CN102433373B - Salmonella characteristic chromogenic liquid nutrient medium, preparation method thereof and rapid detection method of salmonella - Google Patents
Salmonella characteristic chromogenic liquid nutrient medium, preparation method thereof and rapid detection method of salmonella Download PDFInfo
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- CN102433373B CN102433373B CN 201110417455 CN201110417455A CN102433373B CN 102433373 B CN102433373 B CN 102433373B CN 201110417455 CN201110417455 CN 201110417455 CN 201110417455 A CN201110417455 A CN 201110417455A CN 102433373 B CN102433373 B CN 102433373B
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- salmonellas
- salmonella
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- 241000607142 Salmonella Species 0.000 title claims abstract description 131
- 239000007788 liquid Substances 0.000 title claims abstract description 64
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000001514 detection method Methods 0.000 title abstract description 28
- 239000000758 substrate Substances 0.000 claims abstract description 39
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims abstract description 22
- 229960003964 deoxycholic acid Drugs 0.000 claims abstract description 21
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims abstract description 21
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 20
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 18
- 239000000843 powder Substances 0.000 claims abstract description 18
- 241000894006 Bacteria Species 0.000 claims description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 31
- 150000002148 esters Chemical class 0.000 claims description 30
- 239000002131 composite material Substances 0.000 claims description 26
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- 239000012153 distilled water Substances 0.000 claims description 18
- -1 combination promotor Substances 0.000 claims description 17
- 238000004659 sterilization and disinfection Methods 0.000 claims description 17
- 108010046845 tryptones Proteins 0.000 claims description 17
- 239000012452 mother liquor Substances 0.000 claims description 15
- 239000001888 Peptone Substances 0.000 claims description 13
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- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 claims description 12
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 12
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- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 claims description 12
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- 229910052708 sodium Inorganic materials 0.000 claims description 12
- 239000011734 sodium Substances 0.000 claims description 12
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical group [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 12
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- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 9
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- WTFCBWXBJBYVOS-UHFFFAOYSA-L lithium;sodium;dichloride Chemical compound [Li+].[Na+].[Cl-].[Cl-] WTFCBWXBJBYVOS-UHFFFAOYSA-L 0.000 claims description 9
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of safety monitoring of food microorganisms, and discloses a salmonella characteristic chromogenic liquid nutrient medium, a preparation method thereof and a rapid detection method of salmonella. The salmonella characteristic chromogenic liquid nutrient medium comprises the following main components: tryptone, yeast powder, sodium chloride, lithium chloride, sodium deoxycholate, dipotassium phosphate, combined inhibitor, combined accelerator, characteristic enzymolysis substrate and cosolvent. The rapid detection method disclosed by the invention comprises two steps, i.e. pre-enrichment culture and chromogenic identification, and is characterized in that salmonella characteristic enzyme hydrolyzes corresponding substrates to result in that the culture medium is purple, thus rapidly judging the existence of salmonella; and the addition of the accelerator contributes to recovering damaged cells of salmonella and promoting growth of salmonella; and the added inhibitor can selectively inhibit the growth of other competitors so as to reduce the interference on detection by the competitors. The detection method disclosed by the invention has the advantages of short detection period, strong specificity and high accuracy, is simple to operate and is suitable for large-throughput detection of salmonella in food.
Description
Technical field
The present invention relates to a kind of Salmonellas feature colour developing liquid nutrient medium and preparation method thereof, relate in particular to Salmonellas feature colour developing liquid nutrient medium of a kind of principle that develops the color based on enzymolysis and preparation method thereof.Simultaneously, the invention still further relates to a kind of Salmonellas method for quick, relate in particular to a kind of feature enzyme that utilizes Salmonellas Salmonellas is carried out the method for rapid detection, belong to food-borne pathogens safety monitoring field.
Background technology
Salmonellas is the Gram-negative sporeless bacterium, the viability of this bacterium in external environment is stronger, the some months of in water, cow's milk and meat-based food, surviving, the optimum temperuture of its breeding is 37 ℃, wherein modally causes that the Salmonellas of food poisoning has Salmonella typhimurium, Salmonella choleraesuls, Salmonella enteritidis etc.Salmonella does not generally reduce lactose, sucrose and saligenin; Decomposition glucose produces sour aerogenesis (except the salmonella typhi), most H2S that form, do not produce indole, liquefy gelatin not, decomposing urea does not produce acetyl methyl carbinol, majority can utilize Chinese holly Citron hydrochlorate, the nitrate that can reduce is nitrite, does not grow no phenylalanine deaminase on potassium cyanide medium.Salmonellas is not strong to the resistibility of heat, and 60 ℃ of heating 15min are namely dead.
Salmonellas is the infecting both domestic animals and human cause of disease bacterium of a kind of serious threat mankind and animal life health, can cause the outer focal infection of humans and animals acute gastroenteritis, typhoid fever, septicemia and intestines etc., is classified as the category-A pathogenic micro-organism by FAO.Over the years, salmonellal food poisoning is often listed in the umber one of bacterial food poisoning.China disease prevention and control center has classified Salmonellas as one of bacterium index that requires control in the food.
Detection means accurately and timely is the key that prevention and control pathogenic micro-organism are propagated.The methods that adopt traditional microorganism culturing of the detection of Salmonellas at present more, increase steps such as bacterium, selective enrichment, plate isolation, biochemical test and serological identification before mainly comprising, the experiment that relates to is more, the operation is more loaded down with trivial details, sense cycle long (generally need 4～7d), preparation and tailing in work is heavy, can't satisfy the real needs of pathogenic bacterium rapid detection in the present stage food.The substratum that selective enrichment uses mainly contains the brilliant green enrichment liquids of four sulphur hydrochlorates (TTB), selenite cystine broth (SC), magnesium chloride Victoria Green WPB enrichment liquid (MM) etc.When assorted bacterium amount was big, these substratum were not ideal to the selectivity inhibition of the non-Salmonellas of enterobacteriaceae, bring serious interference to plate isolation.Traditional plate isolation general using Salmonellas produces characteristics such as H2S, nonfermented lactose, judge suspicious bacterium colony by indicator colour-change and melanin deposition, the main substratum that adopts is sulfurous acid bismuth (BS) agar, HE agar and xylose lysine deoxidation cholate (XLD) agar etc.Use these substratum to occur the false positive bacterium colony easily, need a large amount of follow-up discriminating work, so specificity and selectivity effect remain to be improved.
Because Salmonellas can produce the stronger monooctyl ester enzyme of specificity, some have appearred in the market based on the dull and stereotyped color developing culture medium of Salmonellas of enzymolysis colour developing principle, such substratum specificity effect is better, can be directly used in evaluation, but price is more expensive, and to the selectivity of assorted bacterium suppress ability a little less than, wrong judgement appears when assorted bacterium amount is big easily.The MUCAP of Salmonellas (4-methylumbelliferyl-caprylate, 4-methyl umbelliferone monooctyl ester) fluorescent method has been applied in meat, egg product, sewage, medicine and the clinical diagnosis at present.This method is that enrichment liquid is streak culture on the selectivity flat board, drips MUCAP reagent at suspicious single bacterium colony, can be discharged the fluorescent substance 4-methyl umbelliferone by the monooctyl ester enzymic hydrolysis, produces the blue-fluorescence differentiation and be the Salmonellas positive under the 366nm ultraviolet lamp.But when fluorescence intensity is more weak, occur false negative result easily, and the use of photofluorometer has also brought inconvenience to experiment.
The method of traditional microorganism culturing remain even to this day world microorganism educational circles the authoritative method of generally accepted examination microorganism.In order to realize the rapid detection of Salmonellas, be necessary traditional method is improved.
Summary of the invention
The present invention is directed to deficiency of the prior art, provide a kind of selective enrichment and colour developing are identified Salmonellas feature colour developing liquid nutrient medium that unites two into one and preparation method thereof.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals:
A kind of Salmonellas feature colour developing liquid nutrient medium, component comprises distilled water, Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, characteristic enzymolysis substrate, solubility promoter, distilled water; Every 100mL distilled water needs other components contents to be: Tryptones 0.8～1.2g, yeast powder 0.4～0.8g, sodium-chlor 0.4～0.8g, lithium chloride 0.3～0.5g, sodium deoxycholate 0.1～0.3g, dipotassium hydrogen phosphate 0.1～0.2g, combination promotor 0.02～0.04g, composite restrainer 0.1～0.2g, characteristic enzymolysis substrate 0.02～0.04g, solubility promoter 4～6mL, pH7.2 ± 0.2.
Wherein, Tryptones and yeast powder provide bacterial growth and metabolism to produce required carbon source and the nitrogenous source of enzyme; Sodium-chlor provides balanced osmotic pressure; Lithium chloride is mainly used in suppressing intestinal bacteria, Shigellae and genus bacillus etc.; Sodium deoxycholate can be used for suppressing gram-positive microorganism, and Salmonellas is had certain promotion; Dipotassium hydrogen phosphate provides buffering for culture system, and non-Salmonellas is had certain inhibition; Characteristic enzymolysis substrate is directly colour developing after the effect of Salmonellas feature enzyme.
As preferably, make up promotor and comprise Sodium.alpha.-ketopropionate and niacinamide.Can promote the recovery of Salmonellas damaged cell, enzyme is produced in the dominant growth and the metabolism that are conducive to Salmonellas.
As preferably, the weight ratio of Sodium.alpha.-ketopropionate and niacinamide is 1: 1～1: 3.
As preferably, composite restrainer comprises Trisodium Citrate and acarbose.Be mainly used in suppressing gram-positive microorganism and part Gram-negative bacteria.
As preferably, the weight ratio of Trisodium Citrate and acarbose is 2: 1～1: 3.
The action effect of inhibitor and promotor depends on its concentration.For example, sodium deoxycholate can promote the growth of Salmonellas when concentration is low, but the restraining effect of gram-positive microorganism is weakened.Add composite restrainer and can assist the inhibition gram-positive microorganism, simultaneously Shigellae, genus bacillus etc. is also had good inhibition.Therefore, suppressing assorted bacterium is the synergistic results of various culture medium additive selectivity with the growth that promotes Salmonellas.
As preferably, characteristic enzymolysis substrate is monooctyl ester class substrate, contains the colour developing group, directly colour developing after the effect of Salmonellas feature enzyme.
As preferably, monooctyl ester class substrate is the indoles monooctyl ester.
As preferably, solubility promoter is methyl-sulphoxide.Do not need emulsification, directly promote characteristic enzymolysis substrate to be dispersed in the liquid nutrient medium.
The preparation method of described Salmonellas feature colour developing liquid nutrient medium, may further comprise the steps: Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water are mixed in proportion, heating for dissolving, pH7.2 ± 0.2 is transferred in the cooling back, 115 ℃ of sterilization 15min, be cooled to room temperature, as mother liquor, standby; With the fully dissolving in solubility promoter in proportion of characteristic enzymolysis substrate, filtration sterilization adds in the mother liquor, shakes up, and becomes liquid nutrient medium, puts refrigerator and preserves.
Another object of the present invention is to provide a kind of feature enzyme by Salmonellas that substrate is carried out the enzymolysis colour developing for the Salmonellas method for quick on basis, reduces the probability that false positive and false negative occur, and improves the accuracy of detection efficiency and detected result.Increase bacterium cultivation and colour developing before comprising and identified for two steps.Salmonellas can produce the stronger feature enzyme of specificity, i.e. monooctyl ester enzyme, and the feature enzyme can carry out the enzymolysis colour developing to substrate.
The Salmonellas method for quick uses Salmonellas feature colour developing liquid nutrient medium, may further comprise the steps:
(a) precedingly increase bacterium and cultivate: the 25g sample is placed the 225mL buffered peptone water, mix back 37 ℃ of static cultivation 8～10h, get enrichment liquid;
(b) colour developing is identified: get the 1mL enrichment liquid and add in the 9mL Salmonellas feature colour developing liquid nutrient medium, mix back 37 ℃ of static cultivation 1～2d, if Salmonellas feature colour developing liquid nutrient medium is purple, illustrate that there is Salmonellas in this sample.
Selective enrichment and colour developing evaluation are united two into one, and selective enrichment is conducive to the growth of Salmonellas and produces enzyme, reduces the interfering factors that detects; The specificity of colour developing identification and utilization enzyme adopts the feature substrate, by the enzymolysis direct judged result that develops the color.
According to technical scheme of the present invention, the present invention is directed to the enzymolysis property of Salmonellas feature enzyme, develop Salmonellas feature colour developing liquid nutrient medium, selective enrichment and colour developing evaluation are united two into one, and set up the Salmonellas method for quick based on this, this method does not need special instruments and equipment, only with the naked eyes judged result, workable, be suitable for the detection of big flux sample.The present invention is easy to operate, high specificity, accuracy height, and sense cycle shortens to 2～3d by 4～7d of traditional detection method, has reduced a large amount of human and material resources consumption, and detection efficiency improves greatly, can be widely used in fields such as food sanitation, environmental monitoring.Salmonellas feature colour developing liquid nutrient medium provided by the invention preparation is simple, is easy to industrialization production.
Embodiment
Describe in further detail below by the present invention of embodiment.
Embodiment 1
Salmonellas feature colour developing liquid nutrient medium, filling a prescription is: Tryptones 0.8g, yeast powder 0.4g, sodium-chlor 0.4g, lithium chloride 0.3g, sodium deoxycholate 0.1g, dipotassium hydrogen phosphate 0.1g, combination promotor 0.02g, composite restrainer 0.1g, characteristic enzymolysis substrate 0.02g, solubility promoter 4mL, distilled water 100mL.
The preparation method of Salmonellas feature colour developing liquid nutrient medium: above-mentioned Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water are mixed in proportion, heating for dissolving, pH7.2 is transferred in the cooling back, 115 ℃ of sterilization 15min, be cooled to room temperature, as mother liquor, standby; With the fully dissolving in solubility promoter in proportion of characteristic enzymolysis substrate, filtration sterilization adds in the mother liquor, shakes up, and becomes liquid nutrient medium, 4 ℃ of stored refrigerated.
When sterilising temp is elevated to 121 ℃, when sterilization time surpassed 15min, combination promotor and composite restrainer took place partially modified easily, the influence effect.Therefore, the sterilising temp of mother liquor is not higher than 115 ℃, and sterilization time is no more than 15min.
Wherein, combination promotor is Sodium.alpha.-ketopropionate and niacinamide, weight ratio, Sodium.alpha.-ketopropionate: niacinamide=1: 1; Composite restrainer is Trisodium Citrate and acarbose, weight ratio, and Trisodium Citrate: acarbose=1: 1, characteristic enzymolysis substrate is the indoles monooctyl ester, solubility promoter is methyl-sulphoxide.
The indoles monooctyl ester is instability when temperature is higher, directly adds mother liquor and sterilizes and can generation degrade voluntarily, influences result's judgement.So the mode degerming of filtration sterilization is adopted in substrate dissolving back, adding is cooled in the mother liquor of room temperature then.
Monooctyl ester class substrate is lipid-soluble substance, is the oil droplet shape in the aqueous solution, is unfavorable for the effect of monooctyl ester enzyme.Methyl-sulphoxide is a kind of perviousness protective material, to the almost non-toxic property of cell, can mix with arbitrary proportion with water, and can dissolve general organic solvent, is described as " menstruum universale ".So, with methyl-sulphoxide dissolving monooctyl ester class substrate, it is dissolved in the feature colour developing liquid nutrient medium fully.
Salmonellas feature colour developing liquid nutrient medium can selectivity suppress non-Salmonellas, promote the recovery of Salmonellas and produce enzyme, selective enrichment and colour developing simultaneously identified and united two into one, shorten detection time, colour developing is identified with characteristic enzymolysis substrate, produce monooctyl ester enzymic hydrolysis colour developing by Salmonellas, judge the existence of Salmonellas.
Embodiment 2
Salmonellas feature colour developing liquid nutrient medium, filling a prescription is: Tryptones 1.0g, yeast powder 0.6g, sodium-chlor 0.6g, lithium chloride 0.4g, sodium deoxycholate 0.2g, dipotassium hydrogen phosphate 0.15g, combination promotor 0.03g, composite restrainer 0.15g, characteristic enzymolysis substrate 0.03g, solubility promoter 5mL, distilled water 100mL.
Wherein, combination promotor is Sodium.alpha.-ketopropionate and niacinamide, weight ratio, Sodium.alpha.-ketopropionate: niacinamide=1: 2; Composite restrainer is Trisodium Citrate and acarbose, weight ratio, and Trisodium Citrate: acarbose=1: 3, characteristic enzymolysis substrate is the indoles monooctyl ester, solubility promoter is methyl-sulphoxide.
The preparation method of Salmonellas feature colour developing liquid nutrient medium: above-mentioned Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water are mixed in proportion, heating for dissolving, pH7.4 is transferred in the cooling back, 115 ℃ of sterilization 15min, be cooled to room temperature, as mother liquor, standby; With the fully dissolving in solubility promoter in proportion of characteristic enzymolysis substrate, filtration sterilization adds in the mother liquor, shakes up, and becomes liquid nutrient medium, 4 ℃ of stored refrigerated.
Embodiment 3
Salmonellas feature colour developing liquid nutrient medium, filling a prescription is: Tryptones 1.2g, yeast powder 0.8g, sodium-chlor 0.8g, lithium chloride 0.5g, sodium deoxycholate 0.3g, dipotassium hydrogen phosphate 0.2g, combination promotor 0.04g, composite restrainer 0.2g, characteristic enzymolysis substrate 0.04g, solubility promoter 6mL, distilled water 100mL.
Wherein, combination promotor is Sodium.alpha.-ketopropionate and niacinamide, weight ratio, Sodium.alpha.-ketopropionate: niacinamide=1: 3; Composite restrainer is Trisodium Citrate and acarbose, weight ratio, and Trisodium Citrate: acarbose=2: 1, characteristic enzymolysis substrate is the indoles monooctyl ester, solubility promoter is methyl-sulphoxide.
The preparation method of Salmonellas feature colour developing liquid nutrient medium: above-mentioned Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water are mixed in proportion, heating for dissolving, pH7.0 is transferred in the cooling back, 115 ℃ of sterilization 15min, be cooled to room temperature, as mother liquor, standby; With the fully dissolving in solubility promoter in proportion of characteristic enzymolysis substrate, filtration sterilization adds in the mother liquor, shakes up, and becomes liquid nutrient medium, 4 ℃ of stored refrigerated.
Embodiment 4
A kind of Salmonellas method for quick is core with Salmonellas feature colour developing liquid nutrient medium.
Increase bacterium cultivation and colour developing before the Salmonellas method for quick specifically comprises and identified for two steps, whole testing process needs 2～3d.Before increase bacterium and cultivate recovery and the propagation mainly be to promote Salmonellas, be beneficial to the product enzyme in later stage.It is the enzymolysis property of utilizing Salmonellas feature enzyme (monooctyl ester enzyme) that colour developing is identified, colourless monooctyl ester class substrate is added in the Salmonellas feature colour developing liquid nutrient medium, discharges the colour developing group through the monooctyl ester enzymic hydrolysis, judges existing of Salmonellas with this.
Before increase bacterium and cultivate concrete steps and be: the 25g sample is placed the 225mL buffered peptone water, mixes back 37 ℃ of static cultivation 8h, get enrichment liquid.
Buffered peptone water is that Salmonellas detects preceding enrichment medium commonly used, the buffered peptone water non-selectivity, and (18～24h) are unfavorable for the control of assorted bacterium in the selective enrichment process, and effectively incubation time control is at 8～10h to increase the bacterium time before long.Buffered peptone water comprises peptone, sodium-chlor, phosphate buffered saline buffer, and wherein peptone provides carbon source and nitrogenous source to satisfy the demand of bacterial growth, and sodium-chlor can be kept balanced osmotic pressure, and phosphate buffered saline buffer provides the pH buffering to be beneficial to the growth of Salmonellas.
Colour developing identifies that concrete steps are: get the 1mL enrichment liquid and add in the 9mL Salmonellas feature colour developing liquid nutrient medium, mix back 37 ℃ of static cultivation 1d, if substratum is purple, illustrate that this sample is the Salmonellas positive, namely exists Salmonellas; If the substratum nondiscoloration illustrates that this sample is the Salmonellas feminine gender.Developing time is relevant with salmonella-polluted degree.
Utilize Salmonellas feature colour developing liquid nutrient medium, make selective enrichment and colour developing identify and unite two into one, can improve specificity and the specificity of detection, save follow-up a large amount of authentication step, shorten sense cycle greatly.Selective enrichment can suppress varied bacteria growing, is conducive to the dominant growth of Salmonellas and produces enzyme, reduces the interfering factors that detects.Selective enrichment can suppress the growth of Serratia, eliminates it to the interference of Salmonellas enzymolysis colour developing judgement.
Embodiment 5
The Salmonellas method for quick increases bacterium cultivation and colour developing and identifies before comprising.
Before increase bacterium and cultivate concrete steps and be: the 25g sample is placed the 225mL buffered peptone water, mixes back 37 ℃ of static cultivation 9h, get enrichment liquid.
Colour developing identifies that concrete steps are: get the 1mL enrichment liquid and add in the 9mL Salmonellas feature colour developing liquid nutrient medium, mix back 37 ℃ of static cultivation 1.5d, if substratum is purple, illustrate that this sample is the Salmonellas positive; If the substratum nondiscoloration illustrates that this sample is the Salmonellas feminine gender.
Embodiment 6
A kind of Salmonellas method for quick is core with Salmonellas feature colour developing liquid nutrient medium.
Before increase bacterium and cultivate concrete steps and be: the 25g sample is placed the 225mL buffered peptone water, mixes back 37 ℃ of static cultivation 10h, get enrichment liquid.
Colour developing identifies that concrete steps are: get the 1mL enrichment liquid and add in the 9mL Salmonellas feature colour developing liquid nutrient medium, mix back 37 ℃ of static cultivation 2d, if substratum is purple, illustrate that this sample is the Salmonellas positive; If the substratum nondiscoloration illustrates that this sample is the Salmonellas feminine gender.
Embodiment 7
Specificity test of the present invention:
Insert intestinal bacteria, Salmonellas, streptococcus aureus, shigella flexneri, bacillus cereus, subtilis, Listeria monocytogenes, proteus vulgaris, Serratia in the buffered peptone water respectively, increase bacterium before carrying out and cultivate, obtain enrichment liquid.Enrichment liquid is inserted in the Salmonellas feature colour developing liquid nutrient medium, cultivate 1～2d, carry out plate count and colour developing observation respectively, the results are shown in Table 1.
Conclusion: the present invention has obvious specificity to the detection of Salmonellas, and except proteus vulgaris, other assorted bacterium almost all is suppressed, and is conducive to the growth of Salmonellas and produces enzyme.Salmonella (comprising each subgenus) all produces the monooctyl ester enzyme, and this performance is that enterobacteriaceae removes sand that respectively to belong to bacterium not available for outer other of thunder Bordetella.Serratia is suppressed in the selective enrichment process, can not disturb the detection of Salmonellas.It is purple that Salmonellas monooctyl ester enzyme acts on monooctyl ester class substrate generation characteristic color.
The specificity test-results of table 1 Salmonellas method for quick
| The test bacterium | Inhibition | The colour developing situation |
| Intestinal bacteria | Almost completely suppress | Do not have |
| Salmonellas | Almost there is not influence | Purple |
| Streptococcus aureus | Suppress fully | Do not have |
| Shigella flexneri | Suppress fully | Do not have |
| Bacillus cereus | Suppress fully | Do not have |
| Subtilis | Suppress fully | Do not have |
| Singly increase the special bacterium of Li Shi | Suppress fully | Do not have |
| Proteus vulgaris | Part suppresses | Do not have |
| Serratia | Suppress fully | Do not have |
Annotate: inhibition is contrast with the result that enrichment liquid inserts nutrient broth medium.The colour developing situation is contrast with the result of the enrichment liquid access Salmonellas feature colour developing liquid nutrient medium of deactivation.
Embodiment 8
The comparison test of the present invention and traditional detection method:
The Salmonellas method for quick: the preceding bacterium 8～10h that increases, 1～2d is identified in colour developing.
Traditional detection method (with reference to GB 4789.4-2010): the preceding bacterium 8～18h that increases, TTB and SC selective enrichment 18～24h, BS and XLD (or HE, color developing culture medium) plate isolation 1～2d, the suspicious bacterium colony of picking carries out TSI, Methionin, NA, indole, urea, KCN and tests 1～2d, still be suspicious behind the preliminary judgement, continue serological test, finally obtain the result.
From the dining room, supermarket and market gathers 56 parts of actual samples such as livestock and poultry meat, vegetables, milk-product, adopts Salmonellas method for quick of the present invention and traditional detection method to carry out the Salmonellas detection respectively, the results are shown in Table 2.
Conclusion: the present invention is consistent with traditional detection method (National Standard Method) detected result: all detect 2 parts of positive in the livestock and poultry meat, with making a living beef and live chickens; All detect 1 part of positive in the milk-product, be all sweet milk.Tradition Salmonellas detection method whole process takes at least 4～7d, and the present invention only needs 2～3d.Illustrate that using the present invention to detect Salmonellas has higher accuracy, detection efficiency improves greatly simultaneously.
The detected result of Salmonellas in table 2 actual sample
Embodiment 9
The research of Salmonellas selective enrichment medium:
Because Salmonellas often with intestinal bacteria, streptococcus aureus contaminated food products, is added selective substances when increasing bacterium, can avoid non-object bacteria dominant growth to the competitive inhibition of Salmonellas.
Preliminary with the representative of intestinal bacteria as Gram-negative bacteria, with the representative of streptococcus aureus as gram-positive microorganism.26 kinds of additives are added respectively among the basic medium LB with different concns, study them to promotion or the retarding effect of three kinds of bacterium.
To promote that Salmonella growth and/or inhibition intestinal bacteria and staphylococcus aureus growth are that the target screening obtains 10 kinds of selective additives, further to shigella flexneri, Bacillus cereus, singly increase the special bacterium of Li Shi, proteus vulgaris, Serratia and carry out bacteriostatic experiment, final definite 7 kinds of selective additives are respectively: niacinamide, Sodium.alpha.-ketopropionate, Trisodium Citrate, acarbose, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate.
Form combination promotor with niacinamide and Sodium.alpha.-ketopropionate, Trisodium Citrate and acarbose are formed composite restrainer.Combination promotor and composite restrainer are optimized combination, to lithium chloride, sodium deoxycholate, that dipotassium hydrogen phosphate carries out concentration is preferred, acquisition determines that the selective enrichment medium prescription is: Tryptones 0.8～1.2g, yeast powder 0.4～0.8g, sodium-chlor 0.4～0.8g, lithium chloride 0.3～0.5g, sodium deoxycholate 0.1～0.3g, dipotassium hydrogen phosphate 0.1～0.2g, combination promotor 0.02～0.04g, composite restrainer 0.1～0.2g, distilled water 100mL to the optimal inhibition effect of non-Salmonellas with to the best accelerating effect of Salmonellas.
When sterilising temp is 121 ℃, when sterilization time surpassed 15min, combination promotor and composite restrainer took place partially modified easily, the influence effect.Therefore, this medium preparation method is: Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water are mixed in proportion, heating for dissolving, cooling back are transferred pH7.2 ± 0.2,115 ℃ sterilization 15min.
Embodiment 10
The research of Salmonellas feature colour developing liquid nutrient medium:
Carry out color developing culture medium research at the feature enzyme (monooctyl ester enzyme) of Salmonellas.Select the indoles monooctyl ester as the feature substrate.
Because the indoles monooctyl ester is insoluble in the aqueous solution, directly add the judgement that can influence the result in the liquid nutrient medium because of skewness.Selected tween 20, tween-80 and methyl-sulphoxide to carry out the hydrotropy test respectively.
Tween 20 and tween-80 belong to tensio-active agent.Substrate and tensio-active agent mix, and high speed homogenization forms emulsion, in the bringing Selection In property enrichment medium.Emulsion is not very stable, the easy breakdown of emulsion of static cultivation.Methyl-sulphoxide can dissolve each other with multiple organic solvent and water, when the 0.01g substrate adds in the 2mL methyl-sulphoxide, need not homogeneous and dissolves at once.Select methyl-sulphoxide as solubility promoter, and optimize best hydrotropy concentration.
Investigate the relation between concentration of substrate and the colour developing degree, the comprehensive detection cost, determine that Salmonellas feature colour developing liquid culture based formulas is: Tryptones 0.8～1.2g, yeast powder 0.4～0.8g, sodium-chlor 0.4～0.8g, lithium chloride 0.3～0.5g, sodium deoxycholate 0.1～0.3g, dipotassium hydrogen phosphate 0.1～0.2g, combination promotor 0.02～0.04g, composite restrainer 0.1～0.2g, monooctyl ester class substrate 0.02～0.04g, solubility promoter 4～6mL, distilled water 100mL, pH7.2 ± 0.2.
The indoles monooctyl ester is instability when temperature is higher, directly sterilizes in the bringing Selection In property enrichment medium degraded voluntarily can take place, and influences result's judgement.Therefore, this medium preparation method is: Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water is mixed in proportion, and heating for dissolving, pH7.2 ± 0.2 is transferred in the cooling back, 115 ℃ of sterilization 15min are as mother liquor.With substrate fully dissolving in solubility promoter in proportion, filtration sterilization, adding is cooled in the mother liquor of room temperature, shakes up.
Embodiment 11
The research of Salmonellas method for quick:
Increase bacterium cultivation and colour developing before the Salmonellas method for quick comprises and identified for two steps, sense cycle is 2～3d.
For recovery and the propagation that promotes Salmonellas, increase bacterium before need carrying out and cultivate.Before increase the bacterium cultural method with reference to National Standard Method (GB 4789.4-2010).Adopting buffered peptone water is preceding enrichment medium.This substratum non-selectivity, (18～24h) make assorted bacterium amount excessive, and the selectivity in the qualification process that is unfavorable for developing the color suppresses to increase the bacterium time before long.Therefore, the preceding bacterium cultivation concrete steps that increase are: the 25g sample is placed the 225mL buffered peptone water, mix back 37 ℃ of static cultivation 8～10h.
Based on feature enzymolysis colour developing principle, in the selective enrichment evaluation that develops the color simultaneously.Selective enrichment can suppress varied bacteria growing, is conducive to the dominant growth of Salmonellas and produces enzyme, reduces the interfering factors that detects.It is the enzymolysis property of utilizing Salmonellas feature enzyme (monooctyl ester enzyme) that colour developing is identified, colourless bringing Selection In property of monooctyl ester class substrate liquid culture is concentrated, and discharges the colour developing group through the monooctyl ester enzymic hydrolysis, judges existing of Salmonellas with this.Salmonellas feature colour developing liquid nutrient medium has merged selective enrichment and colour developing identification mark.Pollution level is different with the cell extent of damage, and the required product enzyme time there are differences.Therefore, colour developing identifies that concrete steps are: get the 1mL enrichment liquid and add in the 9mL feature colour developing liquid nutrient medium, mix back 37 ℃ of static cultivation 1～2d, if substratum is purple, illustrate that there is Salmonellas in this sample.
In a word, the above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to the covering scope of patent of the present invention.
Claims (3)
1. Salmonellas feature colour developing liquid nutrient medium, it is characterized in that component comprises distilled water, Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, characteristic enzymolysis substrate, solubility promoter, distilled water;
Every 100mL distilled water needs other components contents to be: Tryptones 0.8～1.2g, yeast powder 0.4～0.8g, sodium-chlor 0.4～0.8g, lithium chloride 0.3～0.5g, sodium deoxycholate 0.1～0.3g, dipotassium hydrogen phosphate 0.1～0.2g, combination promotor 0.02～0.04g, composite restrainer 0.1～0.2g, characteristic enzymolysis substrate 0.02～0.04g, solubility promoter 4～6mL, pH7.2 ± 0.2;
Combination promotor is Sodium.alpha.-ketopropionate and niacinamide, and the weight ratio of Sodium.alpha.-ketopropionate and niacinamide is 1:1;
Composite restrainer is Trisodium Citrate and acarbose, and the weight ratio of Trisodium Citrate and acarbose is 1:1;
Characteristic enzymolysis substrate is the indoles monooctyl ester, and solubility promoter is methyl-sulphoxide.
2. the preparation method of Salmonellas feature according to claim 1 colour developing liquid nutrient medium, it is characterized in that, may further comprise the steps: Tryptones, yeast powder, sodium-chlor, lithium chloride, sodium deoxycholate, dipotassium hydrogen phosphate, combination promotor, composite restrainer, distilled water are mixed in proportion, heating for dissolving, pH7.2 ± 0.2,115 ℃ sterilization 15min is transferred in the cooling back, is cooled to room temperature, as mother liquor, standby; With the fully dissolving in solubility promoter in proportion of characteristic enzymolysis substrate, filtration sterilization adds in the mother liquor, shakes up, and becomes liquid nutrient medium, preserves standby.
3. the Salmonellas method for quick of a non-diagnostic purpose is characterized in that, right to use requires 1 described Salmonellas feature colour developing liquid nutrient medium, may further comprise the steps:
(a) precedingly increase bacterium and cultivate: the 25g sample is placed the 225mL buffered peptone water, mix back 37 ℃ of static cultivation 8～10h, get enrichment liquid;
(b) colour developing is identified: get the 1mL enrichment liquid and add in the 9mL Salmonellas feature colour developing liquid nutrient medium, mix the static cultivation 24～48h in back, if Salmonellas feature colour developing liquid nutrient medium is purple, illustrate that there is Salmonellas in this sample.
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| CN103641912B (en) * | 2013-12-10 | 2016-04-06 | 山东出入境检验检疫局检验检疫技术中心 | Citric acid bacillus polyclonal antiserum, preparation method and the application in detection Salmonellas thereof |
| CN104894040B (en) * | 2015-06-30 | 2019-07-12 | 刘少伟 | Salmonella in Food one-step method selective enrichment medium and preparation method thereof |
| CN106086155B (en) * | 2016-06-27 | 2020-01-17 | 浙江省农业科学院 | Strawberry surface salmonella typhimurium prediction model under room temperature condition and establishment method thereof |
| CN106434844A (en) * | 2016-11-29 | 2017-02-22 | 上海市农业科学院 | Co-enrichment medium SSEL |
| CN108485953B (en) * | 2018-04-23 | 2023-08-01 | 上海新型烟草制品研究院有限公司 | Bacteria culture device and method for testing antibacterial performance of buccal cigarettes |
| CN108715883A (en) * | 2018-04-24 | 2018-10-30 | 中国检验检疫科学研究院 | A kind of method of rapid screening foodborne bacterial pathogens |
| CN108660186B (en) * | 2018-05-25 | 2019-12-10 | 无锡市赛微生物技术有限公司 | Chromogenic medium for detecting salmonella and preparation method thereof |
| CN109679862A (en) * | 2018-12-10 | 2019-04-26 | 赵嘉咏 | A kind of Salmonella Hantigen inducing composition, soft agar and preparation method thereof |
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