CN101787389A - Method for predicting staphylococcus aureus growth and toxin production in milk - Google Patents
Method for predicting staphylococcus aureus growth and toxin production in milk Download PDFInfo
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- CN101787389A CN101787389A CN200910217427A CN200910217427A CN101787389A CN 101787389 A CN101787389 A CN 101787389A CN 200910217427 A CN200910217427 A CN 200910217427A CN 200910217427 A CN200910217427 A CN 200910217427A CN 101787389 A CN101787389 A CN 101787389A
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Abstract
The invention discloses a method for predicting staphylococcus aureus growth and toxin production in milk, which relates to a method for predicting staphylococcus aureus growth and toxin production through the principle of predictive microbiology. The invention solves the problems that the method for testing the number of staphylococcus aureus and staphylococcus aureus enterotoxin in milk is complex to operate, and the test time is long. In the method: first, the maximum growth rate is obtained according to 7.1917 * 10-5* T2 +0.022782* T-0.32889; second, the bacterial count is obtained according to log N (t) = A + C (exp (-exp (-y (t-M)))); third, the time corresponding to the bacterial count 106.4cfu/mL is combined with F=0.0018543 * T2-0.050277* T +0.32629 to obtain the toxin production speed rate, that is, the prediction of the staphylococcus aureus growth and toxin production is realized. The invention has the advantages of simple operation and short test time.
Description
Technical field
The present invention relates to a kind of staphylococcus aureus growth and method of producing poison predicted.
Background technology
The detection method that whether exists for the Ruzhong streptococcus aureus at present mainly contains two kinds, be the colony counting method and the PCR detection method of GB, but these two kinds of methods all are the streptococcus aureus quantity of measuring in the sample of that time point of sampling, if know just operation repeatedly of the quantity of streptococcus aureus down At All Other Times in this sample, complicated operation.The method of existing mensuration Staphylococcus aureus enterotoxin mainly is an ELISA method and based on the detection kit of this method, though the sensitivity of detection kit is fine, but can only provide qualitative or semiquantitative result, will cause the hazardness size that accurately to assess breast like this.
Summary of the invention
The present invention is in order to solve long problem of the existing method complicated operation that detects Ruzhong streptococcus aureus bacterium number and Staphylococcus aureus enterotoxin, detection time, and a kind of Ruzhong staphylococcus aureus growth and method of producing poison predicted is provided.
The method of prediction Ruzhong staphylococcus aureus growth and product poison is carried out according to following steps: one, detect growth temperature of Ruzhong streptococcus aureus and the initial bacterium number of Ruzhong streptococcus aureus, then with testing sample temperature substitution y=7.1917 * 10
-5* T
2+ 0.022782 * T-0.32889 formula calculates the maximum growth rate of Ruzhong streptococcus aureus, and wherein T represents the growth temperature of streptococcus aureus, and y represents the maximum growth rate of Ruzhong streptococcus aureus; Two, initial bacterium number of Ruzhong streptococcus aureus in the step 1 and the maximum growth rate of Ruzhong streptococcus aureus are updated to formula log N (t)=A+C[exp (exp (y (t-M)))] in, calculate the quantity of Ruzhong streptococcus aureus when time t, wherein exp=e
xT represents the growth time of Ruzhong streptococcus aureus, the quantity of log N (t) Ruzhong streptococcus aureus when being illustrated in t, A is the initial bacterium number [log10 (cfu/mL)] of Ruzhong streptococcus aureus in the step 1, C represents that the Ruzhong streptococcus aureus is at the growth bacterium number [log10 (cfu/mL)] of stationary phase, M represents the pairing time of maximum growth rate, and y is at the maximum growth rate of time M [(log10[cfu/mL])/h]; Three, the Ruzhong streptococcus aureus that step 2 is calculated is 10 at the bacterium number
6.4Pairing time t is the time of origin that toxin produces during cfu/mL, again in conjunction with formula F=0.0018543 * T
2The malicious speed of the resulting product of-0.050277 * T+0.32629, the linearity that finally obtains Ruzhong streptococcus aureus when temperature T is produced malicious curve, F represents that the Ruzhong streptococcus aureus produces malicious speed, T represents the growth temperature of streptococcus aureus, promptly realized the Ruzhong staphylococcus aureus growth and produced malicious model construction, can dope following detected sample Ruzhong staphylococcus aureus growth and the malicious situation of product put any time accurately according to this structure model and detected sample detection data.
Obtain the growth data of streptococcus aureus under the differing temps according to colony counting method, calculate maximum growth rate at each temperature, finally obtain the curvilinear equation of growth velocity, be equation y=7.1917 * 10 in the step 1 with respect to temperature
-5* T
2+ 0.022782 * T-0.32889.
Log N (t)=A+C[exp in the step 2 of the present invention (exp (y (t-M)))] be the Gompertz formula.
Use the pure enterotoxin sample of kit measurement, thereby set up the quantitative criterion curve of measuring toxin.Use kit measurement to go out corresponding staphylotoxin at each temperature, and utilize typical curve to calculate accurate content, thereby construct at each temperature product poison model (being relation conefficient) greater than 0.99 linear model, and set up product poison Rate Models with respect to temperature according at each temperature product poison speed, be the equation F=0.0018543 * T in the step 3
2-0.050277 * T+0.32629.
According to the growth of being measured at each temperature with produce malicious curve, find that time that toxin begins to produce is streptococcus aureus bacterium number and reaches 10
6.4The time of cfu/mL, and temperature independent.
The method amount of lightening one's labor of the present invention is also saved time, only just can know thereafter At All Other Times quantity and toxin according to the streptococcus aureus quantity of a time point of institute's working sample, simple to operate, the accuracy height, shortened the time of detecting greatly, Forecasting Methodology of the present invention has solved long problem of existing method complicated operation, detection time.
Description of drawings
Fig. 1 is the growth curve illustration of Gompertz model-fitting in the embodiment one; Fig. 2 is a SEA canonical plotting in the embodiment one; Fig. 3 is the malicious curve illustration of the suitableeest product of MATLAB7.6 match in the embodiment one; Fig. 4 is S.aureus growth and the graph of a relation that produces poison in the embodiment one; Fig. 5 is the growth velocity graphic representation of S.aureus in the embodiment one; Fig. 6 is the product poison rate profile of S.aureus in the embodiment one.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the method for present embodiment prediction Ruzhong staphylococcus aureus growth and product poison is carried out according to following steps: detect growth temperature of Ruzhong streptococcus aureus and the initial bacterium number of Ruzhong streptococcus aureus, then with testing sample temperature substitution y=7.1917 * 10
-5* T
2+ 0.022782 * T-0.32889 formula calculates the maximum growth rate of Ruzhong streptococcus aureus, and wherein T represents the growth temperature of streptococcus aureus, and y represents the maximum growth rate of Ruzhong streptococcus aureus; Two, initial bacterium number of Ruzhong streptococcus aureus in the step 1 and the maximum growth rate of Ruzhong streptococcus aureus are updated to formula log N (t)=A+C[exp (exp (y (t-M)))] in, calculate the quantity of Ruzhong streptococcus aureus when time t, wherein exp=e
xT represents the growth time of Ruzhong streptococcus aureus, the quantity of log N (t) Ruzhong streptococcus aureus when being illustrated in t, A is the initial bacterium number [log10 (cfu/mL)] of Ruzhong streptococcus aureus in the step 1, C represents that the Ruzhong streptococcus aureus is at the growth bacterium number [log10 (cfu/mL)] of stationary phase, M represents the pairing time of maximum growth rate, and y is at the maximum growth rate of time M [(log10[cfu/mL])/h]; Three, the Ruzhong streptococcus aureus that step 2 is calculated is 10 at the bacterium number
6.4Pairing time t is the time of origin that toxin produces during cfu/mL, again in conjunction with formula F=0.0018543 * T
2The malicious speed of the resulting product of-0.050277 * T+0.32629, the linearity that finally obtains Ruzhong streptococcus aureus when temperature T is produced malicious curve, F represents that the Ruzhong streptococcus aureus produces malicious speed, T represents the growth temperature of streptococcus aureus, promptly realized the Ruzhong staphylococcus aureus growth and produced malicious model construction, can dope following detected sample Ruzhong staphylococcus aureus growth and the malicious situation of product put any time accurately according to this structure model and detected sample detection data.
The method amount of lightening one's labor of present embodiment is also saved time, only just can know thereafter At All Other Times quantity and toxin according to the streptococcus aureus quantity of a time point of institute's working sample, simple to operate, the accuracy height, shortened the time of detecting greatly, the Forecasting Methodology of present embodiment has solved long problem of existing method complicated operation, detection time.
Obtain the growth data of streptococcus aureus under the differing temps according to colony counting method, calculate maximum growth rate at each temperature, finally obtain the curvilinear equation of growth velocity, be equation y=7.1917 * 10 in the step 1 with respect to temperature
-5* T
2+ 0.022782 * T-0.32889.
The initial bacterium number of Ruzhong streptococcus aureus in the present embodiment step 1 is meant the Ruzhong streptococcus aureus bacterium number when incubation time is 0h, and unit is log10 (cfu/mL).
Log N (t)=A+C[exp in the present embodiment step 2 (exp (y (t-M)))] be the Gompertz formula.
According to the growth of being measured at each temperature with produce malicious curve, find that time that the toxin in the present embodiment step 3 begins to produce is streptococcus aureus bacterium number and reaches 10
6.4The time of cfu/mL, and temperature independent.
Use the pure enterotoxin sample of kit measurement, thereby set up the quantitative criterion curve of measuring toxin.Use kit measurement to go out corresponding staphylotoxin at each temperature, and utilize typical curve to calculate accurate content, thereby construct at each temperature product poison model (being relation conefficient) greater than 0.99 linear model, and set up product poison Rate Models with respect to temperature according at each temperature product poison speed, be the equation F=0.0018543 * T in the step 3
2-0.050277 * T+0.32629.
The concrete experimental implementation of the equation of above-mentioned acquisition step 1 and step 3 is as follows: one, earlier pasteurization breast (market is bought and obtained) is carried out S.aureus and SEA check, require the pasteurization Ruzhong S.aureus and the SEA that are used to inoculate all negative; Pure growth to S.aureus (streptococcus aureus reference culture (ATCC 13565) comes from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) carries out enumeration then, get a certain amount of bacterium liquid according to the enumeration result and be inoculated in satisfactory S.aureus and the negative pasteurization of SEA Ruzhong, make the inoculation final concentration be positioned at 10
2～10
3Between the cfu/mL,, be sub-packed in the screw-cap test tube, under 15 ℃, 18 ℃, 20 ℃, 23 ℃, 25 ℃, 30 ℃ and 37 ℃ of temperature, cultivating respectively with the amount of 5mL with the abundant mixing of postvaccinal cow's milk; From design temperature, take out the screw-cap test tube of packing according to the timed interval of design at every turn and also put into mixture of ice and water rapidly, then the sample in the screw-cap test tube is carried out suitable gradient dilution and is coated with the NB flat board; Each extent of dilution do three parallel, coated flat board is put in 37 ℃ cultivates and counting, obtain growth data; Two, with 15 ℃, 18 ℃, 20 ℃, 23 ℃, 25 ℃, the growth data of 30 ℃ and the 37 ℃ Ruzhong S.aureus that measure is down carried out the match of 2 kinds of models with MATLAB7.6 software, get the average MSE value of each model-fitting, the average MSE of Gompertz model is 0.0294, the average MSE of Logistic model is 0.0298, be that culture temperature is 15 ℃, 18 ℃, 20 ℃, 23 ℃, 25 ℃, the growth curve of streptococcus aureus under 30 ℃ and the 37 ℃ of conditions, the growth curve of the streptococcus aureus of the Gompertz model-fitting when wherein, culture temperature is 20 ℃ as shown in Figure 1; Three, with concentration is 0.125ng/mL, 0.25ng/mL, 0.5ng/mL, 1.0ng/mL and the SEA standardized solution of 2.0ng/mL (use buy obtain standard substance and be configured from U.S. Sigma-Aldrich company) pollutes the Ruzhong, with SET2 kit measurement (obtaining) from the purchase of French Biomerieux SA, the miniVIDAS instrument provides corresponding reading (TV value), drawing out with SEA concentration then is ordinate zou, the TV value is the SEA typical curve of X-coordinate, typical curve as shown in Figure 2, the typical curve relational expression is: SEA (ng/mL)=1.2089 * TV+0.0081, and relation conefficient is 0.9994; Measure 15 ℃, 18 ℃, 20 ℃, 23 ℃, 25 ℃, 30 ℃ and 37 ℃ of SEA concentration (ng/mL) that following Ruzhong S.aureus produces respectively, SEA concentration at each temperature all becomes the better linearity relation with the time, and the relation conefficient (R of the malicious model of the suitableeest product of each temperature
2) all more than 0.99, be shown in Table 1, wherein use 23 ℃ the malicious curve (R of the suitableeest product of MATLAB7.6 match
2=0.9914) as shown in Figure 3; Four, by in the step 1 at each temperature growth data and step 3 at each temperature product poison data, cell concentration and the relation of producing poison as can be known, when the S.aureus in Ruzhong reaches finite concentration, just begin to produce SEA, and it is irrelevant to produce the required S.aureus Pressure, Concentration, Temperature of SEA, and Ruzhong S.aureus begins to produce malicious concentration and is about 10
6.4Cfu/mL, cell concentration and the relation of producing poison as shown in Figure 4, from Fig. 4 and other cell concentrations that do not provide with produce the malicious graph of a relation and can determine, when the concentration of Ruzhong S.aureus less than 10
6.4Do not produce toxin during cfu/mL, promptly streptococcus aureus bacterium number reaches 10
6.4Streptococcus aureus begins to produce toxin during cfu/mL; Five, according to the suitableeest growth model of Ruzhong S.aureus at each temperature with produce malicious Model Calculation and go out the maximum growth rate constant and produce malicious rate constant, utilize MATLAB7.6 software to carry out match, obtain the growth velocity model between 15 ℃～37 ℃ and produce malicious Rate Models; Wherein, the quadratic function model as shown in Figure 5, is equation y=7.1917 * 10 in the present embodiment step 1 as the suitableeest growth velocity model
-5* T
2+ 0.022782 * T-0.32889, the malicious Rate Models of the suitableeest product also is the quadratic function model, as shown in Figure 6, is the equation F=0.0018543 * T in the present embodiment step 3
2-0.050277 * T+0.32629.
Table 1 malicious model of the suitableeest product and corresponding R at each temperature
2Value
| Temperature | The malicious model of the suitableeest product | ??R 2 |
| ??15℃ | ??z＝0.0112m-0.5555 | ??0.9965 |
| ??18℃ | ??z＝0.0215m-1.1035 | ??0.9918 |
| ??20℃ | ??z＝0.031m-0.8123 | ??0.9978 |
| ??23℃ | ??z＝0.0683m-1.2689 | ??0.9914 |
| ??25℃ | ??z＝0.3343m-4.5729 | ??0.9913 |
| ??30℃ | ??z＝0.4795m-4.3495 | ??0.9973 |
| ??37℃ | ??z＝0.9986m-5.2686 | ??0.9963 |
Annotate: m represents the time, and unit is hour (h); Z represents SEA content, and unit is ng/mL.
Simultaneous test: first group is to utilize the method prediction Ruzhong staphylococcus aureus growth of present embodiment and produce poison, and the growth temperature of streptococcus aureus is 21 ℃, and initial bacterium number is 10
2.845Cfu/mL, growth time is 26h, by the measurable growth velocity that goes out the Ruzhong streptococcus aureus of the calculation formula of step 1 is 0.18, then according to the formula of step 2 (according to the growth curve of streptococcus aureus as can be known the C in the formula be 7.1[log10 (cfu/mL)], M is 20h) it is measurable that to go out the bacterium number of the 26th hour Ruzhong streptococcus aureus under 21 ℃ be 10
7.9Cfu/mL, and then according to step 3 the measurable product poison speed that goes out the Ruzhong streptococcus aureus of formula be 0.0882, reach 10 in conjunction with the bacterium number
6.4The time of cfu/mL is 22h, is 0.3528ng/mL thereby calculate the 26th hour content of toxins.
Second group is that to utilize existing colony counting method and SET2 test kit to detect culture temperature be 21 ℃, and initial bacterium number is 10
2.845Cfu/mL, the bacterium number and the product poison that grow into the streptococcus aureus of 26h are measured, and detected result demonstration Ruzhong streptococcus aureus is 10 at the 26th hour bacterium number
7.4Cfu/mL, the Ruzhong streptococcus aureus is 0.3257ng/mL the 26th hour product poison amount; By these two groups of result of experiment method accuracy of the present invention as can be seen height.
Claims (1)
1. predict Ruzhong staphylococcus aureus growth and the method for producing poison, it is characterized in that predicting the Ruzhong staphylococcus aureus growth and producing malicious method and carry out according to following steps: one, growth temperature of detection Ruzhong streptococcus aureus and the initial bacterium number of Ruzhong streptococcus aureus, then with testing sample temperature substitution y=7.1917 * 10
-5* T
2+ 0.022782 * T-0.32889 formula calculates the maximum growth rate of Ruzhong streptococcus aureus, and wherein T represents the growth temperature of streptococcus aureus, and y represents the maximum growth rate of Ruzhong streptococcus aureus; Two, initial bacterium number of Ruzhong streptococcus aureus in the step 1 and the maximum growth rate of Ruzhong streptococcus aureus are updated to formula log N (t)=A+C[exp (exp (y (t-M)))] in, calculate the quantity of Ruzhong streptococcus aureus when time t, wherein exp=e
xT represents the growth time of Ruzhong streptococcus aureus, the quantity of log N (t) Ruzhong streptococcus aureus when being illustrated in t, A is the initial bacterium number [log10 (cfu/mL)] of Ruzhong streptococcus aureus in the step 1, C represents that the Ruzhong streptococcus aureus is at the growth bacterium number [log 10 (cfu/mL)] of stationary phase, M represents the pairing time of maximum growth rate, and y is at the maximum growth rate of time M [(log10[cfu/mL])/h]; Three, the Ruzhong streptococcus aureus that step 2 is calculated is 10 at the bacterium number
6.4Pairing time t is the time of origin that toxin produces during cfu/mL, again in conjunction with formula F=0.0018543 * T
2--the malicious speed of the resulting product of 0.050277 * T+0.32629, the linearity that finally obtains Ruzhong streptococcus aureus when temperature T is produced malicious curve, F represents that the Ruzhong streptococcus aureus produces malicious speed, T represents the growth temperature of streptococcus aureus, promptly realized the Ruzhong staphylococcus aureus growth and produced malicious model construction, can dope following detected sample Ruzhong staphylococcus aureus growth and the malicious situation of product put any time accurately according to this structure model and detected sample detection data.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825549A (en) * | 2018-12-20 | 2019-05-31 | 青岛农业大学 | The risk assessment of fresh milk staphylococcus aureus and quick early warning system |
| CN111127239A (en) * | 2020-01-13 | 2020-05-08 | 吉林大学 | Method for establishing staphylococcus aureus growth prediction model in spinach juice |
-
2009
- 2009-12-25 CN CN200910217427A patent/CN101787389A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825549A (en) * | 2018-12-20 | 2019-05-31 | 青岛农业大学 | The risk assessment of fresh milk staphylococcus aureus and quick early warning system |
| CN111127239A (en) * | 2020-01-13 | 2020-05-08 | 吉林大学 | Method for establishing staphylococcus aureus growth prediction model in spinach juice |
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Inventor after: Jiang Yujun Inventor after: Zhao Feng Inventor after: Wang Kexin Inventor after: Qu Xingguang Inventor after: Lv Xuena Inventor after: Xue Yuqing Inventor after: Xie Kunhao Inventor after: Yang Shiqin Inventor after: Yao Liyan Inventor before: Jiang Yujun Inventor before: Zhao Feng Inventor before: Qu Xingguang Inventor before: Lv Xuena Inventor before: Xue Yuqing Inventor before: Xie Kunhao Inventor before: Yang Shiqin Inventor before: Yao Liyan |
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Free format text: CORRECT: INVENTOR; FROM: JIANG YUJUN ZHAO FENG QU XINGGUANG LV XUENUO XUE YUQING XIE KUNHAO YANG SHIQIN YAO LIYAN TO: JIANG YUJUN ZHAO FENG WANG KEXIN QU XINGGUANG LV XUENUO XUE YUQING XIE KUNHAO YANG SHIQIN YAO LIYAN |
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Application publication date: 20100728 |