CN104745671A - Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte - Google Patents
Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte Download PDFInfo
- Publication number
- CN104745671A CN104745671A CN201310751813.XA CN201310751813A CN104745671A CN 104745671 A CN104745671 A CN 104745671A CN 201310751813 A CN201310751813 A CN 201310751813A CN 104745671 A CN104745671 A CN 104745671A
- Authority
- CN
- China
- Prior art keywords
- content
- detection
- lactic acid
- escherichia coli
- substratum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a determination method of the content of Escherichia coli for resisting interference of lactic acid zymophyte, and belongs to the technical field of microbiological detection. The invention comprises a dedicated culture medium for determining the content of Escherichia coli and a determination method thereof. The method is simple, fast, accurate, and efficient in operation, and can be widely applied to the detection of Escherichia coli in industries of food, medicine, rear poultry and the like, and is especially suitable for detecting the content of Escherichia coli in a microbiological preparation product capable of fermenting to produce the lactic acid.
Description
Technical field
The present invention relates to a kind of E. CoIi content measuring method of anti-lactic acid fermenting bacteria interference, be specifically related to a kind of detection method for E. CoIi content in the industries such as food, medicine, herding, particularly relate to a kind of detection method for E. CoIi content in the probiotics of lactic acid producing of fermenting.。
Background technology
Intestinal bacteria are one of coliform, and it is subject to the Bacteria Indicators of enteropathic fungi pollution as evaluation, be one of important indicator of evaluating water quality and food hygiene quality, are also one of important indicators evaluating probiotics quality product quality.
Current country mainly contains two standards to coil determination in food service industry, i.e. standard GB/T/T4789.3-2008 and import-export commodity inspection industry standard SN/T0169-2010.What adopt in GB/T4789.3-2008 is that MPN counting process and colony counting method measure coliform count, is mainly used in food service industry; MPN counting process goes through just fermentation, recurrence ferment confirms the positive pipe number of LST, more most probable intestinal bacteria quantity tried to achieve by corresponding most probable number key (MPN table).Colony counting method by VRBA culture medium culturing, again by BGLB meat soup pipe aerogenesis confirm calculate intestinal bacteria quantity.Be that MPN counting process or colony counting method all exist the problems such as length consuming time (4-5 days), complicated operation, most possible numerical value especially that MPN counting process obtains not is a definite numerical value.Just on the basis of GB GB/T4789.3-2008, beta-glucoside Macrophages and filter membrane/MUG method is added in industry standard SN/T 0169-2010.These two kinds of methods need the equipment of specialty to assist, and testing cost is high, more difficult for general enterprise's operation, is suitable only for the enterprises and institutions that there are certain economic strength in scientific research institutions, colleges and universities etc.The colibacillary implementation standard HJ/T347-2007 detecting employing in water quality.For filter membrane method and 15 pipe fermentation methods, filter membrane method be first by water sample after filter membrane suction filtration, then filter membrane sheet be put in substratum cultivate, reach a conclusion after proof test is carried out to typical case or suspicious bacterium colony, 2 days consuming time; 15 pipe fermentation method operations are divided into three steps, three days consuming time of rear two step confirmatory tests.Therefore being not difficult to find out, obviously there is the deficiencies such as loaded down with trivial details, the consuming time length of testing sequence, testing cost are high, detected result out of true in above various methods.Even more noteworthy, above-mentioned all methods are only applicable to colibacillary detection in general conventional food, water quality, and are not suitable for the detection of some specialty products, such as: probiotics product.
Probiotics is that a class is by one or more active bacteria formulation products formed wherein such as milk-acid bacteria, yeast, genus bacillus, it can improve the health level of the mankind, livestock and poultry host or plant host, or promote normal microflora growth in host or pin main body, microbial state treatment in adjustable host, keeps microecological balance.And intestinal bacteria are one of sanitary indexs of important control in probiotics product.Intestinal bacteria quantity number direct affect probiotics quality product quality.Intestinal bacteria exceed standard directly causing people to have loose bowels, enteritis, the diseases such as animal diarrhea.Therefore need to find a kind of detection method for detecting E. CoIi content in probiotics product.
Probiotics product on sale is on the market all gram-positive microorganism at present, as: milk-acid bacteria, yeast, bacillus category etc.In milk-acid bacteria and genus bacillus, some are planted is can the gram-positive microorganism of ferment lactose lactic acid producing.By judging whether produce sour aerogenesis to verify whether have colibacillary existence in fermentation tube in MPN counting process in GB GB/T4789.3-2008, and some bacterial classification (Bacillus coagulans, milk-acid bacteria etc.) is also also be can produce sour aerogenesis by ferment lactose in anaerobic environment in Tiny ecosystem product, therefore colibacillary detected result can be disturbed; Add cholate, Viola crystallina and toluylene red to carry out colibacillary detection as developer in colony counting method, colibacillary content is judged by colour developing reflection, and milk-acid bacteria in probiotics and Bacillus coagulans also can by the mode metabolism lactic acid of fermentation thus the pH reducing substratum causes indicator to develop the color, thus interference detection results.Therefore colony counting method is the same with MPN counting process does not take into full account that in probiotics product, viable bacteria is to the interference of coil determination result, can such as genus bacillus grow on the substratum of colony counting method? whether the probiotic bacterium (as lactic acid bacteria class) that can produce acid can disturb colibacillary detection, etc. ..., all existing E. CoIi content detection method of all these critical problems is not considered.Therefore, existing E. coli detection method can not be used for the E. coli detection in probiotics product.
Patent CN1796568 has invented a kind of E. coli detection method, adopts Sodium Lauryl Sulphate BP/USP to join in substratum as fungistat, can be used for detecting the intestinal bacteria in tap water, environment ArsenazoⅢ.But the use range of patent CN1796568 is limited, tap water, ambient water and microorganism in food content are few, particularly the genus bacillus quasi-microorganism of anti-Sodium Lauryl Sulphate BP/USP is few especially, and probiotics product is the active bacteria formulation that a class comprises the microorganisms such as genus bacillus, milk-acid bacteria, yeast, its microbe population is far longer than the microbe population in tap water, environment ArsenazoⅢ.To sum up, patent CN1796568 does not consider that in probiotics product, a large amount of viable bacteria is on the impact of E. CoIi content detected result, and the E. CoIi content be not suitable in probiotics detects.Up to the present, document inspection does not find a kind of effective detection method being directed to E. CoIi content in probiotics product.For above all situations, need to find a kind of substratum being suitable for E. CoIi content detection in probiotics product.Therefore, we propose a kind of detection method being specifically designed to E. CoIi content in probiotics product.
Summary of the invention
The present invention proposes a kind of method of counting of E. CoIi content of anti-lactic acid fermenting bacteria interference, obviously can promote the practicality of E. CoIi content detection method, adopt the method can detect colibacillary exact amount in 24 hours.The present invention obviously can abandon the interference of lactic acid fermenting bacteria to detected result, reduces the probability of false positive and false negative appearance, improves the accuracy of detection efficiency and detected result.
E. coli detection substratum of the present invention is by Tryptones 10.0-15.0 g, lactose 10.0-20.0 g, SDS 0.5-1.0 g, massfraction is the TTC solution 3.0-7.0 ml of 2.0%, dipotassium hydrogen phosphate 4.0-6.0g, sodium-chlor 3.0-5.0 g, mass concentration is 1.6 g/100 ml purpurum bromocresolis solution 2.0-6.0 ml, agar powder 10.0-20.0 g or sodium polyacrylate 15.0-25.0 g, distilled water 1 000 ml, final pH are 6.0-6.5.
In substratum, Tryptones is as the basic nutrition material of substratum; Lactose is as the fermentation substrate of E. coli detection; TTC and purpurum bromocresolis are as indicator; SDS adds as the fungistat of gram-positive microorganism, effectively can suppress the growth of lactic acid fermenting bacteria, prevents lactic acid fermenting bacteria ferment lactose lactic acid producing, produces false positive, affects E. coli detection result.
Detection method: coat in E. coli detection special culture media by dilution spread flat band method by measuring samples, optimal temperature is cultivated.
The judgement of detected result and calculating: if the first yellowing of bacterium colony periphery becomes red again, illustrate that this bacterium colony is intestinal bacteria, counting red colonies quantity.
Coliform count in sample=red colonies number × extension rate.
Embodiment
The preparation of embodiment 1, E. coli detection special culture media
1.6 g/100 ml purpurum bromocresolis solution preparations: take purpurum bromocresolis 1.6 g and pour small beaker into, dissolve with a small amount of raw spirit, move to 100 ml volumetric flasks, repeatedly small beaker is washed with raw spirit, washing lotion moves into volumetric flask, is finally settled to 100 ml with raw spirit, shakes up for subsequent use.
The TTC solution preparation of 2%: 2 g TTC are dissolved in 100 ml damping fluids and (dissolve 0.832 g Na in 100 ml distilled water
2hPO
42H
2o and 0.273 g KH
2pO
4, pH7.2).
Take Tryptones 10.0 g, lactose 15.0 g, SDS 0.5 g, dipotassium hydrogen phosphate 4.0 g, sodium-chlor 3.0 g, agar powder 15.0 g, distilled water 500 ml mixes, then adds TTC solution 5 ml that massfraction is 2.0%, and mass concentration is 1.6 g/100 ml purpurum bromocresolis solution 4 ml, mend distilled water to 1 000 ml, regulate pH to 6.0-6.5.
Be sub-packed in triangular flask by above-mentioned substratum and put into high-pressure sterilizing pot 115 DEG C of sterilizing 15 min, system of falling after being cooled to 50 DEG C is dull and stereotyped for subsequent use.
Embodiment 2, substratum practicality are verified
Grope culture medium prescription, find out applicable Escherichia coli Growth, but the formula of the growth of other probiotics in probiotics can be suppressed.
Picking subtilis, bacillus cereus, Bacillus coagulans, Bacillus licheniformis, enterococcus faecalis, milk-acid bacteria, yeast and intestinal bacteria line in the intestinal bacteria special culture media adding different concns SDS, observe colony growth situation on flat board under optimal temperature after cultivating 48 hr.
Table 1 different SDS concentration is on the impact of bacterium/fungal growth
Note: " √ " indicates and can grow; ×, sign can not grow; Zero, represent that growth is not obvious.
Experimental result finds, 1. genus bacillus bacterioid all can not grow higher than during 0.2 g/L in SDS concentration; 2. enterococcus faecalis can not grow higher than during 0.4 g/L in SDS concentration; 3. yeast is not obvious higher than growing phenomenon during 0.2 g/L in SDS concentration; 4. intestinal bacteria are in SDS concentration higher than well-grown during 0.5 g/L, show that SDS does not have restraining effect to intestinal bacteria.Therefore, in intestinal bacteria special culture media, SDS concentration is high can be used for colibacillary detection in probiotics when being 0.5 g/L.
The practicality comparison test of embodiment 3, intestinal bacteria special culture media
Relatively A substratum be generally applicable to the LB substratum that intestinal bacteria cultivate and carry out simultaneous test, see the difference of two kinds of substratum, the whether applicable colibacillary growth of checking A substratum.Getting after the intestinal bacteria cultivating 24hr are diluted to finite concentration is applied on LB and A culture medium flat plate respectively, and each extent of dilution is coated with 3 flat boards, calculates viable count respectively.
Table 2 intestinal bacteria count contrast in LB and A substratum
Experimental result repeatedly shows, intestinal bacteria special culture media is more suitable for colibacillary growth.In intestinal bacteria special culture media, colonial morphology is in yellow after cultivation 12 hr for intestinal bacteria, and after cultivating 24 hr, colonial morphology is for red.Therefore, intestinal bacteria special culture media can be used for the detection of coliform count in probiotics.
Embodiment 4, the inventive method compare with GB flat band method
Get the E. coli broth cultivating 24hr, coat on the substratum of intestinal bacteria special culture media of the present invention and GB flat band method after being diluted to finite concentration, method more of the present invention and GB flat band method detect intestinal bacteria quantity, see whether both have gap.
Table 3 the present invention and plate technique method comparison and detection E. Coli results
Experimental result shows, the inventive method is cultivated after 24hr can the result, and GB flat band method will wait until 48hr, and the even longer time could the result.And from E. CoIi content, what gap is method of the present invention do not have compared with GB middle plateform method, illustrate that method of the present invention can be used for the detection of E. CoIi content, and method of the present invention is operationally imitated and is more better than GB flat band method.
The detection of E. CoIi content in embodiment 5, commercially available feeding micro-ecological preparation
In order to verify whether the present invention is applicable to the detection of E. CoIi content in feeding micro-ecological preparation product, and called in several product of feeding micro-ecological preparation targetedly from the market, carried out colibacillary detection by method of the present invention, detected result is as follows:
E. CoIi content detected result in the commercially available feeding micro-ecological preparation product of table 4
Detected result finds, has in part feeding micro-ecological preparation the situation that there is intestinal bacteria quantity and exceed standard.
Embodiment 6, commercially available people are by probiotics E. CoIi content detected result
In order to verify whether the present invention is applicable to the detection of people with E. CoIi content in probiotics product, call in several people targetedly from the market and use probiotics product, carried out the detection of E. CoIi content by method of the present invention, detected result is as follows:
The commercially available people of table 5 is by E. CoIi content detected result in probiotics product
Upper table result shows, refreshing treasured, Si Liankang and the Medilac-Vita of relaxing all does not detect intestinal bacteria.
Claims (4)
1. an E. CoIi content measuring method for anti-lactic acid fermenting bacteria interference, can be used for the E. coli detection in the industries such as food, medicine, herding, the detection of E. CoIi content in the probiotics product of the lactic acid producing that is specially adapted to ferment.
2. the substratum for detecting E. CoIi content according to claim 1, its formula is as follows: Tryptones (or peptone) 10.0-15.0 g, lactose 10.0-20.0 g, SDS 0.5-1.0 g, massfraction is TTC solution 5 ml of 2.0%, dipotassium hydrogen phosphate 4.0 g, sodium-chlor 3.0 g, mass concentration is 1.6 g/100 ml purpurum bromocresolis solution 4.0 ml, agar powder 10.0-20.0 g or sodium polyacrylate 15.0-25.0 g, distilled water 1 000 ml, pH6.0-6.5,115 DEG C of sterilizing 15 min.
3. detection method according to claim 1 is the mensuration for E. CoIi content in probiotics product, arbitrarily its use range of conversion, or carries out on this basis extending and all belong to this rights protection scope.
4. the culture medium prescription provided in claim 2 is a variate-value, changes arbitrarily content that is single or Multiple components in substratum and all belongs to this rights protection scope.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310751813.XA CN104745671A (en) | 2013-12-31 | 2013-12-31 | Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310751813.XA CN104745671A (en) | 2013-12-31 | 2013-12-31 | Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104745671A true CN104745671A (en) | 2015-07-01 |
Family
ID=53585923
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310751813.XA Pending CN104745671A (en) | 2013-12-31 | 2013-12-31 | Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104745671A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107860769A (en) * | 2017-11-08 | 2018-03-30 | 新疆天润生物科技股份有限公司 | A kind of quick determination method, its detection preparation and application for being used to prevent and treat milk beer production pickup dye |
| CN112980919A (en) * | 2019-12-17 | 2021-06-18 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
-
2013
- 2013-12-31 CN CN201310751813.XA patent/CN104745671A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107860769A (en) * | 2017-11-08 | 2018-03-30 | 新疆天润生物科技股份有限公司 | A kind of quick determination method, its detection preparation and application for being used to prevent and treat milk beer production pickup dye |
| CN112980919A (en) * | 2019-12-17 | 2021-06-18 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
| CN112980919B (en) * | 2019-12-17 | 2023-09-12 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Study on spoilage capability and VBNC state formation and recovery of Lactobacillus plantarum | |
| CN102424832B (en) | Chromogenic medium used for detecting esherichia coli O157:H7 | |
| CN105296591B (en) | A kind of culture medium and detection method for detecting difficult cultivation type lactic acid bacteria in food | |
| CN105176874B (en) | Bacillus coagulans FM603 and its application | |
| CN103642891A (en) | Method for inspecting microbes in cosmetics | |
| CN102864204B (en) | Enrichment culture solution for detection of Salmonella in poultry eggs | |
| EP1880014A2 (en) | Chromogenic plating media for the identification of enterobacter sakazakii | |
| CN101186891A (en) | Salmonella color culture medium, detection kit an detection method | |
| CN112481350A (en) | Composite culture medium for rapidly detecting total number of moulds and yeasts and preparation method thereof | |
| Pei et al. | Isolation and identification of fungi found in contaminated fermented milk and antifungal activity of vanillin | |
| CN102286606A (en) | Cronobacter spp. broth medium and detection method | |
| CN104745671A (en) | Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte | |
| CN112375710B (en) | Safe and nontoxic Bacillus belgii PH6 strain for specifically inhibiting MRSA (methicillin-resistant staphylococcus aureus) and application thereof | |
| CN103667417A (en) | Method for detecting microbes in beverage | |
| CN103820373A (en) | Chromogenic medium for separating and detecting enterobacter cloacae | |
| Duffy et al. | The ability of Campylobacter jejuni cells to attach to stainless steel does not change as they become nonculturable | |
| CN102706821A (en) | Method for quickly identifying food-borne pathogen bacterial biofilm formation inhibitor | |
| CN105349613A (en) | Detection method of high-temperature-resistant aerogenesis acidophil in table vinegar | |
| CN106086159B (en) | A kind of zymolyte culture medium that can detect two kinds of fecal pollution indicator bacterias simultaneously and its application | |
| CN107287275B (en) | Culture medium, kit containing culture medium and application of culture medium | |
| CN101643713A (en) | Legionnella selective separation medium | |
| Alo et al. | Microbiological qualities of burukutu produced from a mixture of sorghum and millet | |
| CN106434838A (en) | Selective beer spoilage bacterium culture medium and application thereof | |
| CN102076868A (en) | Culture medium containing a spore germination inhibiting or delaying compound | |
| CN103160563A (en) | Detection method for the Psychrophile content of milk and dairy product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150701 |