CN102749516B - Method for automatically surveying and mapping growth curve of microorganism in food - Google Patents
Method for automatically surveying and mapping growth curve of microorganism in food Download PDFInfo
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- CN102749516B CN102749516B CN201210230446.4A CN201210230446A CN102749516B CN 102749516 B CN102749516 B CN 102749516B CN 201210230446 A CN201210230446 A CN 201210230446A CN 102749516 B CN102749516 B CN 102749516B
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Abstract
The invention discloses a method for automatically surveying and mapping the growth curve of a microorganism in food. The method has the main contributions that a novel method that the microbiological analysis is carried out by surveying and mapping the growth curve of the microorganism is proposed starting with the growth rule of the microorganism in the food and is a technological innovation on the traditional analytical method; the condition that the growth information of the microorganism can be converted into measurable frequency information by a piezoelectric acoustic wave sensor is discovered through experimental study, and thus, a design principle for designing a fast and efficient novel surveying and mapping device is provided; and a device for automatically surveying and mapping the growth curve of the microorganism in the food is designed and constructed under the guidance of the novel method and the design principle. The device constructed according to the method has the advantages that the real-time and automatic surveying and mapping of the growth curve of the microorganism in the food can be realized, and the in-depth study on the microorganism in the food, such as the study of food processing methods, food shelf lives and microbial antimicrobial activity, can be carried out according to the obtained growth curve; and as the device has the characteristics of simplicity and convenience in operation, accuracy in result, and the like, the device is hopeful to be popularized and applied in microorganism-related research of food processing, storage and selling.
Description
Technical field
The present invention relates to a kind of method of microorganism in food growth curve auto-mapping.
Background technology
The globalization of world commerce also brings food safety risk simultaneously.In recent years, repeatedly food safety affair is there is in world wide, as: U.S.'s outburst is infected " the spinach poisoning " of large intestine, infect the peanut butter " sramana's event " of detection of Salmonella, the Seichin milk that Japanese Human fetal cardiomyocytes enterotoxin up to ten thousand causes be poisoning, the rabid ox disease of Britain, France listeria spp infect, the bird flu of Thailand etc.The annual food origin disease that occurs in the whole world is up to billions of example, and the probability that food origin disease occurs in developed country is also quite high.The annual payment for medical care caused by billions of routine food origin disease increases, dangerous food recall and economic loss that the destruction of product brings inestimable! What is more, and due to antibiotic abuse, the antibody-resistant bacterium of food-borne pathogens appears in the whole world again and again, and it increases sharply and diffusion, brings great challenge to the treatment of infections relating and treatment.Microorganism in food pollutes, and especially drug-resistant microorganism pollutes, and is the main cause causing food origin disease to occur and develop.Microorganism feature real-time in monitor food, and obtain its total plate count and drug resistance thereof, to prevention and corntrol food source property infected by microbes, ensure human health, prevent economic loss significant.
At present, the detection method of food-borne pathogens mainly contains culture identification, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), electrical impedance technology and full automatic microorganism analytic system etc.Culture identification method is that the sample through pre-treatment is inoculated in nutrient broth, and the then colony growth of visual inspection bacterium, carries out negative and positive judgement.For positive sample, then identified by drug sensitive experiment.The method will expend mostly
it time, and program is complicated, and agents useful for same is various, wastes time and energy, and waits result out, and epidemic situation may occur; ELISA is a kind of detection technique that the efficient catalytic effect of immunoreactive for antigen-antibody specificity and enzyme is organically combined, and it both can survey antigen, also can survey antibody.The method sensitivity and specificity are comparatively strong, but required expensive reagents, and need repeatedly wash and incubation; Round pcr adopts external enzymatic Reactive Synthesis DNA fragment specific, then identifies bacterium by amplified production.PCR has highly sensitive, high specificity, the feature such as quick, but false positive and false negative rate too high be the key issue affecting its application.Electrical impedance technology principle is in the process of bacterium growth and breeding in nutrient culture media, the impedance of nutrient culture media will be made to change, by detecting the electrical impedance situation of change of nutrient culture media, judging bacterium growth and breeding characteristic in the medium, corresponding bacterium can be detected.This method has that detection speed is fast, sensitivity advantages of higher, but due to the stability problem of circuit, causes false positive rate too high; One of Bacteria Identification instrument that the Vitek-AMS automatic microbe detection system that biological Mei Liai group company of France produces belongs to the most advanced in the world today, automaticity is the highest.It need not be separated through microorganism and cultivate and purge process, just directly can detect special microbe species from sample and flora comes.But this system and matched reagent expensive and need dependence on import, seriously constrain the raising of China's food-borne pathogens research level.Therefore, the research of sensitive, quick, accurate and low-cost food-borne pathogens real-time watch device is extremely urgent.
Summary of the invention
The object of the invention is to overcome existing method weak point and a kind of automatic, quick, accurate and real-time mapping method of visual microorganism in food growth curve is provided.
In order to realize above-mentioned technical purpose, technical scheme of the present invention is, a kind of method of microorganism in food growth curve auto-mapping, comprises the following steps:
Step one: get 25g food samples and be placed in and fill in the aseptic homogeneous cup of 225mL phosphate buffer, carry out homogeneous 2min with the speed of 8000r/min, make the even liquid of sample of l:10;
Step 2: set up microbial growth curve auto-mapping device, comprise sensor-based system, frame of reference and display system three part, wherein sensor-based system comprises the detector tube with gland bonnet, temperature regulating device, two electrodes, piezoelectric sound wave sensor and autoexcitation circuit, described detector tube is located in temperature regulating device, two described electrodes insert in detector tube by bottom, the wherein input end of an Electrode connection autoexcitation circuit, the input end of another Electrode connection piezoelectric sound wave sensor, the output terminal of piezoelectric sound wave sensor is connected to autoexcitation circuit, described frame of reference comprises a point frequency meter, driving circuit and single-chip microcomputer, the input end of described point frequency meter is connected to autoexcitation circuit, the output terminal of autoexcitation circuit is connected to driving circuit, described single-chip microcomputer is connected to autoexcitation circuit and driving circuit respectively, described display system comprises display, described display is connected to single-chip microcomputer by MAX232 serial ports,
Step 3: get the even liquid of sample that 9mL nutrient culture media and 1mL obtain by step one and be added in the detector tube of microbial growth curve auto-mapping device, tighten gland bonnet;
Step 4: inserted by detector tube in temperature regulating device, regulates temperature regulating device temperature to be (37 ± 0.2) DEG C;
Step 5: start microbial growth curve auto-mapping device;
Step 6: the electrical quantity that in sensor-based system, piezoelectric sound wave sensor pair is connected in series cultivating system in object detection pipe with it changes sensitive frequency response, the frequency signal of the cultivating system detected is converted to the discernible digital signal of computing machine and sends to single-chip microcomputer by frame of reference, single-chip microcomputer exports cultivating system response curve in frequency displacement-time mode in real time by display, when there being growth of microorganism in detector tube, the macromolecular substances of inertia electric in nutrient culture media can be decomposed into electroactive small-molecule substance, when microorganism concn reaches 10 by microorganism
6during about cfu/mL, in detector tube there is comparatively marked change in the electrical quantity of cultivating system, and cause the frequency displacement of piezoelectric sound wave sensor to occur significantly to change, obtain the real-time growth curve of microorganism in food, real time data is preserved with TXT form automatically.
The method of described a kind of microorganism in food growth curve auto-mapping, nutrient culture media is fluid nutrient medium, described fluid nutrient medium is wide spectrum fluid nutrient medium, described wide spectrum Liquid Culture based component is powdered beef 3g, yeast extract 1.5g, glucose 10g, growth factor, distilled water 1000mL, pH value is 7.0 ~ 7.2.
The method of described a kind of microorganism in food growth curve auto-mapping, described growth factor comprises L-PROLINE 20mg, Serine 20mg, ALANINE 40mg, ILE 70mg, L-Leu 98mg, L-ASPARTIC ACID 50mg, TYR 5mg, Pidolidone 15mg, L-Phe 100mg, L-arginine hydrochloride 100mg, L lysine HCL 80mg, Valine 70mg, L-threonine 50mg, L-Histidine hydrochloride 50mg, L-Trp 15mg, METHIONINE 45mg, CYSTINE 2mg, glycocoll 150mg.
The method of described a kind of microorganism in food growth curve auto-mapping, electrode is that a pair stainless steel bar through Passivation Treatment is directly enclosed in polycarbonate, and end face is polished, obtain being positioned at conplane a pair circular flat electrode, the electrode constant of electrode is 0.58cm ~ 1.00cm, distance between electrode constant=planar electrode area/circular flat electrode, distance refers to the distance between two centers of circle.
The method of described a kind of microorganism in food growth curve auto-mapping, temperature regulating device be use resistance wire to make around one with red copper material sleeve on.
The method of the described real-time auto-mapping of a kind of microorganism in food growth curve, plane electrode and piezoelectric sound wave sensor pass through wired in series.
The present invention contrasts prior art, from microorganism in food growth rhythm, proposes the new method of carrying out microbiological analysis by mapping microbial growth curve, is the technological innovation to traditional analysis; Research finds that growth of microorganism convert information can be the frequency information that can survey by piezoelectric sound wave sensor by experiment, thus provides design concept for designing novel drawing apparatus fast and efficiently.Under the guidance of new detection method and design concept, microorganism in food growth curve auto-mapping device is produced in design.
Technique effect of the present invention is:
1, the use of piezoelectric sound wave sensor avoids the impact of electric double layer capacitance, improves the stability of electric signal, enhances signal to noise ratio (S/N ratio);
2, piezoelectric sound wave sensor significantly improves the degree of accuracy of detection after being combined with plane electrode.
3, the combination of sensor-based system, frame of reference and display system can the automatic business processing of implement device, can obtain the frequency displacement-time plot of microorganism in food in real time, rapidly, for the further investigation of microorganism in food provides theoretical foundation.
Accompanying drawing explanation
The structural representation of Fig. 1 apparatus of the present invention;
Wherein: I-sensor-based system, II-frame of reference, III-display system
1 is that (1-1 is gland bonnet to pick-up unit, and 1-2 is detector tube, and 1-3 is microorganism, and 1-4 is nutrient culture media, 1-5 is temperature regulating device, and 1-6 is electrode), 2 is piezoelectric sound wave sensor, and 3 is autoexcitation circuit, 4 is a point frequency meter, and 5 is driving circuit, and 6 is single-chip microcomputer, and 7 is MAX232 serial ports;
Fig. 2 is the workflow diagram of this device;
Fig. 3 is the rate of change of microorganism in food growth course conductance (A) and electric capacity (B);
Fig. 4 is that microorganism in food surveys and draws growth curve (A) in real time, blank curve (B) and colony counting method gained growth curve (C);
Fig. 5 is different initial concentration (cfu/mL) microbial growth curve in fresh milk
A-1.1×10
6,B-7.9×10
4,C-9.8×10
3,D-1.3×10
3,E-97,F-12
Fig. 6 is the real-time growth curve of microorganism in ground rice
A-ground rice is through 121 ° of C, 10min process; B, C-ground rice is through 80 ° of C, 2min process; The undressed ground rice of D, E, F-.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Basic thought of the present invention is the real-time mapping realizing microorganism in food growth curve in conjunction with piezoelectric sound wave sensor, plane electrode and peripheral data treating apparatus, is the further investigation based theoretical of microorganism in food.
Fig. 1 is shown in by the real-time plotting board of microorganism in food growth curve, and comprise sensor-based system, frame of reference and display system three part, wherein sensor-based system is made up of piezoelectric sound wave sensor, detection probe, detector tube, temperature regulating device and autoexcitation circuit; Frame of reference is made up of point frequency meter, driving circuit, single-chip microcomputer and a MAX232 data-interface; Display is made up of display interface and software kit; Wherein detection probe is homemade plane electrode, and material is that 316 stainless steel bars of Passivation Treatment embed in polycarbonate, polishes, polishing; Piezoelectric sound wave sensor (9MHz AT-cut type, two sides is silver-plated) is purchased to Beijing 707 factory, and plane electrode and piezoelectric sound wave sensor pass through wired in series.Piezoelectric sound wave sensor has sensitive frequency response to microbial growth in detector tube, for eliminating extraneous factor impact, represents (Δ F=F by method of difference unification frequency shift value
0-F
i, wherein F
0for initial frequency, F
ifrequency values for recording during i), obtain frequency displacement response signal.And the collection of signal is controlled by computing machine, first send instructions to single-chip microcomputer by computing machine by MAX232 data-interface, signal in acquisition testing pipe, computing machine controls collection signal passage, the signal incoming frequency meter gathered, be divided into segment signal and position signal to pass to single-chip microcomputer, single-chip microcomputer passes to display system by data-interface, automatically draws out real-time response curve with frequency displacement-time form.All frequency displacements and time data, preserve automatically with TXT form.
Condition of work:
Environment temperature: 10 ° of C ~ 30 ° C; Relative humidity :≤85%; Atmospheric pressure: 86.0kPa ~ 106.0kPa; Use power supply: AV (220 ± 22) V, 50Hz ± 1Hz.
The macromolecular substances such as glucose, amino acid that can decompose electric inertia due to microorganism in metabolic process is electroactive Small molecular, so character of nutrient culture media system, as conductivity and specific inductive capacity can change in time, microbial growth information has exactly been reacted in this change.Fig. 3 gives 10 of 4192A LF electric impedance analyzer mensuration
5the electrical quantity situation of change of the Escherichia coli of cfu/mL growth course in the medium, its ordinate represents with the rate of change of the change of electrical quantity relative to initial electrical parameter values, horizontal ordinate is the microbial growth time, and curve A and B represent the conductance of microorganism in food growth course and the rate of change of electric capacity respectively.As seen from the figure, microorganism is in growth course, and conductance increases about 16%, and capacitance variations is about 10%.Result shows, the mensuration problem of microbe colony quantity can be converted into the problem of electrical measurement.Electromagnetic oscillation theory shows, the relation of the frequency f of lc circuit and coefficient of self-induction L, electric capacity C is:
From formula, self-induction of loop coefficient, capacitance variations can change oscillation frequency, can record the change of this frequency with corresponding frequency measurement circuit.Because microorganism Liquid Culture system electrical quantity in growth course can change, so can solve that microorganism in food growth information is converted to can the technical matters of measurement information.The oscillator used in side circuit is piezoelectric sound wave sensor, and its principle of work is substantially identical with the principle of LC oscillatory circuit, but stability is better frequently, can reduce the noise in testing process, so, adopt piezoelectric sound wave sensor in the detection system.
Embodiment 1
Utilize microorganism in food growth curve auto-mapping device and as shown in Figure 2 method to 10
5the colibacillary growth course curve (see Fig. 4 curve A, Fig. 4 curve B is blank reference) of cfu/mL is surveyed and drawn in real time and contrasts with the growth curve (see Fig. 4 curve C) that colony counting method obtains.As can be seen from Figure 4, in ab section, Escherichia coli are in lag phase, do not divide in following period of time at once, and colibacillary quantity remains constant, or increases seldom, and the change of properties of nutrient culture media is also not obvious, so frequency displacement does not have significant change; In the bc stage, for coliform index growth period, Escherichia coli physiological metabolism effect makes the carbohydrates in nutrient culture media, lipoid and protein transduction turn to electroactive material, have significantly changed nutrient culture media character, piezoelectric sound wave sensor has sensitive frequency displacement response to this change, whether the timing definition corresponding to point that frequency displacement starts marked change is microorganism detection time (MDT), can be and have growth of microorganism to provide theoretical foundation.The growth curve obtained from colony counting method then needs artificially from static culture device, to cultivate 24h in sterile working absorption bacterium liquid to flat board, drawing a curve needs inoculation for several times, and the time at least wants delayed 48h, time-consuming effort, need mass propgation device, and performance graph cannot be obtained in real time.
Embodiment 2
Utilize microorganism in food growth curve auto-mapping device to determine the typical growth curve of the fresh milk sample of different bacterium initial concentration, its result as shown in Figure 5.As seen from the figure, bacterium initial concentration is larger, and MDT is less, otherwise bacterium initial concentration is less, and MDT is larger.10-10 is about at bacterium initial concentration
6during cfu/mL, there is linear relationship between the logarithm of bacterium initial concentration and MDT, physical relationship formula is as follows:
LgC=7.216-0.432MDT (4)
In formula, C is the initial concentration of the microorganism in fresh milk sample, and MDT is microorganism detection time, its related coefficient about 0.96, illustrates that in MDT and sample, bacterium initial concentration logarithm is linear.Just can extrapolate the bacterial number in sample according to detection gained MDT, and then the quantitative test of microorganism can be carried out.
Embodiment 3
Microorganism in food growth curve auto-mapping device is utilized to survey and draw in real time the microbial growth curve in ground rice, its result is see Fig. 6, curve A is that ground rice is through 121 ° of C, growth curve after 10min process, curve B and C are that ground rice is through 80 ° of C, growth curve after 2min process, curve D, E and F are microbial growth curve in undressed ground rice.As can be seen from Figure 6, curve A changes without obvious frequency displacement, and without microorganism detection time, deducibility ground rice is after 121 ° of C, 10min process, and microorganism wherein can be totally constrained; Curve B and C probably just occur that significant frequency displacement changes after 10 hours, and show that in ground rice, microbial growth is suppressed after 80 ° of C, 2min process, most of microorganism is killed; Just there is significant frequency displacement in curve D, E and F, show that the quantity of microorganism in undressed ground rice is more within 3 hours, can affect to the quality of ground rice.From above result, by can judge the microbial activity state of food object to the analysis of the real-time growth curve of microorganism in food, for the processing mode of food provides theoretical foundation.
Claims (3)
1. a method for microorganism in food growth curve auto-mapping, is characterized in that, comprises the following steps:
Step one: get 25g food samples and be placed in and fill in the aseptic homogeneous cup of 225mL phosphate buffer, carry out homogeneous 2min with the speed of 8000r/min, make the even liquid of sample of l:10;
Step 2: set up microbial growth curve auto-mapping device, comprise sensor-based system, frame of reference and display system three part, wherein sensor-based system comprises the detector tube with gland bonnet, temperature regulating device, two electrodes, piezoelectric sound wave sensor and autoexcitation circuit, described detector tube is located in temperature regulating device, two described electrodes insert in detector tube by bottom, the input end of one of them Electrode connection autoexcitation circuit, the input end of another Electrode connection piezoelectric sound wave sensor, the output terminal of piezoelectric sound wave sensor is connected to autoexcitation circuit, described frame of reference comprises a point frequency meter, driving circuit and single-chip microcomputer, the input end of described point frequency meter is connected to autoexcitation circuit, the output terminal of autoexcitation circuit is connected to driving circuit, described single-chip microcomputer is connected to autoexcitation circuit and driving circuit respectively, described display system comprises display, described display is connected to single-chip microcomputer by MAX232 serial ports,
Step 3: get the even liquid of sample that 9mL nutrient culture media and 1mL obtain by step one and be added in the detector tube of microbial growth curve auto-mapping device, tighten gland bonnet; Nutrient culture media is fluid nutrient medium, and described fluid nutrient medium is wide spectrum fluid nutrient medium, and described wide spectrum Liquid Culture based component is powdered beef 3g, yeast extract 1.5g, glucose 10g, growth factor, distilled water 1000mL, and pH value is 7.0 ~ 7.2;
Step 4: inserted by detector tube in temperature regulating device, regulates temperature regulating device temperature to be (37 ± 0.2) DEG C;
Step 5: start microbial growth curve auto-mapping device;
Step 6: the electrical quantity that in sensor-based system, piezoelectric sound wave sensor pair is connected in series cultivating system in object detection pipe with it changes sensitive frequency response, the frequency signal of the cultivating system detected is converted to the discernible digital signal of computing machine and sends to single-chip microcomputer by frame of reference, single-chip microcomputer exports cultivating system response curve in frequency displacement-time mode in real time by display, when there being growth of microorganism in detector tube, the macromolecular substances of inertia electric in nutrient culture media can be decomposed into electroactive small-molecule substance, when microorganism concn reaches 10 by microorganism
6during about cfu/mL, in detector tube there is comparatively marked change in the electrical quantity of cultivating system, causes the frequency displacement of piezoelectric sound wave sensor to occur significantly to change, obtain the real-time growth curve of microorganism in food.
2. the method for a kind of microorganism in food growth curve auto-mapping according to claim 1, it is characterized in that, described growth factor comprises L-PROLINE 20mg, Serine 20mg, ALANINE 40mg, ILE 70mg, L-Leu 98mg, L-ASPARTIC ACID 50mg, TYR 5mg, Pidolidone 15mg, L-Phe 100mg, L-arginine hydrochloride 100mg, L lysine HCL 80mg, Valine 70mg, L-threonine 50mg, L-Histidine hydrochloride 50mg, L-Trp 15mg, METHIONINE 45mg, CYSTINE 2mg, glycocoll 150mg.
3. the method for a kind of microorganism in food growth curve auto-mapping according to claim 1, it is characterized in that, two electrodes are that a pair stainless steel bar through Passivation Treatment is directly enclosed in polycarbonate, and end face is polished, obtain being positioned at conplane a pair circular flat electrode, the electrode constant of electrode is 0.58cm ~ 1.00cm.
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| WO2015022306A1 (en) * | 2013-08-12 | 2015-02-19 | Dupont Nutrition Biosciences Aps | Methods for classification of microorganisms from food products |
| CN106290162B (en) * | 2016-10-10 | 2019-09-06 | 承启医学（深圳）科技有限公司 | A kind of device and method of continuous detection bacterium growth curve |
| CN113984835A (en) * | 2021-10-14 | 2022-01-28 | 江苏农林职业技术学院 | Packaging can for monitoring food quality in real time and monitoring method |
| CN115612611B (en) * | 2022-10-27 | 2023-05-30 | 青岛众瑞智能仪器股份有限公司 | Microorganism growth analyzer |
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| Title |
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| 利用压电换能器快速鉴别潲水油;任佳丽 等;《食品与机械》;20110331;第27卷(第2期);全文 * |
| 压电传感器真菌鉴定中的应用及镍和柠檬酸配位研究;钟鸣;《中国优秀硕士学位论文全文数据库》;20091231;全文 * |
| 新型压电微生物传感仪及其在临床分析中的应用研究;任佳丽;《中国博士学位论文全文数据库》;20091231;第28、48、115页、附录B * |
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