CN103776807A - Method for detecting residual bacteria in flushed water of CIP (cleaning in place) cleaning system - Google Patents
Method for detecting residual bacteria in flushed water of CIP (cleaning in place) cleaning system Download PDFInfo
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- CN103776807A CN103776807A CN201410030608.9A CN201410030608A CN103776807A CN 103776807 A CN103776807 A CN 103776807A CN 201410030608 A CN201410030608 A CN 201410030608A CN 103776807 A CN103776807 A CN 103776807A
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Abstract
The invention discloses a method for detecting residual bacteria in flushed water of a CIP (cleaning in place) cleaning system. The method comprises the following steps: (1) taking at least three parts of flushed water of the CIP cleaning system, placing into a sterile reagent bottle, weighing flushed water of the CIP cleaning system with unequal quantity respectively, filtering respectively by sterile filtering membranes, placing the sterile filtering membranes into a sample cup after filtration, then dropwise adding composite fluorescent reagents on the sterile filtering membranes, and then placing the sample cup into an ATP (Adenosine Triphosphate) fluorescence detector for determining each fluorescence value; (2) weighing flushed water of the CIP cleaning system with the quantity equal to that in the step (1), filtering by the sterile filtering membranes, eluting bacteria on the sterile filtering membranes after filtration, and determining the total bacterial count in flushed water of the CIP cleaning system so as to obtain a standard curve between the total bacterial count and the fluorescence value; and (3) taking to-be-detected flushed water of the CIP cleaning system, thereby obtaining the total bacterial count in the flushed water of the to-be-detected CIP cleaning system according to the fluorescence value obtained in the step (1) and the standard curve. The method provided by the invention is an innovation on a determination mode, simplifies the operation procedure, and reduces error source.
Description
Technical field
The method that the present invention relates to residual bacterium in a kind of CIP of detection purging system wash-down water, belongs to microorganism detection field, food safety detection aspect.
Background technology
Food processing enterprises, in production process, need to relate to the health cleaning of equipment, pipeline etc. industries such as dairy products, beverage, drinks, the carelessness of any one link all may bring the economic loss that is difficult to retrieve and severe social influence.CIP(Cleaning In Place) clean on the spot, refer to and need not take apart or mobile device, adopt the detergent remover of high temperature, high concentration, to apparatus forceful action in addition, cleaning with the surface of contact of food, health rank is required to cleaning, the purification of stricter production equipment, be that food-processing industry was adaptation large-scale production in recent years, meet high-quality hygienic requirements, reduce the cleaning technique that cleans cost and grow up.Along with the progressively raising of food enterprise up-to-dateness, CIP purging system will be applied in the middle of varieties of food items processing enterprise future.After CIP work, requirement will be to smell, the visual appearance of equipment, the several aspects of microbial contamination are evaluated, wherein the inspection of microbial contamination is difficult point, also be the health critical control point of food processing, if clean complete after in CIP system wash-down water residual bacterium too high, can bring the hidden danger of microbial contamination to later stage food processing, and after CIP process, residual bacterium mostly is thermoduric bacteria, the high-temperature sterilization measure in food processing later stage is also difficult to it to kill completely, therefore, in CIP system wash-down water, for food production processing health, management and control has great directive significance in the detection of residual bacterium, must strictly control.
CIP cleaning performance requires to reach cleanness degree in microbes or aseptic cleanliness, according to investigation, after CIP process, in the internal control index request CIP system residuary water of food enterprise, total number of bacteria is lower than the sensing range of total plate count national standard method (GB4789.2), so national standard method cannot direct-detection and the microbes contamination of evaluating after CIP cleans, so will find other detection method.In industry, the detection method of the residual bacterium of CIP system is had to two kinds at present: the one, after large volume suction filtration, carry out again flat board and cultivate counting.The advantage of the method is to utilize filter membrane enrichment of bacterial, but membrane retention bacterium still will proceed to and on nutrient culture media, carry out 48 little cultivations, although result is comparatively accurate, efficiency is too low, and CIP can not allow process equipment within idle 48 hours, wait for measurement result after cleaning.The 2nd, Microscopical Method For Detection, this method is to be also combined with large volume suction filtration, the advantage of method is to have shortened detection time, but Microscopical Method For Detection is placed on filter membrane in appropriate sterilized water, shake, vibration, and then by the quantity of microorganism in microscopic examination eluting water, program complexity is easily polluted by inoculating microbe, the error of bringing in wash-out membrane retention bacterium and transfer process is large and be difficult to avoid.From said method, lack in the industry at present the standard method of measuring the residual bacterium of CIP rinse water, and also there is the shortcoming that efficiency is low or program complex operations error is larger in alternative method, therefore, be still necessary to develop a kind of method that can fast, accurately, simply detect residual bacterium in CIP system wash-down water.
Summary of the invention
The object of this invention is to provide the method for residual bacterium in a kind of CIP of detection purging system wash-down water, method of the present invention can Fast Measurement CIP system wash-down water etc. residual total number of bacteria in the extremely low fluid sample of bacteria containing amount, can be in time to CIP system cleaning performance assess, to health, cleaning is instructed.
A kind of method that detects residual bacterium in CIP purging system wash-down water provided by the present invention, Bao draws together the Sudden as Xia Bu:
(1) be taken to few 3 parts of CIP purging system wash-down waters and be placed in aseptic reagent bottle, be designated as respectively headpin, No. 2 bottles and No. 3 bottles; From described headpin, No. 2 bottles and No. 3 bottles, measure respectively the CIP purging system wash-down water of inequality, filter through aseptic filter membrane respectively, after filtration, described aseptic filter membrane is put into sample cup, then on described aseptic filter membrane, drip composite fluorescence reagent, more described sample cup is placed in to ATP fluorescence detector measures each fluorescent value;
(2) from described headpin, No. 2 bottles and No. 3 bottles, measure respectively and the CIP purging system wash-down water of step (1) moderate, filter through described aseptic filter membrane, after filtration, described in wash-out, the bacterium on aseptic filter membrane obtains eluent, measures the total plate count in described eluent; And then obtain the typical curve between total plate count and fluorescent value;
(3) get CIP purging system wash-down water to be measured, filter through described aseptic filter membrane, after filtration, described aseptic filter membrane is put into described sample cup, then on described aseptic filter membrane, drip described composite fluorescence reagent, more described sample cup is placed in to described ATP fluorescence detector measures fluorescent value; Then obtain total plate count in CIP purging system wash-down water to be measured according to the typical curve between described fluorescent value and described total plate count.
In above-mentioned method, in step (1), can from described headpin, described No. 2 bottles and described No. 3 bottles, measure respectively CIP purging system wash-down water described in 10mL, 50mL and 100mL.
In above-mentioned method, in step (1), the aperture of described aseptic filter membrane can be 0.45 μ m.
In above-mentioned method, the principal ingredient of described composite fluorescence reagent is bacteria lysis agent and luciferase, the composite fluorescence reagent that the production code member that Instrument Ltd. as scientific and technological in middle matter match good fortune (Beijing) produces is ZF0011.
In above-mentioned method, in step (2), the solvent of the bacterium described in wash-out on aseptic filter membrane can be sterile saline.
Tool of the present invention has the following advantages:
The new method that the present invention proposes first and applied cracking on film, measured with film.The inventive method, for the innovation of the mode of mensuration, has been simplified running program, in the process of microorganism detection, reduces single stepping program, has just reduced by a source of error.And for enterprise the method have significant economic benefit method (the present invention can allow enterprise in time to CIP clean program carry out handle control, fast detecting can also shorten the evaluation time and improve the production efficiency of enterprise, in guaranteeing cleaning performance, save to greatest extent the resources such as water, cleaning fluid, electricity, can also save the input of manpower and nutrient culture media, culture device etc., obviously reduce and clean cost.The timely of CIP cleaning performance can reduce control the waste that enterprise brings because cleaning residual microbial product microbial contamination, cuts the waste and is equal to extra earning.), the present invention simultaneously also has good social benefit, and (the present invention can help the bacterium in Corporate Identity CIP cleaning process residual, reduce the possibility that production run is subject to microbial contamination, obvious to improving food safety standard meaning, the raising of security status of food is significant for social stability.) (in this method, do not relate to poisonous harmful reagent with environmental benefit, on film, cracking can, by whole bacterium cracking, not have residual viable bacteria, can not bring harm to operating personnel, testing process does not have accessory substance to produce yet, and monitoring utensil and medical disposable material neither do not pollute the environment with special processing yet.）。
Accompanying drawing explanation
Fig. 1 is fluorescent value (RLU) and liquid total number of bacteria linear relationship chart to be measured on the film of measuring in the embodiment of the present invention 1.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The mensuration of total number of bacteria in embodiment 1, CIP system residuary water
Instrument and consumptive material:
ATP fluorescence detector (SF0012 type);
Asepsis injector (10mL～50mL);
Filtrator (reusable, first use 75% alcohol-pickledly, repeatedly rinse with sterilized water, under aseptic condition, dry or dry after for subsequent use)
Tweezers (plastics, the alcohol-pickled aseptic water washing of using again for plastic material, dries rear for subsequent use);
Aseptic filter membrane (aperture: 0.45 μ m, size: diameter 13mm);
Composite fluorescence reagent (middle matter is matched the scientific and technological Instrument Ltd. of good fortune (Beijing), and production code member is ZF0011), the principal ingredient of this composite fluorescence reagent is bacteria lysis agent and luciferase.
Glass test tube (filling 5mL stroke-physiological saline solution after sterilizing for subsequent use);
(1) formulation of typical curve
Get water after last one program of CIP system finishes as detected object, with 5 aseptic reagent bottles of 500mL, numbering 1,2,3,4 and 5, reception is surveyed water and is stored for future use.Get 10 filtrators, with tweezers, aseptic filter membrane is put into filtrator, install for subsequent use.With water 10mL in syringe absorption headpin, filter with mounted aseptic filter membrane and filtrator, filter and complete aseptic filter membrane tweezers are taken out and put into cleaned sample cup, on aseptic filter membrane, drip composite fluorescence reagent, put into ATP fluorescence detector and measure its fluorescent value, getting water 10mL in headpin with same syringe again filters again, filter and completely filter membrane is taken out and puts into the glass test tube that 5mL sterile saline is housed and shake wash-out bacterium with aseptic nipper, then detect total plate count according to GB47892-2010 method.
Then from No. 2 bottles, get 50mL respectively, in No. 3 bottles, get 100mL, in No. 4 bottles, get 150mL, in kingpin, get 200mL, measure respectively each fluorescent value and total plate count according to aforesaid operations step, five groups of corresponding datas are done to linear regression analysis and obtain typical curve equation Y=aX+b, wherein Y representative records fluorescent value, and X represents total plate count value (CFU/mL).
(2) mensuration of sample and calculating
Getting the mounted aseptic filter membrane of 50mL water to be measured and filtrator filters, filter and complete aseptic filter membrane tweezers are taken out and put into cleaned sample cup, on aseptic filter membrane, drip composite fluorescence reagent, put into ATP fluorescence detector and measure fluorescent value, obtain fluorescent value Y, bring Y value into above-mentioned typical curve equation, can calculate total number of bacteria content in this sample according to extension rate.
In order to verify operability and the accuracy of this method, select a Dairy Enterprise to verify on the spot.Measure according to aforesaid operations method.
Test parameters:
Concentrated: No. 1 blank 10mL sterilized water is concentrated, No. 2 10mL water to be measured is concentrated, and No. 3 100mL water to be measured is concentrated, and No. 4 200mL water to be measured is concentrated.
Wash-out parameter: 5mL stroke-physiological saline solution wash-out.
Result is as shown in table 1:
Table 1 actual sample measurement result analyzes-1
Can find out from the gradient relation of eluent total number of bacteria, along with the increase of concentrated volume, on filter membrane, concentrated pressure becomes large, cause wash-out difficulty, eluting rate is along with the increase of concentrated volume reduces gradually, so select the concentrated relatively easy elution samples gained GB data of 10mL and multiple thereof to calculate as typical curve computing reference value, result is as shown in table 2:
Table 2 actual sample measurement result analyzes-2
Do linear dependence analysis with fluorescent value on film (RLU) and liquid total number of bacteria to be measured (CFU/mL), (corresponding typical curve equation is result: y=0.0351x-0.034) as shown in Figure 1, two groups of data dependences are better, illustrate the method can Accurate Determining water to be measured in total number of bacteria, coordinate axis only initial point is owing to having in water to be measured due to the free ATP of part.
Claims (5)
1. detect a method for residual bacterium in CIP purging system wash-down water, bag is drawn together as lower step Sudden:
(1) be taken to few 3 parts of CIP purging system wash-down waters and be placed in aseptic reagent bottle, be designated as respectively headpin, No. 2 bottles and No. 3 bottles; From described headpin, No. 2 bottles and No. 3 bottles, measure respectively the CIP purging system wash-down water of inequality, filter through aseptic filter membrane respectively, after filtration, described aseptic filter membrane is put into sample cup, then on described aseptic filter membrane, drip composite fluorescence reagent, more described sample cup is placed in to ATP fluorescence detector measures each fluorescent value;
(2) from described headpin, No. 2 bottles and No. 3 bottles, measure respectively and the CIP purging system wash-down water of step (1) moderate, filter through described aseptic filter membrane, bacterium on aseptic filter membrane described in wash-out after filtration, measures the total plate count in CIP purging system wash-down water; And then obtain the typical curve between total plate count and fluorescent value;
(3) get CIP purging system wash-down water to be measured, filter through described aseptic filter membrane, after filtration, described aseptic filter membrane is put into described sample cup, then on described aseptic filter membrane, drip described composite fluorescence reagent, more described sample cup is placed in to described ATP fluorescence detector measures fluorescent value; Then obtain total plate count in CIP purging system wash-down water to be measured according to the typical curve between described fluorescent value and described total plate count.
2. method according to claim 1, is characterized in that: in step (1), measure respectively CIP purging system wash-down water described in 10mL, 50mL and 100mL from described headpin, described No. 2 bottles and described No. 3 bottles.
3. method according to claim 1 and 2, is characterized in that: in step (1), the aperture of described aseptic filter membrane is 0.45 μ m.
4. according to the method described in any one in claim 1-3, it is characterized in that: the principal ingredient of described composite fluorescence reagent is bacteria lysis agent and luciferase.
5. according to the method described in any one in claim 1-4, it is characterized in that: in step (2), the solvent of the bacterium described in wash-out on aseptic filter membrane is physiological saline.
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| CN201410030608.9A CN103776807B (en) | 2014-01-22 | 2014-01-22 | A kind of method detecting remaining bacteria in CIP purging system wash-down water |
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Cited By (4)
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| CN106338301A (en) * | 2016-04-10 | 2017-01-18 | 天津海河标测技术检测有限公司 | Test method for confirming cleaning mode of reusable medical instrument |
| CN109295158A (en) * | 2018-09-30 | 2019-02-01 | 军事科学院系统工程研究院卫勤保障技术研究所 | A kind of quick sterilization effect evaluation method |
| CN111718976A (en) * | 2020-06-29 | 2020-09-29 | 四川新华西乳业有限公司 | Method for detecting thermophilic bacteria in CIP cleaning residual water |
| CN112051204A (en) * | 2020-08-14 | 2020-12-08 | 一汽奔腾轿车有限公司 | Method for detecting filtering cleanliness of water-based colored paint |
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| CN111718976A (en) * | 2020-06-29 | 2020-09-29 | 四川新华西乳业有限公司 | Method for detecting thermophilic bacteria in CIP cleaning residual water |
| CN112051204A (en) * | 2020-08-14 | 2020-12-08 | 一汽奔腾轿车有限公司 | Method for detecting filtering cleanliness of water-based colored paint |
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