CN110591906B - Coliform group counting test piece, preparation method and application thereof - Google Patents
Coliform group counting test piece, preparation method and application thereof Download PDFInfo
- Publication number
- CN110591906B CN110591906B CN201910987921.4A CN201910987921A CN110591906B CN 110591906 B CN110591906 B CN 110591906B CN 201910987921 A CN201910987921 A CN 201910987921A CN 110591906 B CN110591906 B CN 110591906B
- Authority
- CN
- China
- Prior art keywords
- coliform
- test piece
- counting
- cold water
- water soluble
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A coliform group counting test piece, a preparation method and application thereof belong to the technical field of food detection. The plastic floor is composed of a transparent plastic upper layer film with a sticky lower surface, a plastic middle layer plate with a hollow area and a bottom plate printed with squares in sequence from top to bottom; the method is characterized in that: the adhesive lower surface of the transparent plastic upper layer film is coated with compound cold water soluble gel powder, and a hollow area of the plastic middle layer film is filled with the following components in percentage by mass (7-9): 3, preparing a coliform counting and developing detection culture medium and compounding cold water soluble gel powder. The coliform bacteria in food is detected by utilizing the specific color development system, the bubble formation counting system, the culture medium design system and the gel system of the coliform bacteria counting test piece, the process is simple and convenient, the color development of the colonies and the bubbles are clear, and the counting is accurate. The test strip has wide application prospect in the fields of delivery inspection, health and epidemic prevention, market supervision, commodity inspection and import and export quarantine of food production enterprises.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a coliform counting test piece, a preparation method and application thereof in detecting coliform counting in food.
Background
The determination of coliform group is used for judging the sanitary quality of food, can reflect whether the food meets the sanitary requirement of national regulation in the production and transportation process, and makes sanitary evaluation on the detected sample according to the inspection result. Coliform bacteria is a main index in a food hygiene microbial standard system in China, and the current national standard GB4789.3-2016 (national food safety Standard) food microbiology inspection coliform bacteria count has two detection methods for detecting the coliform bacteria, namely an MPN (Multi-path protein polymerase chain reaction) counting method and a plate counting method, and the two methods have the defects of complicated operation steps, long detection period and the like, and are difficult to meet the detection requirements of the modern food industry which is developed at high speed at present. Therefore, the rapid, practical, efficient and convenient detection method is the key research point for carrying out the microbial detection technology in agricultural products and food. The rapid detection technology of coliform group bacteria has great market demand in the fields of factory inspection, health and epidemic prevention, market supervision, commodity inspection and import and export quarantine of food production enterprises.
The method is based on the basic components of a VRBA culture medium in the national standard GB4789.3 food safety national standard food microbiology inspection coliform colony counting, and is used for optimizing four components of tryptone, yeast extract powder, sodium chloride and lactose to achieve an accurate determination effect on the coliform colony test piece so as to obtain the optimal combined amount of the nutrient culture medium of the coliform colony test piece; a color development system of the coliform group test piece is formed by a specific color development substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside, a coloring agent neutral red and crystal violet; simultaneously, adding enzyme activator MgCl into the culture medium 2 And an inducer isopropyl-beta-D-thiogalactopyranoside, so that the reaction rate and the color development time are accelerated, and the color can be developed after culturing for 18-24 h; the compound cold water soluble gel is analyzed through the texture characteristics of the gel of the test piece, and the porous starch and the mannitol which is a surfactant are added to ensure that the gas production characteristics of the coliform test piece are synchronous with the accurate counting of bacterial colonies, the bacterial colonies and air bubbles are uniformly distributed in the test piece, and the coliform determination result is accurate; according to the invention, the coliform group test piece with excellent performance and accurate counting is developed through the design and optimization of a coliform group culture medium system and the study of gas production and color development characteristics.
Disclosure of Invention
One of the purposes of the invention is to provide a coliform group counting test piece which is simple in operation, low in price and accurate in detection, and consists of a transparent plastic upper layer film with a sticky lower surface (such as a PET silicon film which is high in transparency, non-toxic, free of pungent smell, waterproof and breathable and has a thickness of 0.05-0.10 mm, and the lower surface of the PET silicon film has stickiness), a plastic middle layer plate with a hollowed-out area (such as a polypropylene (PP) plastic plate or a PET plastic plate with good hardness and a thickness of 0.15-0.30 mm), and a bottom plate printed with squares (such as waterproof and anti-seepage white coated paper and has a thickness of 0.10-0.20 mm, wherein the size of the squares is 1cm multiplied by 1 cm); the high temperature resistant temperature range of the materials is 130-150 ℃; the viscous lower surface of the transparent plastic upper layer film is coated with compound cold water (the temperature range is 20-45 ℃) soluble gel, the hollow area of the plastic middle layer plate is of a circular, oval or square structure, and the proportion range of the area of the hollow area to the area of the plastic middle layer plate is 1: 1.6-2.7, filling a coliform counting and developing detection culture medium and compound cold water soluble gel powder which are prepared according to a certain proportion in the hollow area, wherein the compound cold water soluble gel powder and the coliform counting and developing detection culture medium are mixed according to the mass ratio of 3:7 to 9.
The components of the compound cold water soluble gel powder are as follows: mixing the xanthan gum and the guar gum which are ground and then pass through a standard sieve of 100-150 meshes, wherein the mass ratio of the xanthan gum to the guar gum is 1:1 to 1.5.
The coliform counting and color developing detection culture medium comprises the following components in parts by weight: 15-20 g of tryptone, 2-4 g of yeast extract powder, 6-8 g of lactose, 2-6 g of sodium chloride, 0.2-0.4g of bile salt, 0.3-0.5 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.5-0.7 g of isopropyl-beta-D-thiogalactopyranoside, 1.5-3 g of magnesium chloride, 0.07-0.1 g of neutral red, 0.004-0.009 g of crystal violet, 2-4 g of soybean peptone, 1-2 g of mannitol and 0.1-0.3 g of porous starch.
The invention also aims to provide a preparation method of the coliform bacteria counting test piece, which comprises the following steps:
(1) Preparation of coliform flora counting and color development detection culture medium
Weighing 15-20 g of tryptone, 2-4 g of yeast extract powder, 6-8 g of lactose, 2-6 g of sodium chloride, 0.2-0.4g of bile salt, 0.3-0.5 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.5-0.7 g of isopropyl-beta-D-thiogalactopyranoside, 1.5-3 g of magnesium chloride, 0.07-0.1 g of neutral red, 0.004-0.009 g of crystal violet, 2-4 g of soybean peptone, 1-2 g of mannitol and 0.1-0.3 g of porous starch, and uniformly mixing to obtain the coliform counting and developing detection culture medium powder.
(2) Preparation of compound cold water soluble gel powder
Mixing the ground xanthan gum and guar gum which pass through a standard sieve of 100-150 meshes to obtain compound cold water soluble gel powder, wherein the mass ratio of the xanthan gum to the guar gum is 1:1 to 1.5.
(3) Preparation of coliform group counting test piece
Cutting a transparent plastic upper layer film, a plastic middle layer plate with a hollow area and a bottom plate printed with squares into the same size, wherein the length is 7-11 cm, the width is 6-10 cm, and the side, which is adhered with a label, of the transparent plastic upper layer film, the plastic middle layer plate with the hollow area and the bottom plate printed with the squares is sealed and bonded by utilizing polyacrylic resin pressure-sensitive adhesive (transparent, nontoxic, tasteless and sterile), and the bonding width is 3-7 mm;
uniformly coating the compounded cold water soluble gel powder on the adhesive lower surface of the transparent plastic upper layer film, wherein the thickness is 0.10-0.20 mm;
uniformly coating polyacrylic resin pressure-sensitive adhesive on the bottom plate exposed out of the hollow area of the plastic middle-layer plate, wherein the thickness of the pressure-sensitive adhesive is 0.05-0.10 mm, and mixing the compound cold water soluble gel powder and coliform group counting and color developing detection culture medium powder according to the mass ratio of 3: 7-9, uniformly coating the obtained mixed powder in a hollow area of a plastic middle layer plate and compacting; and finally, carrying out vacuum packaging, and sterilizing by adopting an ethylene oxide method to obtain the coliform bacteria counting test piece.
(4) Operating procedure for determining coliform group bacteria by using said coliform group bacteria counting test piece
Solid or semi-solid sample: weighing 25g of sample (the sample can be rice flour product, cake food or nut product), adding 225mL of sterile diluent (distilled water, physiological saline or phosphate buffer), fully shaking or beating with a beating type homogenizer for 1-2 min, and preparing into 1:10 of the diluted homogenate.
Liquid sample: sucking 25mL of sample (the sample can be beverage) by using a sterile sucker, placing the sample in a container or a sterile homogenizing bag containing 225mL of sterile diluent (distilled water, physiological saline or phosphate buffer), fully shaking or beating by using a beating type homogenizer for 1-2 min, and preparing into a product 1:10 of the diluted homogenate.
1mL of the above 1:10, adding the diluted solution into a test tube containing 9mL of sterile diluent, and uniformly mixing on a vortex mixer to obtain a mixture of 1:100 parts of diluted and homogenized solution; sequentially preparing 10 times of gradient dilution homogenate to obtain 10 -1 、10 -2 、10 -3 、10 -4 CFU/mL 4 dilutions were pooled and ready for use.
According to the estimation of the sample pollution condition, 2-3 diluted uniform solutions with proper dilution are selected, the transparent plastic upper layer film of the coliform group counting test piece is opened, 1mL of the diluted uniform solution of the sample is added to the area of the middle layer with the coliform group counting chromogenic detection culture medium, so that the diluted uniform solution of the sample is uniformly distributed in the whole culture area; slowly dropping the plastic upper layer film, avoiding extrusion, standing for 30 s-1 min until the compound cold water soluble gel is solidified with water, putting the plastic upper layer film into a constant temperature incubator to be cultivated at 36 +/-1 ℃ for 18-24 h, and counting according to the national food safety standard GB4789.3 'food safety national standard food microbiology inspection coliform bacteria count'. Meanwhile, 1mL of physiological saline was used as a blank control.
The counting and color developing detection culture medium of the test piece for coliform group count developed by the invention is composed of compound cold water soluble gel (prepared by taking the mass ratio w/w of xanthan gum to guar gum as 1-1.5), a basic culture medium (tryptone, yeast extract powder, lactose, sodium chloride, bile salt and soybean peptone), porous starch, a surfactant mannitol, a specific color developing substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside, a coloring agent neutral red and crystal violet, and an enzyme activator MgCl 2 And inducer isopropyl-beta-D-thiogalactopyranoside. Compared with similar products, the characteristic is that the coliform bacteria generate gas on the test piece and the color system of the test piece is designed andand (6) optimizing. The invention realizes the synchronization and accuracy of the air bubbles generated by the coliform on the test piece and the counting of the bacterial colonies, and the color development of the bacterial colonies is clear. Mainly comprising 2 important technical features.
Important technical features 1: according to the invention, the cold water soluble gel is compounded through the analysis of the texture and the gel characteristics of the cold water soluble gel of the coliform group test piece for the first time, and the porous starch and the mannitol as the surfactant are added into the culture medium, so that coliform group colonies and air bubbles are uniformly distributed, and specific chromogenic colonies and gas production characteristics can be displayed on the test piece.
Through the texture analysis of the gel agent of the coliform test tablet, selecting xanthan gum and guar gum to be matched, wherein the mass ratio w/w of the xanthan gum to the guar gum is 1:1 to 1.5; and a proper amount of porous starch is added, so that pores of the culture medium powder are uniform, and uniform distribution and maintenance of bubbles generated by the growth of coliform groups are facilitated. Optimizing the addition amount of the porous starch in the culture medium, and determining the addition concentration of the porous starch to be 0.1-0.3 g/L; the surfactant plays an important role in the growth and gas production effects of coliform groups, and can be used for controlling the shape of bubbles produced by the growth of the coliform groups on the test piece and maintaining the effect by properly adding the surfactant, and simultaneously can improve the solubility and dispersibility of a liquid sample after being added into a culture medium of the test piece and accelerate the absorption and uniform diffusion of bacteria liquid. Optimizing the addition amount of the surfactant mannitol in the culture medium, and determining the addition concentration of the mannitol to be 1-2 g/L.
Important technical features 2: the test piece color development system integrates a coliform bacteria enzyme specificity color development system and a coloring agent. The color development system of the test piece consists of a specific enzyme color development system (substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside, enzyme activator MgCl 2 And inducer isopropyl-beta-D-thiogalactopyranoside), neutral red of the coloring agent and crystal violet together form a special color development effect, and the color development of the bacterial colony is clear.
As the coliform bacteria can produce beta-galactosidase in the growth process, the beta-galactosidase can react with a substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside to generate blue precipitates, and the existence of bacterial colonies can be judged. The invention adopts the enzyme specificity color development principle to improve the bacterial colony in the test pieceThe color development time, the color development effect and the reaction rate are increased, and an enzyme activator MgCl is added 2 And an enzyme inducer isopropyl-beta-D-thiogalactopyranoside, and the color development is good after the culture for 18 to 24 hours, so that the reaction rate and the color development time are accelerated. The color development effect is obviously improved under the combined action of the neutral red as a background color, crystal violet and an enzyme substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside.
According to the coliform group counting test piece prepared based on the components, after a diluent of a food sample to be tested is added into a middle-layer plate culture area, culture medium powder is mixed with the upper-layer compound cold water soluble gel, and a stable gel colloid with moderate pores and compact net-shaped space is formed in the gel water absorption process, so that the coliform group counting test piece is particularly suitable for coliform group growth and colony formation under the test piece environment. The coliform bacteria in food is detected by utilizing the specific color development system, the bubble formation counting system, the culture medium design system and the gel system of the coliform bacteria counting test piece, the process is simple and convenient, the color development of the colonies and the bubbles are clear, and the counting is accurate. The test strip has wide application prospect in the fields of delivery inspection, health and epidemic prevention, market supervision, commodity inspection and import and export quarantine of food production enterprises.
Drawings
FIG. 1: the structure schematic diagram of the coliform group counting test piece is shown in the invention;
as shown in fig. 1, each part is: the label comprises a label 1, an upper layer of transparent PET silica gel film 2, a transparent polypropylene plastic middle layer plate 3 with a hollow area in the middle, a culture area 4 and white coated paper 5.
FIG. 2 is a schematic diagram: the gas production and distribution effect diagram of the bacterial colony after the basal medium is added with the surfactant mannitol;
FIG. 3: the gas production and distribution effect diagram of the bacterial colony after the basic culture medium is added with the porous starch and the surfactant mannitol;
as can be seen from FIGS. 2 and 3, under the combined action of mannitol and porous starch, the distribution of bubbles generated by coliform bacteria is more uniform and clear, and the counting is convenient.
FIG. 4: a color development effect graph of the 5-bromo-4-chloro-3-indole-beta-D-galactoside with different concentrations on the test piece;
as shown in FIG. 4, when the concentration of the chromogenic substrate is 0.1 g-0.2 g/L, the bacterial colony is light blue, and is not easy to be distinguished from the color of the test piece negative; when the concentration of the chromogenic substrate is 0.3 g-0.5 g/L, the bacterial colony is blue, the chromogenic effect is obvious, and the counting is convenient.
FIG. 5 is a schematic view of: influence graphs of crystal violet with different concentrations on the color development effect of the test piece;
as shown in FIG. 5, when the concentration of crystal violet is 0.006g/L, the background color of the test piece is light purple, and the bacterial colony is clear and convenient for counting. The crystal violet has bacteriostatic action, and the colony number at the concentration of 0.009g/L is less than that of the former gradient (0.006 g/L); when the concentration of the crystal violet is 0.012g/L, the colony count is obviously less than the standard 0.006g/L, and the interpretation is wrong. Therefore, 0.006g/L was selected as the optimum concentration of crystal violet.
FIG. 6: influence graphs of neutral red with different concentrations on the background color effect of the test piece;
as shown in FIG. 6, when the concentration of neutral red was 0.06g/L, both the background red and the colony blue were light and not easily observed. When the concentration of neutral red is 0.09g/L, the background color is red, and the bacterial colony is blue, so that the bacterial colony is clear and easy to count. When the concentration of neutral red is 0.12g/L or 0.15g/L, the background red color deepens, but the blue complex of the bacterial colony does not deepen any more, so that the bacterial colony is difficult to observe. Finally, 0.09g/L was determined as the optimum neutral red concentration.
FIG. 7 is a schematic view of: crystal violet, neutral red, 5-bromo-4-chloro-3-indole-beta-D-galactoside synergistic effect picture;
as shown in figure 7, under the combined action of crystal violet, neutral red and 5-bromo-4-chloro-3-indole-beta-D-galactoside, the background color of the test piece is mauve, the bacterial colony is blue, the color development effect of the test piece is optimal, and the counting and observation are convenient.
FIG. 8: the coliform counting test piece regression equation and the correlation coefficient curve are adopted;
FIG. 8 shows that the test result of the test piece for coliform bacteria developed by the invention has no difference significance with the national standard flat plate method, R 2 Reaches 0.999, and has good correlation.
Detailed Description
EXAMPLE 1 preparation of culture Medium for coliform count assay
The coliform count detection culture medium comprises the following raw components in parts by weight: 17.0g of tryptone, 3.0g of yeast extract powder, 7.5g of lactose, 4.0g of sodium chloride, 0.3g of bile salt, 0.4g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.6g of isopropyl-beta-D-thiogalactopyranoside, 2.28g of magnesium chloride, 0.09g of neutral red, 0.006g of crystal violet, 3g of soybean peptone, 1.5g of mannitol and 0.3g of porous starch.
The preparation method of the coliform bacteria counting detection culture medium comprises the following steps: weighing 17.0g of tryptone, 3.0g of yeast extract powder, 7.5g of lactose, 4.0g of sodium chloride, 0.3g of bile salt, 0.4g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.6g of isopropyl-beta-D-thiogalactopyranoside, 2.28g of magnesium chloride, 0.09g of neutral red, 0.006g of crystal violet, 3g of soytone, 1.5g of mannitol and 0.3g of porous starch, and uniformly mixing to obtain culture medium powder for later use.
EXAMPLE 2 preparation of coliform count test piece
1. The test piece consists of three layers, namely an upper PET silica gel film, a middle polypropylene plastic plate with a hollow area and a bottom layer printed with faint yellow 1cm multiplied by 1cm square white square copper plate paper. Cutting the three parts into the same size, length 9cm and width 8cm, and sealing and bonding the side with label with polyacrylic resin pressure sensitive adhesive (transparent, nontoxic, odorless, and sterile) with bonding width of 5mm.
2. The compounded cold water soluble gel powder is evenly sprayed on the inner side of the film of the test piece, the thickness is 0.15mm, and the mass of the cold hydrogel of each test piece is 0.2g.
3. Firstly, uniformly coating 0.2g of transparent polyacrylic resin pressure-sensitive adhesive on a bottom plate corresponding to the hollow area of the middle layer, preparing the compound cold water soluble gel powder and coliform group counting, developing and detecting culture medium powder according to a mass ratio of 3.
4. The test pieces were vacuum packed.
5. The test piece was sterilized by ethylene oxide sterilization.
EXAMPLE 3 study of gas production System by coliform test piece
Screening and preparation of cold water soluble gel
The cold water soluble gel of the coliform test piece was screened by gel property analysis, and the results are shown in table 1. As can be seen from Table 1, the water absorption of xanthan gum is 45.4mL/g, that of guar gum is 56.21mL/g, that of sodium polyacrylate is 149.12mL/g, and that other cold water soluble gels have poor water retention and are not suitable as coagulants for test pieces. Wherein the diffusion time of guar gum and sodium polyacrylate is 1.0s and 1.2s respectively, the turbidity of the sodium polyacrylate is 96.3%, but the water absorption speed is too high, the gel flow speed is slow, so that the sample liquid cannot be uniformly diffused, and the agglomeration is easily formed; the turbidity of the guar gum is 73.9%, the gel flow speed after water absorption is moderate, the guar gum can be fully hydrated in cold water to form smooth colloid, and the transparency of the xanthan gum is the highest among the three, so the guar gum and the xanthan gum are selected as the cold water soluble gel of the test piece, and the optimal mass ratio of the guar gum to the xanthan gum is 1:1.
table 1: gel characterization data
The preparation method of the compound cold water soluble gel comprises the following steps: respectively grinding cold water soluble gel guar gum and xanthan gum into powder, sieving with a 120-mesh molecular sieve, and mixing the guar gum and the xanthan gum according to a mass ratio of 1:1, mixing well for standby.
(II) Effect of porous starch and surfactant mannitol on air bubbles in coliform determination
Coliform bacteria produce gas during the lactose decomposition process, and generate bubbles due to the specific surface tension of the liquid. The addition of the surfactant in the basic culture medium of the test piece has an important effect on the gas production effect, can be used for controlling the shape and the effect of bubbles generated by the growth of coliform groups on the test piece, can improve the solubility and the dispersibility of products, and can accelerate the absorption and the diffusion of bacteria liquid. And the porous starch is added, so that the pores of the culture medium powder are uniform, and uniform distribution and maintenance of bubbles generated by the growth of coliform bacteria are facilitated.
The coliform group bacteria test piece basic culture medium (tryptone 17.0g, yeast extract powder 3.0g, lactose 7.5g, sodium chloride 4.0g, bile salt 0.3g, soy peptone 3 g) and the culture medium added with porous starch and different surfactants are compared in the structure characteristics shown in Table 2.
Table 2: comparison of texture characteristics of test piece basic culture medium and culture medium added with porous starch and surfactant
As is clear from Table 2, the culture medium matrix structure properties in which the surfactant mannitol and the porous starch were added were superior to those of the other culture medium matrix. The compounding of the mannitol and the porous starch can improve the solubility and the dispersibility of the product and can accelerate the absorption and the diffusion of the bacteria liquid.
The addition amounts of mannitol and porous starch in the coliform group test piece culture medium are optimized, and the optimal addition concentration of mannitol is determined to be 1.5g/L, and the optimal addition amount of porous starch is determined to be 0.3g/L. The test piece is used for detecting coliform groups, the coliform groups are cultured for 18-24 h at 36 +/-1 ℃, the bubbles generated by the coliform groups in the test piece are uniformly and clearly distributed, the effect is kept to be optimal, and the effect of adding the porous starch and the mannitol serving as the surfactant is shown in a figure 2 and a figure 3.
As can be seen from FIGS. 2 and 3, under the combined action of mannitol and porous starch, the distribution of bubbles generated by coliform bacteria is more uniform and clear, and the counting is convenient.
EXAMPLE 4 investigation of color development System of coliform test piece
(I) optimization of 5-bromo-4-chloro-3-indole-beta-D-galactoside concentration
As the coliform bacteria can produce beta-galactosidase in the growth process, the beta-galactosidase can react with a substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside to generate blue precipitates, and the existence of bacterial colonies can be judged.
In the manufacturing process of the test piece, as shown in fig. 4, when the concentration of the chromogenic substrate is 0.1 g-0.2 g/L, the bacterial colony is light blue, and is not easy to be distinguished from the color of the negative film of the test piece; when the concentration of the chromogenic substrate is 0.3-0.5 g/L, the bacterial colony is blue, the chromogenic effect is obvious, and the counting is convenient. In combination with FIG. 4 and economic aspects, the substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside concentration of 0.4g/L was selected as the optimum concentration to be added to the culture medium to prepare test pieces.
(II) enzyme activator MgCl 2 And the influence of the inducer isopropyl-beta-D-thiogalactopyranoside on the color development effect of the test piece
According to the requirement of the optimal condition of the enzyme catalytic reaction, a certain amount of accessory factors should be added in principle. Cofactor refers to a non-protein component required for the activity of an enzyme, including coenzymes, prosthetic groups, and metal ion activators. The invention adopts the enzyme specificity color development principle, adds the enzyme activator MgCl for improving the color development time, the color development effect and the reaction rate of the bacterial colony in the test piece 2 And inducer isopropyl-beta-D-thiogalactopyranoside, and the color development is good after culturing for 18-24 h, thereby quickening the reaction rate and the color development time. MgCl 2 The final addition amount of the enzyme activator is 2.28g/L, and the addition amount of the inducer isopropyl-beta-D-thiogalactopyranoside is 0.6g/L; (III) test piece ground color optimization
1. Influence of Crystal Violet on color development Effect of test piece
The effect of different concentrations of crystal violet on the color development of the test pieces is shown in FIG. 5. As shown in FIG. 5, when the concentration of crystal violet is 0.006g/L, the background color of the test piece is light purple, and the bacterial colony is clear and convenient for counting. Because crystal violet has an antibacterial effect, the colony count at the concentration of 0.009g/L is less than that of the former gradient (0.006 g/L); when the concentration of the crystal violet is 0.012g/L, the colony count is obviously less than the standard 0.006g/L, and the interpretation is wrong. Therefore, 0.006g/L was selected as the optimum concentration of crystal violet.
2. Influence of neutral Red on color development Effect of test piece
The effect of different concentrations of neutral red on the background color effect of the test piece is shown in FIG. 6. When the concentration of neutral red is 0.06g/L, the background red and the colony blue are lighter and difficult to observe. When the concentration of the neutral red is 0.09g/L, the background color is red, and the bacterial colony is blue, so that the bacterial colony is clear and easy to count. When the concentration of neutral red is 0.12g/L or 0.15g/L, the background red color deepens, but the blue complex of the bacterial colony does not deepen any more, so that the bacterial colony is difficult to observe. Finally, 0.09g/L was determined as the optimum neutral red concentration.
3. The synergistic effect of crystal violet, neutral red and 5-bromo-4-chloro-3-indole-beta-D-galactoside on the color development of the test piece is shown in figure 7.
As shown in figure 7, under the combined action of crystal violet, neutral red and 5-bromo-4-chloro-3-indole-beta-D-galactoside, the background color of the test piece is mauve, the bacterial colony is blue, the color development effect of the test piece is optimal, and the counting and observation are convenient.
Example 5 method for measuring coliform count in food Using test piece
1. Weighing 25g of a food sample to be detected (the sample to be detected can be solid food such as rice and flour products, biscuits and cakes) or 25mL of the food sample to be detected (the sample to be detected can be liquid food such as drinks) by aseptic operation, adding the sample to be detected into an aseptic homogenizing bag of 225mL of diluent, and beating the sample for 1-2 min by using a homogenizer to prepare a 1. The sample homogeneous solution is subjected to 10-fold serial gradient dilution to obtain 10 -1 、10 -2 、10 -3 、10 -4 And 4 dilution homogenizing solutions of CFU/mL, selecting 2-3 dilutions according to the pollution degree of the sample, and preparing two parallel samples for each dilution.
2. Taking 1mL of diluent of a food sample to be detected, lifting the upper membrane of the test piece, adding the diluent into the culture area, uniformly distributing the sample liquid in the whole culture area, slowly dropping the upper membrane to avoid the extrusion of fingers, standing for 30s for curing gel, and putting the gel into an incubator to culture for 18-24 h at 36 +/-1 ℃. Two replicates of each dilution were made, while 1mL of saline was used as a blank.
3. And (4) putting the test piece subjected to enrichment culture into an aseptic operation table, observing whether the test piece has blue-green colonies or not, and counting the blue-green colonies. The counting method is carried out according to GB4789.3-2016 coliform group count for food safety national standard food microbiology inspection.
If the colony number of the test piece of all the dilutions is more than 150CFU, the test piece with the highest dilution is counted, other test pieces can be recorded as more than or not, and the result is calculated by multiplying the average colony number by the highest dilution factor.
If the colony number of the test strip at all dilutions is less than 15CFU, the colony number is calculated as the average colony number at the lowest dilution multiplied by the dilution factor.
If all dilutions (including the liquid sample stock) test pieces were grown aseptically, they were calculated as less than 1 times the lowest dilution factor.
If the colony number of the test piece of all the dilutions is not between 15CFU and 150CFU, and a part of the colony numbers is less than 15CFU or more than 150CFU, the colony number is calculated by multiplying the average colony number closest to 15CFU or 150CFU by the dilution factor.
EXAMPLE 6 evaluation of the Effect of measurement of coliform count test piece
1. Coliform count test pieces were prepared as in example 1 and example 2 and were ready for use.
2. Determination of detection limit and sensitivity of test strip
The experimental strains are Escherichia coli ATCC25922, citrobacter freundii ATCC43864, klebsiella pneumoniae CMCC (B) 46117 and Enterobacter cloacae CMCC45301.
Respectively taking single colonies of Escherichia coli, citrobacter freundii, klebsiella pneumoniae and Enterobacter cloacae, inoculating the single colonies into 1000mL LB liquid medium (tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL, pH value 7.0), placing the single colonies into a shaking table, carrying out shaking culture at 36 +/-1 ℃ for 18h at 200r/min, taking 1mL of bacterial liquid, adding 9mL of normal saline, mixing uniformly to prepare diluent, repeating the above operations, diluting the bacterial liquid to a dilution degree (the concentration is 10) suitable for counting -4 、10 -5 、10 -6 CFU/mL), 1mL of the bacterial solution was taken and the colony count of Escherichia coli was determined using the test piece according to the method in example 5. And simultaneously, carrying out coliform counting determination according to GB4789.3-2016 (national food safety Standard food microbiology test coliform count), making two parallel samples for each dilution, and taking an average value of detection results.
Test node with lowest dilution colony numberIf the result is used as the lowest detection limit, the lowest detection limit is used as the detection standard of the sensitivity of the test piece, the test result which is larger than the lowest detection limit is positive, the test result which is smaller than the lowest detection limit is negative, and the sensitivity = positive/(positive + negative), the experimental result shows that the two methods can reach the detection limit of the same level, the detection limit is 4CFU/mL, and the sensitivity is 100%. The detection result of the test piece for coliform group developed by the invention has no difference significance (P) from the national standard flat plate method>0.05)，R 2 Reaching 0.999 with good correlation. See table 3, table 4 and fig. 8.
Table 3: accuracy detection result of coliform bacteria test piece
Table 4: coliform group bacteria test piece sensitivity detection result
3. Test strip specificity detection
Selecting bacterial suspensions of Escherichia coli, citrobacter freundii, klebsiella pneumoniae, enterobacter cloacae, bacillus subtilis, vibrio parahaemolyticus, salmonella typhimurium, pseudomonas aeruginosa, shigella flexneri, salmonella typhi, bacillus cereus, shigella sonnei and Staphylococcus aureus, diluting the bacterial suspensions by 10 times of gradient with physiological saline, respectively inoculating the diluents on a VRBA plate and a test piece, and placing the plate and the test piece in an incubator at the culture temperature of 37 ℃ for 18-24 h. And observing the growth condition of each colony, picking typical and suspicious colonies, further inoculating the typical and suspicious colonies into BGLB broth for confirmation experiment, and culturing at 37 ℃ for 24-48 h to prevent false positive and false negative results. The results are shown in Table 5.
Table 5: result of detecting specificity of coliform test piece
As can be seen from the above table, no bacterial colony appears after the main pathogenic bacteria of other genera such as salmonella typhi and staphylococcus aureus except the coliform flora are cultured on the test piece for 24 hours, and the results of various standard strains on the test piece are completely consistent with those on a VRBA flat plate, so that the test piece has good selectivity and specificity for detecting the coliform flora.
4. Application of test piece in food sample detection
Weighing 25g or sucking 25mL of food sample, adding 225mL of sterile physiological saline, beating and uniformly mixing by using a beating type homogenizer to prepare a 1. And adding 1mL of coliform bacteria suspension into the treated food sample homogenizing solution to carry out artificial pollution on the food sample. The artificially contaminated sample solution was serially diluted 10 times, 1mL of a 1 -4 、10 -5 、10 -6 CFU/mL), total coliform colony count was determined according to the method of example 5, and the conformity of the coliform test piece counting method of the present invention to the measurement results of GB4789.3-2016 "food safety national standard food microbiology test coliform colony count" method was compared.
Table 6: result comparison and coincidence rate of food sample detection by test piece method and national standard method
The coliform group test piece counting method and the national standard GB4789.3-2016 coliform group counting method for food safety national standard food microbiology inspection are used for respectively detecting 120 artificially contaminated food samples, the inspection results are shown in Table 6, the data show that the coincidence rate of the two methods is more than 95%, the detection results of the two methods have no difference significance, and the results show that: the coliform counting test piece is suitable for detecting coliforms in food.
Claims (7)
1. A coliform group count test piece, from top to bottom by have viscidity lower surface transparent plastic upper strata membrane, have plastic middle layer board, print have bottom plate of square sequentially; the method is characterized in that: the adhesive lower surface of the transparent plastic upper layer film is coated with compound cold water soluble gel powder, and a hollow area of the plastic middle layer film is filled with the following components in percentage by mass (7-9): 3, preparing a coliform counting chromogenic detection culture medium and compound cold water soluble gel powder; the compound cold water soluble gel powder is prepared by mixing xanthan gum and guar gum which are ground and then pass through a standard sieve of 100-150 meshes, wherein the mass ratio of the xanthan gum to the guar gum is 1: (1-1.5) preparing; the weight components of the coliform group counting and color development detection culture medium are 15-20 g of tryptone, 2-4 g of yeast extract powder, 6-8 g of lactose, 2-6 g of sodium chloride, 0.2-0.4g of bile salt, 0.3-0.5 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.5-0.7 g of isopropyl-beta-D-thiogalactopyranoside, 1.5-3 g of magnesium chloride, 0.07-0.1 g of neutral red, 0.004-0.009 g of crystal violet, 2-4 g of soybean peptone, 1-2 g of mannitol and 0.1-0.3 g of porous starch.
2. The coliform count test strip of claim 1, wherein: the transparent plastic upper layer film is a PET silica gel film with the thickness of 0.05 mm-0.10 mm, and the lower surface of the PET silica gel film is sticky.
3. The coliform count test piece of claim 1, wherein: the plastic middle layer plate with the hollow area is a polypropylene plastic plate or a PET plastic plate, and the thickness is 0.15 mm-0.30 mm.
4. The coliform count test strip of claim 1, wherein: the bottom plate printed with the grids is white coated paper, and the thickness of the bottom plate is 0.10-0.20 mm; the size of the squares is 1cm by 1cm.
5. The coliform count test piece of claim 1, wherein: the temperature range of the compound cold water soluble gel is 20-45 ℃.
6. A method for preparing a coliform count test piece as claimed in any one of claims 1 to 5, which comprises the steps of:
(1) Preparation of coliform flora counting and color development detection culture medium
Weighing 15-20 g of tryptone, 2-4 g of yeast extract powder, 6-8 g of lactose, 2-6 g of sodium chloride, 0.2-0.4g of bile salt, 0.3-0.5 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.5-0.7 g of isopropyl-beta-D-thiogalactopyranoside, 1.5-3 g of magnesium chloride, 0.07-0.1 g of neutral red, 0.004-0.009 g of crystal violet, 2-4 g of soybean peptone, 1-2 g of mannitol and 0.1-0.3 g of porous starch, and uniformly mixing to obtain coliform counting and developing detection culture medium powder;
(2) Preparation of compound cold water soluble gel powder
Mixing the ground xanthan gum and guar gum which pass through a standard sieve of 100-150 meshes to obtain compound cold water soluble gel powder, wherein the mass ratio of the xanthan gum to the guar gum is 1: (1-1.5);
(3) Preparation of coliform bacteria counting test piece
Cutting the transparent plastic upper layer film, the plastic middle layer plate with the hollow area and the bottom plate printed with the grids into the same size, and sealing and bonding one side of the transparent plastic upper layer film, the plastic middle layer plate with the hollow area and the bottom plate with the grids by utilizing polyacrylic resin pressure-sensitive adhesive; uniformly coating the compound cold water soluble gel powder on the adhesive lower surface of the transparent plastic upper layer film; uniformly coating polyacrylic resin pressure-sensitive adhesive on the bottom plate exposed out of the hollowed area of the plastic middle-layer plate, and mixing the compound cold water soluble gel powder with coliform group counting, developing and detecting culture medium powder according to a mass ratio of 3: (7-9), uniformly coating the obtained mixed powder in a hollowed-out area of a plastic middle-layer plate, and compacting; and finally, carrying out vacuum packaging, and sterilizing by adopting an ethylene oxide method, thereby obtaining the coliform bacteria counting test piece.
7. Use of a coliform counting test piece as claimed in any one of claims 1 to 5 for coliform counting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910987921.4A CN110591906B (en) | 2019-10-17 | 2019-10-17 | Coliform group counting test piece, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910987921.4A CN110591906B (en) | 2019-10-17 | 2019-10-17 | Coliform group counting test piece, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110591906A CN110591906A (en) | 2019-12-20 |
| CN110591906B true CN110591906B (en) | 2023-02-03 |
Family
ID=68850639
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910987921.4A Active CN110591906B (en) | 2019-10-17 | 2019-10-17 | Coliform group counting test piece, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110591906B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811404A (en) * | 2017-01-22 | 2017-06-09 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of quick detection coliform and preparation method thereof, detection method |
| CN111088156B (en) * | 2020-01-06 | 2021-03-30 | 山东省海洋资源与环境研究院 | Coliform group testing piece for marine environment monitoring and accessory device used in cooperation with coliform group testing piece |
| CN111727906A (en) * | 2020-07-06 | 2020-10-02 | 吉林农业大学 | Ball-type movable collision experimental device for testing attention of experimental animal |
| CN111733069B (en) * | 2020-07-11 | 2023-07-18 | 吉林农业大学 | Colony counting test piece for dual detection of mold and yeast fluorescence/visible light and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008017712A (en) * | 2006-07-10 | 2008-01-31 | Chisso Corp | Culture medium for microorganism |
| CN101215597A (en) * | 2007-12-26 | 2008-07-09 | 广东省微生物研究所 | Bacillus cereus specificity color biochemical fast detection kit and detection method |
| CN102311990A (en) * | 2011-09-16 | 2012-01-11 | 广州绿洲生化科技有限公司 | Chromogenic medium of coliform group and quick detection card thereof |
| CN102604963A (en) * | 2011-01-24 | 2012-07-25 | 华中农业大学 | Separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene |
| CN105754849A (en) * | 2016-03-31 | 2016-07-13 | 吉林农业大学 | Total bacterial count testing slip and preparation method and application thereof |
| CN108220384A (en) * | 2017-12-29 | 2018-06-29 | 广东海大畜牧兽医研究院有限公司 | A kind of vibrios testing piece and preparation method thereof |
-
2019
- 2019-10-17 CN CN201910987921.4A patent/CN110591906B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008017712A (en) * | 2006-07-10 | 2008-01-31 | Chisso Corp | Culture medium for microorganism |
| CN101215597A (en) * | 2007-12-26 | 2008-07-09 | 广东省微生物研究所 | Bacillus cereus specificity color biochemical fast detection kit and detection method |
| CN102604963A (en) * | 2011-01-24 | 2012-07-25 | 华中农业大学 | Separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene |
| CN102311990A (en) * | 2011-09-16 | 2012-01-11 | 广州绿洲生化科技有限公司 | Chromogenic medium of coliform group and quick detection card thereof |
| CN105754849A (en) * | 2016-03-31 | 2016-07-13 | 吉林农业大学 | Total bacterial count testing slip and preparation method and application thereof |
| CN108220384A (en) * | 2017-12-29 | 2018-06-29 | 广东海大畜牧兽医研究院有限公司 | A kind of vibrios testing piece and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110591906A (en) | 2019-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110591906B (en) | Coliform group counting test piece, preparation method and application thereof | |
| US8846334B2 (en) | Detection of acid-producing bacteria | |
| ANDERSON et al. | A rapid and direct plate method for enumerating Escherichia coli biotype I in food | |
| JP7115806B2 (en) | Articles and methods for detecting aerobic bacteria | |
| JP6920212B2 (en) | Culture device for anaerobic microorganisms | |
| CN103290094B (en) | A kind of streptococcus aureus color developing culture medium and testing plate thereof | |
| JP2016533727A (en) | Built-in anaerobic environment-generating culture device and method of use | |
| CN109182445B (en) | Mould and yeast counting test piece, preparation method and application thereof | |
| CN104673876B (en) | A kind of transparent water absorbent gel and detection plate for microorganism detection | |
| EP3607083B1 (en) | Rapid detection of e. coli in a thin film culture device | |
| CN112724474B (en) | Cold hydrogel and application thereof | |
| CN108456711B (en) | Cold water soluble gel matrix resistant to mesophilic bacteria decomposition and application thereof | |
| JP3630737B2 (en) | Simple medium | |
| CN107287275B (en) | Culture medium, kit containing culture medium and application of culture medium | |
| CN111733069B (en) | Colony counting test piece for dual detection of mold and yeast fluorescence/visible light and preparation method thereof | |
| CN112111436A (en) | Method for separating and screening bacillus from western Hunan sour meat | |
| US20030044882A1 (en) | Composition and method for detecting and early and differentiated counting of gram-negative microorganisms | |
| CN113846003A (en) | Research, preparation method and application of bifidobacterium colony counting test piece | |
| CN118109553A (en) | Coliform group rapid counting test piece and preparation method and application thereof | |
| CN113249431B (en) | Test piece for penicillin G residue in milk, preparation method and application thereof | |
| CN116948803A (en) | Anti-liquefaction bacterial colony total number test piece and preparation method and application thereof | |
| CN101270383A (en) | Test method for main necrosis microorganism of tomato paste | |
| JP7506986B2 (en) | Self-contained anaerobic environment generating culture device and method of use | |
| Alagoa et al. | Surface bacterial flora of Tilapia (Oreochromis niloticus) in open markets of Yenagoa, Bayelsa state | |
| Cai et al. | The single-tube quantitative rapid detection of coliform bacteria based on enzyme-specific technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |