Chromogenic culture medium for detecting mould and yeast and test board of chromogenic culture medium

Abstract

The invention discloses a chromogenic culture medium for detecting mould and yeast and a test plate thereof, the culture medium comprises a nutrient substance mixture, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gel, the composite inhibitor is chloramphenicol, ofloxacin, ceftazidime and 3 # cholate, and the cold water gel is a water-soluble gel. And the compound chromogenic enzyme substrate is 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic ester, and 5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosamine, and the compound chromogenic enzyme substrate is 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic ester and 5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosamine. The chromogenic culture medium and the test plate prepared from the chromogenic culture medium can accurately and quickly observe and distinguish bacterial colonial morphology of mould and yeast, have the characteristics of good specificity, high sensitivity, high accuracy and convenience in observation and counting, and have huge development potential and wide application prospect in microbiological detection.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a color development culture medium for detecting molds and yeasts and a test plate thereof. Background Art

[0002] Mold is a type of fungus that can form various tiny spores and easily contaminate food. Food contamination caused by mycotoxins can turn food into toxic substances, posing a serious challenge to food safety. About 25% of the harvested crops in the world are contaminated by mycotoxins each year, resulting in billions of dollars in agricultural and industrial losses, posing safety hazards to human and livestock health, and posing a major health risk. Yeast is a single cell and has been well used in the brewing, food and other industries, but some yeasts can cause diseases in humans, animals and plants. Therefore, it is very important to monitor mold and yeast in products.

[0003] At present, the detection method for mold and yeast in food mainly adopts GB 4789.15-2016 "National Food Safety Standard Food Microbiological Examination Mold and Yeast Count", but this method has cumbersome detection steps and a long test operation cycle, which can easily lead to problems such as regulatory lags.

[0004] Test pieces and test dishes can save the tedious steps of preparing culture medium. The rapid mold and yeast detection devices on the market mainly include 3M test pieces, Oasis test pieces, Meizheng test pieces, Huankai test pieces and Nissui CompactDry rapid mold and yeast test dishes. Oasis test piece CN03274715.2 is a paper sheet that absorbs the culture medium, which can easily lead to slow absorption of the test bacterial solution, and improper covering film operation will affect the experimental results; 3M, Meizheng and Huankai test pieces cannot observe the morphology of mold and yeast in three dimensions; Nissui CompactDry rapid mold and yeast test dish can observe the morphology of mold and yeast in three dimensions, but due to the use of non-woven fabric, the colony morphology and color are blurred and difficult to distinguish, and some yeasts will not show color, resulting in missed detection; in the patent "A test dish and preparation method for rapid detection of mold and yeast" CN 201510987947.0 and "A mold and yeast counting test piece, preparation method and application" CN201811099187.X, the culture medium inhibitor used in the test dish and test piece only uses chloramphenicol, and the color substrate only uses 5-bromo-4-chloro-3-indolyl-phosphate, resulting in poor specificity and mold spreading. Although test pieces and test dishes can be used for rapid detection of mold and yeast, there are problems such as the inability to accurately observe the morphology of all colonies, difficulty in distinguishing between mold and yeast, difficulty in counting due to the spread of mold, missed detection and false positives.

[0005] In summary, the existing mold and yeast detection methods have many defects, and it is urgent to develop a convenient and highly accurate detection device. The present invention provides a color development culture medium and a test plate for detecting mold and yeast, which can achieve rapid, accurate and clear detection of mold and yeast. Summary of the invention

[0006] To this end, the present invention aims to provide a chromogenic culture medium and a test plate thereof for detecting molds and yeasts which have good specificity, high sensitivity, high accuracy and are convenient for observation and counting.

[0007] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions: An object of the present invention is to provide a chromogenic culture medium for detecting molds and yeasts, characterized in that it comprises a nutrient mixture, a composite inhibitor, and a composite chromogenic enzyme substrate, wherein the composite inhibitor is chloramphenicol, ofloxacin, ceftazidime and bile salt No. 3, and the composite chromogenic enzyme substrate is 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic acid ester, and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidoside.

[0008] Preferably, the nutrient mixture includes 5-30 g of peptone, 10-50 g of yeast extract powder, 1-10 g of glucose, 1-10 g of sodium chloride, and 1-10 g of Fe 2+ 0.005-0.05g, Mn 2+ 0.01-0.1g, Cu 2+ 0.01-0.05g, Zn 2+ 0.01-0.1g, Mg 2+ 0.01-0.1g, linoleic acid 0.001-0.05g, egg yolk lecithin 0.001-0.05g.

[0009] Preferably, the concentrations of the components of the composite inhibitor are 0.01-0.1 g / L of chloramphenicol, 0.001-0.01 g / L ofloxacin, 0.001-0.01 g / L of ceftazidime, and 0.1-1 g / L of bile salt No. 3.

[0010] Preferably, the concentrations of the components of the composite chromogenic enzyme substrate are 0.05-0.5 g / L of 5-bromo-4-chloro-3-indole-toluidine phosphate, 0.05-0.5 g / L of 5-bromo-4-chloro-3-indole-acetate, and 0.05-0.5 g / L of 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase.

[0011] Preferably, the nutrient mixture further comprises cold water gelatin, and the cold water gelatin comprises xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum.

[0012] Preferably, the concentrations of the cold water gelable components are xanthan gum 10-30 g / L, guar gum 10-30 g / L, sodium carboxymethyl cellulose 1-5 g / L, sodium hydroxypropyl cellulose 1-10 g / L, and gellan gum 1-10 g / L.

[0013] Another object of the present invention is to provide a detection plate, which comprises a substrate, and any one of the above-mentioned color development culture media for detecting molds and yeasts configured on the surface of the substrate.

[0014] Preferably, the substrate comprises a transparent covering film, a culture area and a lower circular groove plate from top to bottom, wherein the culture area is arranged in the lower circular groove plate, and the transparent covering film covers the lower circular groove plate.

[0015] Preferably, the material of the transparent coating is one of PE and PET, and the material of the bottom groove plate is one of polymethyl methacrylate, polycarbonate, PET and polystyrene.

[0016] The embodiments of the present invention have the following advantages: 1. The mold and yeast test plate provided by the present invention is a prefabricated culture system, which does not require the agar culture medium to be sterilized in advance by high pressure, takes into account the appropriate pouring temperature, and is easy to carry; 2. The composite inhibitor in the mold and yeast test plate provided by the present invention can not only effectively inhibit the growth of non-target bacteria, but also effectively prevent the spread of mold; the composite color-developing enzyme substrate is more selective for target bacteria, improving the detection rate; the nutrient mixture can promote the growth of mold and yeast and accelerate color development; cold water can gel instead of agar as a coagulant for the culture medium, and the operation is convenient. Therefore, the present invention has the characteristics of high sensitivity, strong specificity, and easy observation and counting, which greatly improves the detection accuracy and reduces the false positive rate and false negative rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0018] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0019] Figure 1 The schematic diagram of the structure of the mold and yeast test plate of the present invention; wherein 1-transparent covering film, 2-circular culture area, 3-lower circular groove plate; Figure 2 The growth of Saccharomyces cerevisiae on the mold and yeast test plate of the present invention; Figure 3 The growth of Aspergillus niger on the mold and yeast test plate of the present invention; Figure 4 Growth of Mucor circinelloides on the mold and yeast test plate of the present invention; Figure 5 The growth of Mucor circinelloides on the 3M Rapid Mold and Yeast Test Plate. DETAILED DESCRIPTION

[0020] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 A chromogenic culture medium for detecting molds and yeasts Each 1000mL contains 5g peptone, 10g yeast extract powder, 1g glucose, 1g sodium chloride, Fe 2+ 0.005g, Mn 2+ 0.01g, Cu 2+ 0.01g, Zn 2+ 0.01g, Mg 2+0.01g, linoleic acid 0.001g, egg yolk lecithin 0.001g. Composite inhibitor 0.112g, composite colorimetric enzyme substrate 0.15g, cold water gel 23g. Among them, the composite inhibitor is composed of chloramphenicol, ofloxacin, ceftazidime, and bile salt No. 3; the composite colorimetric enzyme substrate is composed of 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic acid ester and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase; the 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase is dissolved in dimethylformamide; the cold water gel is composed of xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum. Mix the prepared nutrients, inhibitors, and colorimetric enzyme substrates evenly, add cold water to gel, divide into portions, dry, and then use.

[0022] Example 2 A chromogenic culture medium for detecting molds and yeasts Each 1000mL contains 10g peptone, 12g yeast extract powder, 5g glucose, 4g sodium chloride, Fe 2+ 0.008g, Mn 2+ 0.05g, Cu 2+ 0.03g, Zn 2+ 0.04g, Mg 2+ 0.08g, linoleic acid 0.01g, egg yolk lecithin 0.02g. Composite inhibitor 0.16g, composite chromogenic enzyme substrate 0.35g, cold water gel 40g. Among them, the composite inhibitor is composed of chloramphenicol, ofloxacin, ceftazidime, and bile salt No. 3; the composite chromogenic enzyme substrate is composed of 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetate and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase; the 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase is dissolved in dimethylformamide; cold water gel is composed of xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum. After mixing the prepared nutrients, inhibitors, and chromogenic enzyme substrates, add cold water gel, divide into packages, dry, and then use.

[0023] Example 3 A chromogenic culture medium for detecting molds and yeasts Each 1000mL contains 30g of peptone, 50g of yeast extract powder, 10g of glucose, 10g of sodium chloride, Fe 2+ 0.05g, Mn 2+ 0.1g, Cu 2+ 0.05g, Zn 2+ 0.1g, Mg 2+0.1g, linoleic acid 0.005g, egg yolk lecithin 0.005g. Composite inhibitor 1.12g, composite chromogenic enzyme substrate 1.5g, cold water gel 85g. Among them, the composite inhibitor is composed of chloramphenicol, ofloxacin, ceftazidime, and bile salt No. 3, and the composite chromogenic enzyme substrate is composed of 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic acid ester and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase; the 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase is dissolved in dimethylformamide; the cold water gel is composed of xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum. After mixing the prepared nutrients, inhibitors, and chromogenic enzyme substrates, add cold water gel, divide into packages, dry, and then use.

[0024] Example 4 A test plate for detecting mold and yeast The mold and yeast test plate of this embodiment is as follows Figure 1 As shown, it includes a transparent covering film 1, a circular culture area 2, and a circular groove plate 3. The transparent covering film 1 can cover the circular groove plate 3; wherein the circular culture area is composed of a nutrient mixture, a composite inhibitor, a composite colorimetric enzyme substrate, and a cold water gel, the circular groove plate is made of PS (polystyrene), and the covering film is made of PET (polyethylene terephthalate).

[0025] The colorimetric culture medium for detecting molds and yeasts in the circular culture area contains 5g of peptone, 10g of yeast extract powder, 1g of glucose, 1g of sodium chloride, and Fe 2+ 0.005g, Mn 2+ 0.01g, Cu 2+ 0.01g, Zn 2+ 0.01g, Mg 2+ 0.01g, linoleic acid 0.001g, egg yolk lecithin 0.001g. Composite inhibitor 0.112g, composite color enzyme substrate 0.15g, cold water gel 23g. Among them, the composite inhibitor is composed of chloramphenicol, ofloxacin, ceftazidime, and bile salt No. 3, and the composite color enzyme substrate is composed of 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic acid ester and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase; the 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase is soluble in dimethylformamide; the cold water gel is composed of xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum.

[0026] The above basic components are fully hydrolyzed with 1000 mL, and then cold water is added to gel, stirred evenly, and dispensed into the circular culture area 2 of the test plate, dried, and pasted with a transparent film 1, so as to prepare a test plate for detecting molds and yeasts.

[0027] The test plate for detecting mold and yeast prepared in this embodiment was tested and verified by inoculating positive quality control strains and negative control bacteria. The results showed that the mold was blue-green, large, and the hyphae could be directly seen. Spores of some molds could be observed. Yeast was green, smaller than mold, and had regular, smooth and three-dimensional colonies with edges. Negative quality control bacteria (such as Escherichia coli and Staphylococcus aureus) did not grow.

[0028] Example 5 A test plate for detecting mold and yeast The mold and yeast detection test plate of this embodiment is different from the test plate of Example 4 in that the color development culture medium for detecting mold and yeast in the circular culture area contains 10g of peptone, 12g of yeast extract powder, 5g of glucose, 4g of sodium chloride, and Fe 2+ 0.008g, Mn 2+ 0.05g, Cu 2+ 0.03g, Zn 2+ 0.04g, Mg 2+ 0.08g, linoleic acid 0.01g, egg yolk lecithin 0.02g. Composite inhibitor 0.16g, composite colorimetric enzyme substrate 0.35g, cold water gel 40g. The composite inhibitor is composed of chloramphenicol, ofloxacin, ceftazidime, and bile salt No. 3, and the composite colorimetric enzyme substrate is composed of 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic acid ester and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase; the 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidase is dissolved in dimethylformamide; the cold water gel is composed of xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum.

[0029] The above basic components are fully hydrolyzed with 1000 mL, and then cold water is added to gel, stirred evenly, and dispensed into the circular culture area 2 of the test plate, dried, and pasted with a transparent film 1, so as to prepare a test plate for detecting molds and yeasts.

[0030] The test plate for detecting mold and yeast prepared in this example was tested and verified by inoculating positive quality control strains and negative control bacteria.

[0031] According to GB 4789.28-2013 "Quality Requirements for Food Microbiology Test Culture Media and Reagents of National Food Safety Standard", the quality control strains are set as follows: Saccharomyces cerevisiae ATCC 9763, Aspergillus niger ATCC 16404, Escherichia coli ATCC25922, and Staphylococcus aureus ATCC 6538. The strains were prepared into a bacterial suspension with a concentration of 20-200 CFU / mL, and 1 ml was taken and inoculated on the mold and yeast test plates, and Sabouraud dextrose agar plates (SDA) at the same time, and cultured in a 28°C incubator. The mold and yeast test plates were cultured for 72 hours, and the Sabouraud dextrose agar was cultured for 5 days. The results showed that Saccharomyces cerevisiae was green, smaller than mold, with regular, smooth and three-dimensional colonies with edges. See attached Figure 2 Aspergillus niger grows in three dimensions, is blue-green, is relatively large, and has visible hyphae. Some molds have spores attached to them. Figure 3 ; Bacteria do not grow. The growth rates of Saccharomyces cerevisiae ATCC 9763 and Aspergillus niger ATCC16404 were both greater than 0.7, meeting the quality control assessment standard (PR ≥ 0.7). See Table 1 for details.

[0032] Table 1 Growth of quality control strains

[0033] Note: “-” means the test was not conducted.

[0034] Test Example 1 Detection of mold and yeast test plate specificity experiment The specific performance evaluation was performed using the mold and yeast detection test plate prepared in Example 4 of the present invention: 4 strains of Saccharomyces cerevisiae (numbered: ATCC 9763, ATCC 26602, CICC 1840 and CICC 1796), 2 strains of Candida albicans (numbered: ATCC 14053, CICC 1965), 1 strain of Aspergillus niger (ATCC 16404), 1 strain of Penicillium aureum (CICC4026), 1 strain of Monascus (CICC 40712), 1 strain of Aspergillus flavus (CICC 41668), 1 strain of Aspergillus ochraceus (CICC 2471), 1 strain of Fusarium moniliforme (ATCC 10052), 1 strain of Fusarium graminearum (isolated from milk), 1 strain of Mucor circinelloides (isolated from milk), 1 strain of Escherichia coli (ATCC 25922), 1 strain of Staphylococcus aureus (ATCC 6538), 1 strain of nitrogen-producing Pseudomonas (isolated from milk), 1 strain of Hafnia alvei (isolated from milk), and 1 strain of Serratia marcescens (isolated from milk) were used for specificity experiments.

[0035] The strain was prepared into a bacterial suspension with a concentration of 20-200 CFU / mL, and 1 ml of the bacterial suspension was inoculated into a mold and yeast test plate, a Nissui CompactDry rapid mold and yeast test dish, and a 3M rapid mold and yeast test piece. Mold and yeast were inoculated into SDA at the same time as a positive control, and bacteria were inoculated into 3% sodium chloride tryptone soy agar (TSA) as a positive control. The mold and yeast test plate was cultured in a 28°C incubator for 3 days, SDA was cultured in a 28°C incubator for 5 days, TSA was cultured in a 37°C incubator for 2 days, and Nissui CompactDry rapid mold and yeast test dish and 3M rapid mold and yeast test piece were cultured according to their instructions.

[0036] The results of SDA and TSA showed that all 19 strains were viable and countable, as shown in Table 2. The specific results are as follows: (1) Yeast growth: 6 yeast strains can grow and be counted on mold and yeast test plates, Nissui CompactDry rapid mold and yeast test dishes, and 3M rapid mold and yeast test sheets; (2) Fungal growth: All 8 fungal strains can grow and be counted on the fungal and yeast test plate; the Nissui Compact Dry rapid fungal and yeast test plate did not detect Fusarium graminearum, resulting in missed fungal detection; Mucor circinelloides can grow on the 3M rapid fungal and yeast test plate, but due to the serious spread of the fungal body, it cannot be counted, while on the fungal and yeast test plate, the colonies are distinct, the morphology is clear, and it can be counted, see attached Figure 4 , Attachment Figure 5 ; (3) Bacterial growth: All five bacterial strains did not grow on the mold and yeast test plates; Pseudomonas aeruginosa grew on the Nissui CompactDry rapid mold and yeast test dish and looked like yeast, causing a false positive for yeast; Hafnia alvei and Serratia marcescens grew on the 3M rapid mold and yeast test plate and looked like yeast, causing a false positive for yeast.

[0037] In summary, the specificity results show that the mold and yeast test plate provided by the present invention has stronger detection specificity, distinct colonies, clear morphology, and countability, and can avoid the occurrence of false positives and missed detections to a large extent.

[0038] Table 2 Specificity and quality control results of 19 strains

[0039] Table 2 (continued)

[0040] Note: “+” represents growth, the morphology of molds and yeasts can be distinguished, and the counts can be made; “-” represents no growth; “±” represents growth, but the colonies spread severely, and the morphology cannot be observed or counted.

[0041] Test Example 2: Test for the accuracy of mold and yeast test plates The accuracy performance evaluation was performed using the mold and yeast detection test plate prepared in Example 4 of the present invention: accuracy experiments were performed in accordance with RB / T033-2020 "Guidelines for Confirmation and Validation of Microbial Detection Methods", GB 4789.28-2024 "National Food Safety Standard Quality Requirements for Food Microbiology Examination Culture Media and Reagents" and GB 4789.15-2016 "National Food Safety Standard Food Microbiology Examination Mold and Yeast Count".

[0042] 2.1 Standard strain accuracy experiment Two strains of Saccharomyces cerevisiae (numbered: ATCC 9763, ATCC 26602), two strains of Candida albicans (numbered: ATCC 14053, CICC 1965), one strain of Aspergillus niger (ATCC 16404), and one strain of Penicillium aureum (CICC 4026) were used for the accuracy test of standard strains. The strains were prepared into a bacterial suspension with a concentration of 20-200 CFU / mL, and 1 ml of bacterial suspension was inoculated on the mold and yeast test plate, Nissui CompactDry rapid mold and yeast test dish, 3M rapid mold and yeast test piece, and inoculated on the national standard plate (Bengal Red Plate) at the same time. The mold and yeast test plate was cultured in a 28℃ incubator for 3 days, the national standard plate was cultured in a 28℃ incubator for 5 days, and the Nissui CompactDry rapid mold and yeast test dish and 3M rapid mold and yeast test piece were cultured according to their instructions.

[0043] The results of standard strain detection are shown in Table 3. The |Δlg| values ​​of the mold and yeast test plate, Nissui CompactDry rapid mold and yeast test dish, and 3M rapid mold and yeast test piece for detecting 4 strains of bacteria are all less than 0.5; the |Δlg| values ​​of the mold and yeast test plate are all less than those of the Nissui CompactDry rapid mold and yeast test dish and the 3M rapid mold and yeast test piece. In summary, the mold and yeast test plate provided by the present invention has high accuracy and strong sensitivity in detecting standard strains.

[0044] Table 3 Standard strain detection results

[0045] Note: 1. “lgCFU / ml” refers to the logarithmic value of the colony count of the mold and yeast test plate, Nissui CompactDry rapid mold and yeast test dish, 3M rapid mold and yeast test piece, and the national standard plate; 2. “|Δlg|” refers to the absolute value of the difference between the logarithmic value of the colony count of the mold and yeast test plate, Nissui CompactDry rapid mold and yeast test dish, 3M rapid mold and yeast test piece and the logarithmic value of the colony count of the national standard plate.

[0046] 2.2 Natural Sample Accuracy Experiment Take 1 ml of Sanyuan pure milk, Mengniu pure milk and Yili pure milk and add them to the test plate and the national standard plate in turn, and do two parallel tests; other sample tests are carried out in accordance with GB 4789.15-2016 "National Food Safety Standard Food Microbiology Examination Mold and Yeast Count".

[0047] As shown in Table 4, among the 15 natural samples, 3 samples did not detect mold and yeast, and the results were consistent with the national standard; 2 samples did not detect yeast, and the results were consistent with the national standard; the |Δlg| values ​​of the other samples were all less than 0.5, and the CVs were all less than 10%, indicating that the test results were highly consistent with the national standard plate. In summary, the mold and yeast test plate provided by the present invention has high detection accuracy for natural samples, and the test results are highly consistent with the national standard plate.

[0048] Table 4. Test results of natural samples

[0049] Note: “-” means the colony count is less than 1; “ / ” means no calculation was performed.

[0050] 2.3 Artificially contaminated sample accuracy experiment The three samples of high-protein skim high-calcium milk powder, Meijia Landou cheese sticks and Master Kong instant noodles in Table 4 were used to prepare artificial contaminated samples, and the strains selected were Saccharomyces cerevisiae ATCC 9763 and Aspergillus niger ATCC 16404.

[0051] Preparation of artificial contaminated samples: (1) Take freshly cultured bacterial strains and prepare 10 3 CFU / mL, 10 4 CFU / mL and 10 5 CFU / mL working bacterial suspension. The bacterial suspension was counted according to GB 4789.2 "National Food Safety Standard for Food Microbiology Examination and Determination of Total Colony Count". The concentrations of Saccharomyces cerevisiae ATCC 9763 bacterial suspension were 5.5×10 3 CFU / mL, 6.1×10 4 CFU / mL and 6.8×10 5CFU / mL, and the concentration of Aspergillus niger ATCC 16404 suspension was 3.5×10 3 CFU / mL, 4.2×10 4 CFU / mL and 3.9×10 5 CFU / mL; (2) Take 1 ml of bacterial suspension and make freeze-dried bacterial powder according to relevant requirements; (3) Take a concentration of 5.5×10 3 CFU / mL of Saccharomyces cerevisiae and 3.5×10 3 CFU / mL of Aspergillus niger freeze-dried powder were added to the three samples to prepare low-concentration artificial contamination samples, and the concentration was 6.1×10 4 CFU / mL of Saccharomyces cerevisiae and 4.2×10 4 CFU / mL of Aspergillus niger freeze-dried powder were added to the three samples to prepare medium-concentration artificial contamination samples, and the concentration was 6.8×10 5 CFU / mL of Saccharomyces cerevisiae and 3.9×10 5 CFU / mL of Aspergillus niger freeze-dried powder was added to three samples to prepare high-concentration artificial contamination samples; (4) The artificial contamination samples of high-protein skim high-calcium milk powder and Master Kong instant noodles were stored at 20-25℃ for 2 weeks, and the artificial contamination samples of Meijia Landou cheese sticks were stored at 2-8℃ for 2 weeks.

[0052] The artificially contaminated samples were diluted 10 times, 100 times, and 1000 times. 1 mL of the sample dilution was taken and inoculated into the mold and yeast test plate and the national standard plate.

[0053] The results are shown in Table 5. The |ΔLg| values ​​of the mold and yeast test plates provided by the present invention are all less than 0.5, and the CVs are all less than 10%, indicating that the test results are highly consistent with those of the national standard plates. In summary, the mold and yeast test plates provided by the present invention have high detection accuracy for natural samples, and the test results are highly consistent with those of the national standard plates.

[0054] Table 5 Detection results of artificially contaminated samples

[0055] In summary, the test plate for detecting molds and yeasts provided by the present invention has a high detection rate for strains in natural samples and artificially contaminated samples, and has the characteristics of high sensitivity. Compared with other test dishes on the market, it has the advantages of strong specificity, easy observation and counting, high accuracy of detection results, and low cost. The test plate for molds and yeasts of the present invention overcomes the existing technical barriers and has obvious advantages.

[0056] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A chromogenic culture medium for detecting molds and yeasts, characterized in that: The invention comprises a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is chloramphenicol, ofloxacin, ceftazidime and bile salt No. 3, and the composite chromogenic enzyme substrate is 5-bromo-4-chloro-3-indole-phosphoric acid toluidine salt, 5-bromo-4-chloro-3-indole-acetic acid ester and 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-aminoglucosidoside.

2. A chromogenic culture medium for detecting molds and yeasts according to claim 1, characterized in that: The concentrations of the components of the composite inhibitor are 0.01-0.1 g / L of chloramphenicol, 0.001-0.01 g / L ofloxacin, 0.001-0.01 g / L of ceftazidime, and 0.1-1 g / L of No. 3 bile salt.

3. A chromogenic culture medium for detecting molds and yeasts according to claim 1, characterized in that: The concentration of the composite color-developing enzyme substrate is 0.05-0.5 g / L.

4. A chromogenic culture medium for detecting molds and yeasts according to claim 1, characterized in that: Each 1000mL of the nutrient mixture contains 5-30g of peptone, 10-50g of yeast extract powder, 1-10g of glucose, 1-10g of sodium chloride, 2+ 0.005-0.05g, Mn 2+ 0.01-0.1g, Cu 2+ 0.01-0.05g, Zn 2+ 0.01-0.1g, Mg 2+ 0.01-0.1g, linoleic acid 0.001-0.05g, egg yolk lecithin 0.001-0.05g.

5. A chromogenic culture medium for detecting molds and yeasts as claimed in claim 4, characterized in that: The nutrient component mixture further comprises cold water gel, and the cold water gel is xanthan gum, guar gum, sodium carboxymethyl cellulose, sodium hydroxypropyl cellulose, and gellan gum.

6. A chromogenic culture medium for detecting molds and yeasts according to claim 5, characterized in that: The concentrations of the cold water gelatinizable components are 10-30 g / L xanthan gum, 10-30 g / L guar gum, 1-5 g / L sodium carboxymethyl cellulose, 1-10 g / L sodium hydroxypropyl cellulose and 1-10 g / L gellan gum.

7. A test plate comprising a substrate, and a color development medium for detecting molds and yeasts according to any one of claims 1 to 6 disposed on a surface of the substrate.

8. The test board according to claim 7, characterized in that: The substrate comprises a transparent covering film, a culture area and a lower circular groove plate from top to bottom, wherein the culture area is arranged in the lower circular groove plate, and the transparent covering film covers the lower circular groove plate.

9. The test board as claimed in claim 8, characterized in that The culture area is filled with a culture medium for molds and yeasts, wherein the culture medium comprises a color developing culture medium for detecting molds and yeasts as described in any one of claims 1-6.

10. The test board according to claim 8, characterized in that The material of the transparent coating is PE or PET, and the material of the bottom groove plate is one of polymethyl methacrylate, polycarbonate, PET and polystyrene.

11. Use of the color development medium for detecting molds and yeasts and the test plate thereof according to any one of claims 1 to 10 in detecting the content of molds and yeasts.

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