CN109182445B - Mould and yeast counting test piece, preparation method and application thereof - Google Patents
Mould and yeast counting test piece, preparation method and application thereof Download PDFInfo
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- CN109182445B CN109182445B CN201811099187.XA CN201811099187A CN109182445B CN 109182445 B CN109182445 B CN 109182445B CN 201811099187 A CN201811099187 A CN 201811099187A CN 109182445 B CN109182445 B CN 109182445B
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- 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
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Abstract
A mould and yeast counting test piece, a preparation method and application thereof in detecting mould and yeast counting in food belong to the technical field of food detection. The test piece consists 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 high temperature resistant temperature range of the materials is 130-150 ℃; the lower sticky surface of the transparent plastic upper layer film is coated with compound cold water soluble gel, and the hollow area is filled with the sterilized non-woven fabric which has the same shape as the hollow area and is soaked with a mould and yeast counting and developing detection culture medium. The invention uses the test piece to count and measure the mould and the yeast in the food, the components of the culture medium can accelerate the multiplication and colony formation of mould spores and the yeast, and inhibit the spreading and growth of mould hyphae, thereby solving the technical problems of poor colony coverage, poor counting accuracy and long required time caused by the spreading and growth of the mould hyphae and the slow growth of the yeast.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a mould and yeast counting test piece, a preparation method and application thereof in detecting mould and yeast counting in food.
Background
The mould and yeast counting index is an important index reflecting the food sanitation quality and is used for judging the degree of food contamination. The mold and yeast counting measurement is a detection index of food such as beverages, nut products, rice and flour products, cakes and the like in China, and is also an important monitoring item for food risk prediction and hazard assessment. At present, GB4789.15 national food safety standard food microbiology inspection mould and yeast counting adopts a mould and yeast flat plate counting method, the method needs the work of culture medium preparation, sterilization, glassware cleaning and the like, and has complicated procedures and large workload. The microorganism test sheet detection product is convenient to count, only needs sample preparation, multiplication culture and counting steps when in use, saves a large amount of auxiliary work such as culture medium preparation, disinfection and sterilization, cleaning treatment of culture vessels and the like, and is the best detection product for counting and measuring the microorganism pollution at home and abroad.
In the existing commercial microorganism test piece detection products, the Petrifilm of the American 3M companyTMThe test piece is widely applied all over the world, a series of domestic test piece products are also successively released by a plurality of enterprises such as Beijing land bridge technology limited company and the like in China, and some test pieces are in the stages of research and development and small-range use. Although these test strips have the outstanding advantages of convenience and practicability, there are some key technical bottlenecks to be solved in the test strips for counting mold and yeast. The outstanding problem is focused on three aspects: the mould hyphae spread and grow to influence counting and even cause the counting to be impossible, and the coloration color of the mould bacterial colony covers yeast deeply, so that the counting result has large deviation; the yeast grows slowly, the bacterial colony is small, and the detection period is long; the color development of the bacterial colony cannot indicate all mould fungi and yeasts, and needs to be perfect to prevent missing detection. The invention develops the mould and yeast counting test piece aiming at the existing problems, and adopts two specific enzyme substrate color development methods, namely a specific color development substrate of lipase, namely p-nitrophenylpalmitateAnd a specific chromogenic substrate for alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate; the color development promoting factors of ethanol, copper sulfate, calcium chloride and zinc chloride are added, so that the color development of bacterial colonies is accelerated, and the detection time of the test piece is shortened; the mold hyphae are controlled to spread and grow by the combined use of inorganic salts such as magnesium sulfate, potassium dihydrogen phosphate, ferric sulfate and the like, so that the size of a bacterial colony is moderate, the color of the mold and yeast is clear, and the counting is accurate.
Disclosure of Invention
One of the purposes of the invention is to provide a mould and yeast counting test piece which is simple in operation, low in price and accurate in detection, wherein the test piece sequentially 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, the thickness is 0.05 mm-0.10 mm, the lower surface of the PET silicon film is sticky), a plastic middle layer plate with a hollow area (such as a polypropylene (PP) plastic plate or a PET plastic plate with better hardness, the thickness is 0.15 mm-0.30 mm) and a bottom plate printed with grids (such as waterproof and anti-seepage white coated paper, the thickness is 0.10 mm-0.20 mm, and the size range of the grids is 1 cm-2 cm multiplied by 1 cm-2 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, and filling the hollow area with a sterilized non-woven fabric which is in the same shape as the hollow area and is soaked with a mould and yeast counting and developing detection culture medium.
The compound cold water soluble gel comprises the following components: mixing the sodium polyacrylate and the guar gum which are ground and pass through a standard sieve of 100-150 meshes, wherein the weight percentage of the sodium polyacrylate is 15-25% and the balance is the guar gum, calculated according to the weight sum of 100% of the sodium polyacrylate and the guar gum.
The weight components of the mould and yeast counting and color developing detection culture medium are as follows: 10-20 g of peptone, 10-20 g of glucose, 4-10 mL of ethanol, 0.1-0.3 g of copper sulfate, 0.1-0.3 g of calcium chloride, 0.05-0.25 g of zinc chloride, 2-6 g of magnesium sulfate, 0.3-1.0 g of monopotassium phosphate, 0.3-1.0 g of ferric sulfate, 0.1-0.4 g of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 0.1-0.4 g of p-nitrophenylpalmitate, 0.05-0.2 g of chloramphenicol, and 1000mL of distilled water.
The sterilized non-woven fabric is used as a carrier of a chromogenic detection culture medium, and the specification of the sterilized non-woven fabric is 50-80 g/m in density2The spunlace nonwoven fabric of (1).
The invention also aims to provide a preparation method of the mould and yeast counting test piece, which comprises the following steps:
(1) preparation of culture medium for mould and yeast counting and chromogenic detection
Weighing 10-20 g of peptone, 10-20 g of glucose, 2-8 mL of ethanol, 0.1-0.3 g of copper sulfate, 0.1-0.3 g of calcium chloride, 0.05-0.25 g of zinc chloride, 2-6 g of magnesium sulfate, 0.3-1.0 g of monopotassium phosphate, 0.3-1.0 g of ferric sulfate and 0.05-0.2 g of chloramphenicol, adding 900mL of distilled water, and sterilizing at 121 ℃ for 15-20 min to obtain a liquid culture medium; dissolving 0.1-0.4 g of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in 100mL of distilled water, and filtering and sterilizing the solution by using a disposable filter with the pore diameter of 0.22 mu m; dissolving 0.1-0.4 g of p-nitrophenylpalmitate in 2mL of ethanol; adding the two solutions into a sterilized liquid culture medium to obtain a mixed liquid culture medium; the thickness of 0.10 mm-0.20 mm and the density of 50-80 g/m2The sterilized non-woven fabric is fully soaked in the mixed liquid culture medium for 15-30 min, and then is dried under the conditions of sterility and 45-50 ℃ to obtain the sterilized non-woven fabric containing a mould and yeast counting and developing detection culture medium;
(2) preparation of compound cold water soluble gel
Mixing the sodium polyacrylate which is ground and passes through a standard sieve of 100-150 meshes with guar gum to obtain compound cold water soluble gel powder, wherein the weight percentage of the sodium polyacrylate is 15-25%;
(3) preparation of mould and yeast counting test piece
Cutting the transparent plastic upper layer film, the plastic middle layer plate and the bottom plate printed with the grids into the same size (the length is 9-12 cm, the width is 8-10 cm), and sealing and bonding one side adhered with the label by using polyacrylic resin pressure-sensitive adhesive (transparent, nontoxic, tasteless and sterile), wherein the bonding width is 4-6 mm;
uniformly coating the compound 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 placing and compacting the sterilized non-woven fabric soaked with the mould and yeast counting and developing detection culture medium in the hollow area of the plastic middle-layer plate;
and (4) carrying out vacuum packaging, and then sterilizing by adopting an ethylene oxide sterilization method, thereby obtaining the mould and yeast counting test piece.
(3) Operation steps for measuring mould and microzyme count by test strip method
Solid or semi-solid sample: weighing 25g of sample (the sample can be rice and flour product, cake food or nut product), adding 225mL of sterile diluent (distilled water, physiological saline or phosphate buffer), and fully shaking or beating with a beating type homogenizer for 1-2 min to obtain a product with the weight ratio of 1: 10 of the diluted homogenate.
Liquid sample: sucking 25mL of sample (the sample can be beverage) by a sterile pipette, placing the sample in a container or a sterile homogenizing bag containing 225mL of sterile diluent (distilled water, normal saline or phosphate buffer), fully shaking or beating by a beating type homogenizer for 1 min-2 min, and preparing into a product 1: 10 of the diluted homogenate.
Taking 1mL of 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 uniform solution; sequentially preparing 10 times of gradient dilution homogenizing liquid for later use.
According to the estimation of the sample pollution condition, selecting 2-3 diluted uniform solutions with proper dilution, opening the transparent plastic upper layer film of the mould and yeast counting test piece, adding 1mL of the diluted uniform solution of the sample to a sterilized non-woven fabric of which the middle layer is soaked with a mould and yeast counting chromogenic detection culture medium, and uniformly distributing the diluted uniform solution of the sample to the whole culture area; slowly dropping the plastic upper layer film, avoiding extrusion, standing for 30 s-1 min until a cold water soluble gel and water are compounded and solidified, putting the plastic upper layer film into a constant temperature incubator to be cultured for 40-50 h at 25-30 ℃, and counting according to the national food safety standard GB4789.15 'food safety national standard food microbiology inspection mould and yeast counting'. Meanwhile, 1mL of physiological saline was used as a blank control.
The invented mould and yeast counting test plate is characterized by that its culture medium is composed of basic nutrient culture medium, inorganic salt, bacteriostatic agent, colour-developing agent and colour-developing promoting factor. Mainly comprising 3 important features.
Important technical features 1: according to the invention, through the design and optimization of the composition and the dosage of the inorganic salt, the growth and color rendering properties of the mould and the yeast in the test piece are obviously improved, and the technical problem that the excessive growth of mould hyphae influences counting and the growth of the yeast is slow to cause long detection time is effectively controlled. Test results show that the inhibition of the mold hypha overgrowth and the promotion of the yeast growth can be realized by controlling the concentration of the inorganic salt. Wherein Mg2+Plays an important role in the counting test piece of the mould and the yeast. Mg in conventional microbiological media2+Can be used as a trace element to be utilized by microorganisms, and Mg is contained in a national standard Bengal red culture medium2+The dosage is 0.5g/L, and the experimental research result of the invention shows that 4g/L Mg2+The method has no influence on the formation of mould spores, has correct mould detection counting result, can obviously inhibit the growth of mould hyphae, and effectively solves the problem of poor counting accuracy caused by overlarge bacterial colony for mould spreading growth. Mg (magnesium)2+Also as an activator of the enzyme, Mg2+Has activating effect on alkaline phosphatase, and can enhance the sensitivity of 5-bromo-4-chloro-3-indole phosphate color reaction. In addition, the invention also researches the influence of different concentrations of magnesium sulfate, potassium dihydrogen phosphate and ferric sulfate on the growth state of the mould and yeast, and the optimal combination of the magnesium sulfate, the potassium dihydrogen phosphate and the ferric sulfate, namely 4g/L Mg, is optimized through experimental analysis2+0.2g/L of K+And Fe2+The concentration combination can inhibit the growth of mould hypha to the maximum extent, promote the formation of mould spore and the growth of microzyme, and can also enhance sensitivity of alkaline phosphatase and color-developing substrateThe 5-bromo-4-chloro-3-indolyl phosphate is subjected to color development reaction.
Important technical features 2: based on gel characteristics and texture analysis, the components and concentration of the culture medium and the cold water soluble gel texture characteristics are firstly researched, the influence of inorganic salt ion concentration on the gel texture of the test piece culture medium is researched, the optimal inorganic salt concentration is screened, the culture medium components, the cold water soluble gel and the detection sample liquid of the test piece form a colloid with a net structure, and a good environment is provided for the growth of microorganisms on the test piece. The experimental result shows that the inorganic salt ion concentration has great influence on the gel texture, and the variation trend is consistent with the influence of the inorganic salt ion concentration on the growth rate of the bacterial colony. The gel structure of the optimized test piece has no significant difference with the structure of a national standard method flat plate, and the reticular space structure formed by the gel is beneficial to the growth of mould and saccharomycetes.
Important technical features 3: a test piece for the composite enzyme substrate type mould and yeast is developed. The color developing agent is based on enzyme substrate type color development, selects two color developing substrates of specific enzymes, can indicate the specific enzymes generated by common mould and yeast in food pollution and develop color, and improves the detection accuracy and efficiency. Compared with the similar research, the double-enzyme substrate and the color development promoting factor are compounded, an accurate determination result can be obtained within 48 hours (the detection period of the national standard method is 5 days), the detection time is obviously shortened, and the counting result is more accurate. Wherein the color developing agent is a compound of two color developing agents of 5-bromo-4-chloro-3-indole phosphate (BCIP, a substrate of alkaline phosphatase) and p-nitrophenylpalmitate (a substrate of lipase). Hydrolysis of p-nitrophenylpalmitate with lipase gave a yellow colony. The 5-bromo-4-chloro-3-indolyl phosphate BCIP was hydrolyzed by alkaline phosphatase to make the colonies blue. Counting blue and yellow colonies on the test strip to report the test result; the invention also promotes the enzymatic reaction of the chromogenic enzyme substrate and the specific enzyme by adding the chromogenic promoting factors of ethanol, copper sulfate, calcium chloride and zinc chloride with optimal concentration, so that the color can be developed by the colony generation of the mould and the yeast, the time required by the color development is shortened, the colors presented by the mould and the yeast are not mutually covered, the colony color is clear, and the counting is accurate. Particularly, the ethanol with the optimal concentration plays an important role in the color development effect, and the experimental result shows that the low-concentration ethanol can promote the color development reaction of the mold and the yeast. The color development results of the mold and the yeast after 48 hours of culture show that the mold and the yeast without the ethanol group appear colonies but do not develop the color, and the mold and the yeast with the ethanol group appear colonies and develop the color. The color development effect is good when the concentration is 4-10 mL/L. Meanwhile, the experimental result also shows that the ethanol with the concentration has no inhibition effect on the growth of mould and yeast, which is the first proposal that the low-concentration ethanol is applied to the test piece.
According to the mould and yeast 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, a culture medium on non-woven fabric is mixed with the upper-layer compound cold water soluble gel, and a stable gel colloid with moderate pores and a compact reticular space is formed in the gel water absorption process, so that the mould and yeast counting test piece is particularly suitable for the growth of mould and yeast and the formation of bacterial colonies in the test piece environment. The mould and yeast counting test piece for detecting the total number of mould and yeast colonies in various foods has the advantages of simple and convenient procedure, clear color of the colonies and accurate counting. 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 of the mould and yeast counting test piece is shown schematically;
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: the invention relates to a regression equation and a correlation coefficient curve of a mould and yeast counting test piece;
Detailed Description
Example 1 preparation of culture Medium for the determination of the mold and Yeast count
Counting, developing and detecting the original weight components of the culture medium by mould and yeast: 15g of peptone, 15g of glucose, 10mL of ethanol, 0.2g of copper sulfate, 0.2g of calcium chloride, 0.15g of zinc chloride, 4g of magnesium sulfate, 0.7g of monopotassium phosphate, 0.71g of ferric sulfate, 0.2g of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 0.2g of p-nitrophenylpalmitate, 0.1g of chloramphenicol, and 1000mL of distilled water.
The preparation method of the mould and yeast counting detection culture medium comprises the following steps: adding 15g of peptone, 15g of glucose, 8mL of ethanol, 0.2g of copper sulfate, 0.2g of calcium chloride, 0.15g of zinc chloride, 4g of magnesium sulfate, 0.7g of potassium dihydrogen phosphate and 0.71g of ferric sulfate into 900mL of distilled water, heating, stirring for dissolving, sterilizing at 121 ℃ for 15min under high pressure, and cooling to room temperature; separately, 0.2g of p-nitrophenylpalmitate was dissolved in 2mL of ethanol, 0.2g of BCIP was dissolved in 100mL of distilled water, and the resulting solution was filtered through a disposable filter at 0.22 μm to sterilize the solution, and then the two solutions were added to the above-mentioned liquid medium and mixed well with stirring. The density is 60g/m2And soaking sterilized non-woven fabrics with the diameter of 6cm in the liquid culture medium for 15min, drying by a sterile oven at 45 ℃, and sticking to a culture area for later use.
The preparation method of the compound cold water soluble gel comprises the following steps: respectively grinding cold water soluble gel guar gum and sodium polyacrylate into powder, sieving with a 120-mesh molecular sieve, and fully and uniformly mixing according to the weight percentage of 80% and 20% of guar gum and sodium polyacrylate for later use.
EXAMPLE 2 preparation of mold and Yeast count test pieces
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 plate white square copper plate paper. The three parts are cut into 10.5cm multiplied by 9cm, the middle hollow area (the diameter is 6 cm) is bonded with the bottom plate to form a culture area, and the bottom plate is printed with light orange squares of 1cm multiplied by 1cm, so that counting is convenient. The upper, middle and lower parts are adhered to each other by polyacrylic resin pressure sensitive adhesive, and one side edge of the label is adhered to be 5mm in width.
2) The compounded cold water soluble gel powder is evenly coated 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 0.2 g.
3) At a density of 60g/m2And non-woven fabrics with the diameter of 6cm are used as culture medium carriers.
4) And (3) uniformly coating 0.2g of transparent polyacrylic resin pressure-sensitive adhesive on the bottom plate corresponding to the hollow-out area of the middle layer, and then sticking the non-woven fabric carrying the culture medium to the culture area.
5) The test pieces were vacuum packed.
6) The test piece was sterilized by ethylene oxide sterilization.
Example 3 method for determining mold and yeast counts in food Using test strips
1) Weighing 25g of 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 food sample to be detected (the sample to be detected can be liquid food such as drinks) by aseptic technique, adding the sample into an aseptic homogenizing bag of 225mL diluent, beating for 1-2 min by a homogenizer to prepare 1: sample aliquot of 10. And performing 10-fold serial gradient dilution on the sample homogeneous solution, selecting 2-3 dilutions according to the pollution degree of the sample, and preparing two parallel samples for each dilution.
2) Taking 1mL of the diluent of the 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 be cultured for 48h at 28 ℃. Two replicates of each dilution were made, while 1mL of saline was used as a blank.
3) The test piece having completed the proliferation culture was placed in a sterile operating table, and the presence or absence of yellow and blue colonies was observed, and the yellow and blue colonies were counted. The counting method is carried out according to GB4789.15 (food safety national standard food microbiology inspection mould and yeast count).
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 10CFU, 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 10CFU and 150CFU, and a part of the colony number is less than 10CFU or more than 150CFU, the colony number is calculated by multiplying the average colony number closest to 10CFU or 150CFU by the dilution factor.
Example 4: evaluation of measurement Effect of mold and Yeast count test piece
1) Mold and yeast counting test pieces were prepared as in examples 1 and 2 and were ready for use.
2) Determination of detection limit and sensitivity of test strip
The experimental strains are candida albicans CCIC1965 and aspergillus niger CCIC 2487.
Respectively inoculating single colony of Candida albicans and Aspergillus niger into 100mL YEPD liquid culture medium (peptone 20g, glucose 20g, yeast extract powder 10g, chloramphenicol 0.1g, distilled water 1000mL, pH 7.0), shaking and culturing in shaking table at 28 deg.C and 200r/min for 12h, adding 1mL bacterial solution into 9mL normal saline, mixing to obtain diluent 1, repeating the above steps to dilute the bacterial solution to a dilution (concentration of 10) suitable for counting0～102CFU/mL), 1mL of the bacterial solution was taken and the number of colonies of mold and yeast was determined using the test piece according to the method in example 3. And simultaneously, performing mould and yeast counting determination according to GB 4789.15-2016 (national food safety Standard food microbiology inspection mould and yeast count), making two parallel samples for each dilution, and averaging the detection results.
The detection result of the colony number with the lowest dilution degree 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 detection result which is larger than the lowest detection limit is positive, the detection result which is smaller than the lowest detection limit is negative, the sensitivity is positive/(positive + negative), and the experiment result shows that the two methods can reach the detection limit of the same level, the detection limit is 1CFU/mL, and the sensitivity is 100%. The detection result of the mould and yeast test piece developed by the invention has no difference significance (P) from the national standard flat plate method>0.05)，R2Reaches 0.9985, and has good correlation. See table 1 and fig. 2.
Table 1: test strip accuracy and sensitivity measurement data
2) Test strip specificity detection
Respectively inoculating 23 strains of mould and yeast such as candida albicans and aspergillus niger and the like into 100mLYEPD liquid culture medium (peptone 20g, glucose 20g, yeast extract powder 10g, chloramphenicol 0.1g, distilled water 1000mL, pH value 7.0), and carrying out shake culture on a constant temperature shaking table at 28 ℃ for 12h at 200r/min to prepare a primary bacterial liquid for later use. Respectively inoculating 5 strains of bacteria such as escherichia coli, salmonella, staphylococcus aureus and the like into 100mLLB liquid culture medium (5 g of yeast extract powder, 10g of peptone, 5g of sodium chloride, 1000mL of distilled water and pH value of 7.0), and carrying out shake culture on the bacteria in a constant-temperature shaking table at 37 ℃ for 200r/min for 12h to prepare a stock solution for later use. Taking 25mL of original bacterial liquid, adding 225mL of sterile normal saline, repeating the above operations to prepare 10-time series diluted bacterial liquid, and carrying out colony count determination on 13 strains of saccharomycetes, 10 strains of moulds and 5 strains of bacteria by adopting the method of the invention mould and saccharomycetes test piece example 3 and GB 4789.15-2016 (national food safety Standard food microbiology inspection mould and Yeast count).
The experimental result shows that 13 strains of saccharomycetes and 10 strains of moulds can grow and develop color when cultured for 48 hours, 5 strains of bacteria do not grow, and the specificity of the test piece is good. The test strip method has no difference significance (P is more than 0.05) with the mould and yeast measuring and counting result of GB 4789.15-2016 (national food safety standard food microbiology test mould and yeast counting) and the counting result is accurate. The results are shown in Table 2.
Table 2: test piece specificity test data
3) 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 mixture of 1: 10 samples are homogenized. 1mL of mould and yeast mixed solution is added into the treated food sample homogenizing solution to carry out artificial pollution on the food sample. Carrying out 10-time serial dilution on the artificial pollution sample solution, and taking 1: adding 1mL of 10 diluent into 9mL of physiological saline, and fully and uniformly mixing to prepare a mixture of 1: 100 dilution, and repeating the above procedure to dilute the sample to 3 dilutions (10 concentration) of appropriate counts0～102CFU/mL), the total number of mold and yeast colonies was determined according to the method of example 3, and the test piece measurements were compared for compliance with the results of the national standard method.
Table 3: result comparison and coincidence rate data of food sample detection by two methods
The mould and yeast test piece method and the national standard method are respectively used for detecting 120 artificially polluted food samples, the test results are shown in table 3, the data show that the coincidence rate of the yeast in the two methods can reach 99.0 percent at most, the coincidence rate of the mould can reach 99.0 percent at most, the detection results in the two methods have no difference significance (P is more than 0.05), and the results show that: the mould and yeast counting test piece is suitable for detecting the total number of the moulds and the yeasts in the food.
Claims (8)
1. A mould and yeast counting test piece is characterized in that: the test piece consists 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 high temperature resistant temperature range of the materials is 130-150 ℃; the lower sticky surface of the transparent plastic upper film is coated with compound cold water soluble gel, and the hollow area is filled with sterilized non-woven fabric which has the same shape as the hollow area and is soaked with a mould and yeast counting and developing detection culture medium; the compound cold water soluble gel is prepared by mixing sodium polyacrylate and guar gum which are ground and pass through a 120-mesh standard sieve, wherein the weight percentage of the sodium polyacrylate is 20 percent and the rest is the guar gum according to the weight sum of 100 percent of the sodium polyacrylate and the guar gum; the weight components of the mould and yeast counting and color developing detection culture medium are 15g of peptone, 15g of glucose, 10mL of ethanol, 0.2g of copper sulfate, 0.2g of calcium chloride, 0.15g of zinc chloride, 4g of magnesium sulfate, 0.7g of monopotassium phosphate, 0.71g of ferric sulfate, 0.2g of 5-bromo-4-chloro-3-indole phosphate, 0.2g of p-nitrophenylpalmitate, 0.1g of chloramphenicol and 1000mL of distilled water.
2. A mold and yeast counting test strip according to claim 1, wherein: the transparent plastic upper layer film is a PET silica gel film, the thickness of the transparent plastic upper layer film is 0.05 mm-0.10 mm, and the lower surface of the PET silica gel film is sticky.
3. A mold and yeast counting test strip according to claim 1, wherein: the plastic middle layer plate is a polypropylene plastic plate or a PET plastic plate, and the thickness of the plastic middle layer plate is 0.15 mm-0.30 mm.
4. A mold and yeast counting test strip according to claim 1, wherein: the bottom plate is white coated paper, and the thickness of the bottom plate is 0.10 mm-0.20 mm.
5. A mold and yeast counting test strip according to claim 1, wherein: the non-woven fabric has a density of 50-80 g/m2The spunlace nonwoven fabric of (1).
6. A mold and yeast counting test strip according to claim 1, wherein: the hollow area of plastics median lamina is circular, oval or square structure, and the proportional range of hollow area and median lamina area is 1: 1.6-1: 2.7.
7. a method for preparing a mold and yeast counting test strip according to claim 1, comprising the steps of:
(1) preparation of culture medium for mould and yeast counting and chromogenic detection
Adding 15g of peptone, 15g of glucose, 8mL of ethanol, 0.2g of copper sulfate, 0.2g of calcium chloride, 0.15g of zinc chloride, 4g of magnesium sulfate, 0.7g of potassium dihydrogen phosphate and 0.71g of ferric sulfate into 900mL of distilled water, heating, stirring for dissolving, sterilizing at 121 ℃ for 15min under high pressure, and cooling to room temperature; dissolving 0.2g of p-nitrophenylpalmitate in 2mL of ethanol, dissolving 0.2g of 5-bromo-4-chloro-3-indolyl phosphate in 100mL of distilled water, filtering and sterilizing by using a disposable filter under the condition of 0.22 mu m, adding the two solutions, and uniformly stirring to obtain a liquid culture medium; soaking the sterilized non-woven fabric in a liquid culture medium for 15min, and drying at 45 ℃ by using an aseptic oven to obtain the sterilized non-woven fabric containing a mould and yeast counting and developing detection culture medium;
(2) preparation of compound cold water soluble gel
Mixing the sodium polyacrylate which is ground and passes through a 120-mesh standard sieve with guar gum to obtain compound cold water soluble gel powder, wherein the weight percentage of the sodium polyacrylate is 20%;
(3) preparation of mould and yeast counting test piece
Cutting the transparent plastic upper layer film, the plastic middle layer plate and the base plate printed with the grids into the same size, and sealing and bonding one side adhered with the label by using a 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 hollow area of the plastic middle-layer plate, and placing and compacting the sterilized non-woven fabric soaked with the mould and yeast counting and developing detection culture medium in the hollow area of the plastic middle-layer plate;
and (4) carrying out vacuum packaging, and then sterilizing by adopting an ethylene oxide sterilization method, thereby obtaining the mould and yeast counting test piece.
8. Use of a mold and yeast count test strip according to claim 1 for counting mold and yeast in food products.
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| CN113249431B (en) * | 2021-05-13 | 2022-10-04 | 吉林农业大学 | Test piece for penicillin G residue in milk, preparation method and application thereof |
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| CN105483204A (en) * | 2015-12-25 | 2016-04-13 | 贵州勤邦食品安全科学技术有限公司 | Detection vessel for rapidly detecting moulds and yeasts and preparation method |
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| CN105483204A (en) * | 2015-12-25 | 2016-04-13 | 贵州勤邦食品安全科学技术有限公司 | Detection vessel for rapidly detecting moulds and yeasts and preparation method |
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