number Amount is respectively 3.93 × 106cfu/ml、4.16×106cfu/ml、4.56×106cfu/ml、4.81×106Cfu/ml, with theory Value is compared, and error range is -9.66%～+6.89%.
Embodiment 4：
In the present embodiment 4, bacterium is carried out to certain edible fungus industrial manufacturing enterprise asparagus different batches liquid spawn Quantitative measurement.Specifically include following steps：
1) gradient dilution is carried out to certain edible mushroom factory flammulina velutipes liquid strains sample, each gradients (10 of 200 μ l is taken respectively-1、 10-3、10-5、10-7) dilution bacterium solution coats incubated 24 hours of 28 DEG C of aseptic NB flat boards and count, 3 repetitions of each gradient, Sample of the flat-plate bacterial colony number between 30-300 is used to calculate viable count mean value.
2) take 250ml flammulina velutipes liquid strains carries out suction filtration, centrifugation by aforementioned (step 2.1-2.4 in embodiment 1) method And make polluted bacteria gradient dilution bacteria suspension in liquid spawn.
3) 1mL gradients bacteria suspension (10 is taken respectively-1、10-3、10-5、10-7), it is separately added into 25 μ l SYTO 9+PI (1:1 mixes Close), and be well mixed, room temperature lucifuge dyeing 15min；Using Shanghai rib light F97Xp fluorescent spectrophotometer assay dye liquor fluorescence Intensity, excitation wavelength 470nm, launch wavelength 510nm is excited, emission spectrum bandwidth is 5nm, determines green glow absorbing wavelength (510nm) peak F g.
4) according to bacterial number actual value in simulation germ contamination asparagus in embodiment 2 and 3 and pleurotus eryngii liquid strain With fitting formula calculated value, calculate between actual value and measured value correction coefficient be 1.869, i.e. actual value=measured value × 1.896；And viable count is calculated according to matched curve and correction coefficient.Flammulina velutipes liquid strains different batches sample contamination bacterium Quantity testing result is shown in Table 1.
The flammulina velutipes liquid strains different batches sample contamination bacterial number testing result of table 1
Embodiment 5：
In the present embodiment 5, to bacterial number in certain edible fungus industrial manufacturing enterprise pleurotus eryngii different batches liquid spawn Determine.
Specifically include following steps：
1) gradient dilution is carried out to certain edible mushroom factory pleurotus eryngii liquid strain sample, each gradient dilutions of 200 μ l is taken respectively Bacterium solution (10-1、10-3、10-5、10-7) 28 DEG C of sterilizing NB plating mediums are inoculated in, count after incubated 24 hours, each is dilute Degree of releasing sets 3 repetitions, the same 5.3.1 of computational methods；
2) take 250ml pleurotus eryngii liquid strains by above-mentioned (step 2.1-2.4 in embodiment 1) method carry out suction filtration, from After the heart, washing, precipitation, polluted bacteria gradient dilution bacteria suspension in liquid spawn is made；
3) it is 10 to take dilution factor in graded series bacteria suspension respectively-1、10-3、10-5、10-7Bacteria suspension 1ml, be separately added into 25μl SYTO 9+PI(1:1 mixing), and be well mixed, room temperature lucifuge dyeing 15min；Using Shanghai rib light F97Xp fluorescence point Light photometric determination dye liquor fluorescence intensity, excitation wavelength 470nm, launch wavelength 510nm is excited, emission spectrum bandwidth is 5nm, determines peak F g of green glow absorbing wavelength (510nm)；And according to matched curve and the step 4 of embodiment 4) described in correction Coefficient calculates viable count.Pleurotus eryngii liquid strain different batches sample contamination bacterial number testing result is shown in Table 2.
The pleurotus eryngii liquid strain different batches sample contamination bacterial number testing result of table 2
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and specification this The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, the present invention Claimed scope is by appending claims, specification and its equivalent thereof.

Claims (6)

1. a kind of edible fungi liquid strain germ contamination detection method, it is characterised in that comprise the steps：
1) prepared by calibration curve：
Respectively each dilution polluted bacteria dilution bacteria suspension that represents to obtaining carries out NB flat board culture countings and fluorescent staining Determine, described represents polluted bacteria as edible fungi liquid strain G-And G+It is at least one respectively in class polluted bacteria；Subsequent basis Each dilution factor bacteria suspension plate count result (Bq) and corresponding fluorescent dyeing determination result (Fg) set up calibration curve；
2) use and be adapted to filter the filter membrane of hypha of edible fungus, filter liquid bacteria culture fluid；Subsequently, centrifugal filtrate, discards culture Liquid, and using SPSS washing thalline sediment, after reprecipitation, take sterilized water carries out settling flux to precipitum cell Process, and carry out gradient dilution and form graded series bacteria suspension；
3) to step 2) the middle polluted bacteria graded series bacteria suspension for obtaining is dyeed and is determined fluorescent value, and establishing criteria is bent Polluted bacteria concentration in line computation edible fungi liquid strain.
4) calculating of correction coefficient and updating formula：Step 1 is quantitatively adding by bacterial classification to be measured) the typical contaminated bacteria that used, Formed simulation germ contamination liquid spawn, and using step 2) and 3) detection calculate its polluted bacteria concentration (measured value), and with Polluted bacteria concentration ratio pair is actually mixed in liquid spawn, correction coefficient is obtained；Its updating formula is：Actual value=measured value × Correction coefficient.
2. a kind of edible fungi liquid strain germ contamination detection method as claimed in claim 1, it is characterised in that step 1) In, described represents polluted bacteria as pseudomonad (G-) and bacillus subtilis (G+)。
3. a kind of edible fungi liquid strain germ contamination detection method as described in right 2, it is characterised in that step 3) described in Calibration curve be used polluted bacteria strain calibration curve fit standard curve.
4. a kind of edible fungi liquid strain germ contamination detection method as claimed in claim 3, it is characterised in that step 3) in Described fit standard curve is：Fg=249.6lg (Bq)+232.9, R2=0.996.
5. a kind of edible fungi liquid strain germ contamination detection method as described in right 1, it is characterised in that step 3) described in Calibration curve be used polluted bacteria strain calibration curve fit standard curve.
6. a kind of edible fungi liquid strain germ contamination detection method as described in any one in claim 1-5, its feature It is, step 2) in, the filter membrane of described suitable filtration hypha of edible fungus is 5 μm of nylon wires for aperture.
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Cited By (5)

* Cited by examiner, † Cited by third party
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CN111562210A (en) * 2020-06-16 2020-08-21 北京挑战农业科技有限公司 Method for detecting viable count in pre-coated forage microecological preparation product
CN112961768A (en) * 2021-02-04 2021-06-15 海南微氪生物科技股份有限公司 Leeuwenhoek real-time selective microorganism rapid detection system
CN113151393A (en) * 2021-05-26 2021-07-23 宁德师范学院 Method for judging bacterial contamination condition of liquid strain by combining centrifugal filtration with fluorescent staining
CN113687072A (en) * 2021-09-10 2021-11-23 佛山墨赛生物技术有限公司 Test strip, kit and detection method for quantitative detection of viable bacteria of Escherichia coli O157H 7
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CN111562210A (en) * 2020-06-16 2020-08-21 北京挑战农业科技有限公司 Method for detecting viable count in pre-coated forage microecological preparation product
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CN113151393A (en) * 2021-05-26 2021-07-23 宁德师范学院 Method for judging bacterial contamination condition of liquid strain by combining centrifugal filtration with fluorescent staining
CN113687072A (en) * 2021-09-10 2021-11-23 佛山墨赛生物技术有限公司 Test strip, kit and detection method for quantitative detection of viable bacteria of Escherichia coli O157H 7
CN115572754A (en) * 2022-10-09 2023-01-06 上海市农业科学院 Method for evaluating activity of edible and medicinal fungus liquid strain

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