Method for rapidly and quantitatively detecting viable count of fungi in wheat product and application of method

Abstract

The invention relates to a method for rapidly and quantitatively detecting the viable count of fungi in a wheat product and application of the method, the method comprises the following steps: mixing wheat with sterile water, filtering and taking supernate to obtain primary spore suspension; mixing the primary spore suspension with nisin to obtain a secondary spore suspension; mixing the secondary spore suspension with propionium bromide azide, incubating in a dark place, exposing under illumination, centrifuging, taking precipitate, extracting DNA (deoxyribonucleic acid), performing real-time fluorescent quantitative PCR (polymerase chain reaction) detection, and calculating the viable count of fungi according to a standard curve. Compared with a traditional culture counting method, the method for rapidly and quantitatively detecting the viable count of the fungi in the wheat product has the advantages that the time is short, and the detection result has no significant order of magnitude difference.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and relates to a method for rapid quantitative detection of the number of live fungi in wheat products and an application thereof. Background Art

[0002] Wheat is the second largest food crop in my country after rice. During the process of wheat being processed into wheat flour, about 75% of the wheat is converted into wheat flour, and the remaining 25% appears as by-products, namely 20% wheat bran and 5% waste flour. Wheat grains contain starch, protein, fat and relatively rich trace nutrients, and are a natural culture medium for microorganisms. After wheat is crushed and processed through multiple steps, its cortex will come into contact with the endosperm, so that the microorganisms in the wheat cortex may be transferred to various wheat products. Studies have shown that the microorganisms that contaminate wheat flour in my country mainly include Bacillus, coliforms, molds and yeasts, among which the typical value of the total number of microbial colonies reaches 10 3 -10 7 cfu / g, the typical value of total spore count is 10 2 -10 7 cfu / g, typical value for mold and yeast is 10 2 -10 6 cfu / g, the typical value of coliform bacteria is 10-10 2 cfu / g.

[0003] Fungi (mostly molds) themselves and their metabolites pollute the raw materials, processing and finished products of wheat flour. Under storage conditions with high humidity and temperature, microorganisms present in wheat products will multiply in large numbers, causing mildew, agglomeration and production of toxins, which will seriously affect the hygienic quality of wheat products, causing economic losses and even endangering human health. At present, the counting of stored grain fungi does not distinguish between dead and live bacteria. The traditional method of counting the number of live molds and yeasts is time-consuming and can generally only detect dominant bacteria with a content of more than 1%, and the culture medium is selective for colonies.

[0004] Fungi that survive in wheat products can cause potential harm. Therefore, distinguishing between dead and live fungi and quantitatively detecting the number of live fungi is an important and critical technical issue for the contamination of wheat products, evaluating product quality, and avoiding food safety risks. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for rapid and quantitative detection of the number of viable fungi in wheat products and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for rapid and quantitative detection of the number of viable fungi in wheat products, the method comprising:

[0008] (1) Mixing wheat with sterile water, filtering and collecting the supernatant to obtain a primary spore suspension;

[0009] (2) mixing the primary spore suspension with nisin to obtain a secondary spore suspension;

[0010] (3) The secondary spore suspension was mixed with propidium azidobromide, incubated in the dark and then exposed to light. After centrifugation, the precipitate was collected and DNA was extracted for real-time fluorescence quantitative PCR detection. The number of viable fungi was calculated according to the standard curve.

[0011] The invention provides a method for rapidly and quantitatively detecting the number of live fungi in wheat products. Compared with the traditional culture counting method, the method takes less time and has no significant difference in the detection results.

[0012] Preferably, in step (1), the ratio of wheat to sterile water is 1 g: (10-100) mL.

[0013] The specific point values ​​in (10-100) can be selected as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. Other specific point values ​​within the above numerical range can also be selected, and they will not be described one by one here.

[0014] Preferably, in step (2), the mass ratio of nisin to the primary spore suspension is (0.01-0.1) g:1 kg, wherein the specific point value in (0.01-0.1) can be selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0015] Preferably, the potency of nisin is 900-1000 IU / mg, for example 900 IU / mg, 910 IU / mg, 920 IU / mg, 930 IU / mg, 940 IU / mg, 950 IU / mg, 960 IU / mg, 970 IU / mg, 980 IU / mg, 990 IU / mg, 1000 IU / mg, etc. Other specific points within the above numerical range can be selected, which will not be described one by one here.

[0016] Preferably, the mixing temperature in step (2) is 35-40° C. and the mixing time is 1-4 h.

[0017] The temperature can be selected as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc., and the time can be selected as 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 3.8h, 4h, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0018] Preferably, before the secondary spore suspension is mixed with propidium azidobromide, the concentration of spores in the secondary spore suspension is adjusted to 1×10 3 -1×10 6 / mL, for example 1×10 3 / mL, 2×10 3 / mL, 5×10 3 / mL, 8×10 3 / mL, 1×10 4 / mL, 2×10 4 / mL, 5×10 4 / mL, 8×10 4 / mL, 1×10 5 / mL, 2×10 5 / mL, 5×10 5 / mL, 8×10 5 / mL, 1×10 6 Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0019] Preferably, the final concentration of propidium azidobromide in step (3) is 5-50 μg / mL, for example 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, etc. Other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0020] Preferably, the light-proof incubation time is 10-30 min, for example 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0021] Preferably, the exposure time is 5-10 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0022] The method for establishing the standard curve comprises: preparing a fungal spore suspension of known concentration, performing gradient dilution using sterile water, performing fluorescent quantitative PCR, and establishing a standard curve with the number of live fungal spores as the horizontal axis and the threshold cycle number Ct value as the vertical axis.

[0023] Preferably, the fungi in the fungal spore suspension of known concentration include Aspergillus flavus.

[0024] Preferably, the nucleotide sequences of the primers for real-time fluorescence quantitative PCR detection include the sequences shown in SEQ ID NO:1 and SEQ ID NO:2.

[0025] SEQ ID NO: 1:TCCGTAGGTGAACCTGCGG.

[0026] SEQ ID NO:2:TCCTCCGCTTATTGATATGC.

[0027] Among them, SEQ ID NO: 1 is the upstream universal primer of the ITS gene, and SEQ ID NO: 2 is the downstream universal primer of the ITS gene.

[0028] In a second aspect, the present invention provides a method for rapid and quantitative detection of the number of viable fungi in wheat products and its application in fungus detection in wheat products.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a method for rapid quantitative detection of the number of live fungi in wheat products. Compared with the traditional culture counting method, it takes less time and has no significant difference in the detection results (P < 0.05). The detection method provided by the present invention is of great significance for the rapid and accurate quantification of live fungi in wheat products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a detection flow chart.

[0032] Figure 2 It is the standard curve of Ct value and viable count. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0034] Example 1

[0035] Establish a linear relationship between Ct value and viable count

[0036] Bacteria: Aspergillus flavus, purchased from CGMCC, with the deposit number of CGMCC 3.4410.

[0037] Experimental methods:

[0038] (1) The spores were made into a suspension and the viable bacteria were counted using red Bengal medium according to the national standard "National Food Safety Standard Food Microbiology Examination Mold and Yeast Count" (GB 4789.15-2016); then the suspension was prepared with sterile water to a viable count of 6.5×10 1 CFU / mL, 6.5×10 2 CFU / mL, 6.5×10 3 CFU / mL, 6.5×10 4 CFU / mL, 6.5×10 5 CFU / mL, 6.5×10 6 CFU / mL, 6.5×10 7 CFU / mL of bacterial solution.

[0039] (2) DNA extraction: Add 500 μL DNA extraction solution to a centrifuge tube to form a suspension, add 5 μL RNase, incubate at 37°C in a water bath for 30 min, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix, centrifuge at 12,000 g for 10 min, take the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1), mix, and centrifuge at 12,000 g for 10 min. Take the supernatant, add 2.5 volumes of anhydrous ethanol, mix by inversion, and keep at -20°C for 1 h. Then centrifuge at 10,000 g for 10 min, collect the precipitate, wash with 600 μL of 75% ethanol, and centrifuge at 10,000 g again for 5 min. Dry naturally for 5 min. Dissolve the DNA in 200 μL ultrapure water and store at -20°C for later use.

[0040] DNA extraction solution: 0.2 mol / L Tris-HCl (pH 7.5), 0.5 mol / L NaCl, 10 mmoL / L EDTA, 1% SDS and water.

[0041] (3) qPCR reaction method:

[0042] Reaction system: 10 μL of 1×SYBR Green, 2 μL of DNA sample, 1 μL of upstream and downstream primers, and ddH2O supplemented to a total volume of 20 μL. The sequences of the primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0043] Reaction conditions: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 5 s, annealing and extension at 57°C for 40 s, 40 cycles, collection of fluorescence signals, plotting of melting curves, and obtaining Ct values.

[0044] (4) Experimental results:

[0045] The results are shown in Table 1. The results in Table 1 are plotted into a standard curve. Figure 2 As shown, the horizontal axis is the number of viable bacteria, the vertical axis is the Ct value, and the R 2 =0.9988, good linear relationship.

[0046] Table 1

[0047]

[0048]

[0049] Example 2

[0050] Precision of Quantification of Viable Fungi in Wheat Products (Bran)

[0051] Experimental sample: bran

[0052] Experimental methods:

[0053] (1) Take 25.0 g of sample and place it in a Erlenmeyer flask containing 225 mL of sterile water, vortex thoroughly for 10 min, filter through four layers of gauze, and transfer the liquid to another empty Erlenmeyer flask to prepare a primary spore suspension; add Nisin (nisin) to a final concentration of 0.08 g / kg (sample), place at 37°C for 3 h, and prepare a secondary spore suspension; use a hemocytometer to roughly count the number of spores, centrifuge at 10,000 g for 2 min, and dilute with sterile water to dilute the spore concentration and control it at 10 4 Pieces / mL-10 5 / mL, and three spore suspensions were prepared.

[0054] (2) Take 1 mL of the above three spore suspensions, add PMA (propidium azide bromide) to a final concentration of 25 μg / mL, mix thoroughly, incubate in the dark for 30 min, then expose to light for 15 min, centrifuge at 10,000 g for 2 min, wash three times, discard the supernatant, and store at -20°C.

[0055] (3) The DNA extraction method and qPCR reaction method were the same as in Example 1.

[0056] (4) Experimental results:

[0057] The results are shown in Table 2.

[0058] Table 2

[0059]

[0060]

[0061] As can be seen from Table 2, the method described in the present application can quickly and effectively obtain the content of the number of viable bacteria in the bran, with no order of magnitude difference for the same sample and high precision.

[0062] Example 3

[0063] Comparison of the Quantitative Quantification of Viable Fungi in Wheat Products (Wheat Flour) and the Traditional Culture Method

[0064] Experimental sample: powder

[0065] Experimental methods for this application:

[0066] (1) Take 25.0 g of sample and place it in a Erlenmeyer flask containing 225 mL of sterile water. Vortex thoroughly for 10 min, filter through four layers of gauze, and transfer the liquid to another empty Erlenmeyer flask to prepare a primary spore suspension.

[0067] (2) Nisin was added to the primary spore suspension to a final concentration of 0.10 g / kg (sample) and placed at 37°C for 4 h to prepare a secondary spore suspension. The number of spores was roughly calculated using a hemocytometer, and the suspension was centrifuged at 10,000 g for 2 min. The suspension was diluted with sterile water to dilute or concentrate the spore concentration and control it at about 10 5 Pieces / mL-10 6 / mL, make three spore suspensions

[0068] (3) Take 1 mL of the three spore suspensions, add PMA to a final concentration of 30 μg / mL, mix thoroughly, incubate in the dark for 20 min, expose to light for 15 min, centrifuge at 10,000 g for 2 min, wash three times, discard the supernatant, and store at -20°C for use in extracting fungal DNA.

[0069] The DNA extraction method and qPCR reaction method were the same as in Example 1.

[0070] Traditional cultivation method:

[0071] Take 25.0 g of sample and place it in a conical flask containing 225 mL of sterile water. Vortex it thoroughly for 10 min, filter it through four layers of gauze, and transfer the liquid to another empty conical flask to make a primary spore suspension. The primary spore suspension is completed in accordance with the national standard "National Food Safety Standard Food Microbiological Examination Mold and Yeast Count" (GB 4789.15-2016).

[0072] Table 3

[0073]

[0074] The data of the culture method and the method provided by this patent were compared by T-test, indicating that there was no significant difference in the number of live fungi in wheat products (secondary flour) detected by the two methods (P < 0.05).

[0075] Example 4

[0076] Quantification of live fungi in wheat products (wheat flour) and the necessity of adding Nisin and PMA

[0077] Experimental sample: wheat flour

[0078] (1) Take 25.0 g of sample and place it in a Erlenmeyer flask containing 225 mL of sterile water. Vortex thoroughly for 10 min and filter through four layers of gauze to prepare a primary spore suspension. Transfer an equal amount of the suspension to the other five empty Erlenmeyer flasks, which are recorded as Group 1, Group 2, Group 3, Group 4 and the control group.

[0079] (2) Nisin was added to groups 1 and 3 at a concentration of 0.05 g / kg, while groups 2 and 4 were left untreated to prepare a secondary spore suspension;

[0080] (3) Count the number of spores using a hemocytometer, centrifuge at 10,000 g for 2 min, and dilute with sterile water to dilute or concentrate the spores to about 10. 5 Pieces / mL-10 6 / mL, and three spore suspensions were prepared;

[0081] (4) 1 mL of spore suspension was taken three times from each of the four groups. PMA was added to groups 1 and 2 to a final concentration of 50 μg / mL. Groups 3 and 4 were not treated with PMA. The cells were incubated in the dark for 20 min, exposed to light for 10 min, centrifuged at 10,000 g for 2 min, washed three times, and the supernatant was discarded. The cells were stored at -20°C to prepare samples for fungal DNA extraction.

[0082] The DNA extraction method and qPCR reaction method were the same as in Example 1.

[0083] Groups 1-4 were tested using the method of the present application, with the only difference being that Group 1 was treated with both Nisin and PMA, Group 2 was treated with only PMA without Nison, Group 3 was treated with only Nison without PMA, and Group 4 was treated neither with Nison nor with PMA.

[0084] The control method is to use the traditional culture method for testing, and the counting is completed in accordance with the national standard "National Food Safety Standard Food Microbiology Examination Mold and Yeast Count" (GB 4789.15-2016).

[0085] The results are shown in Table 4.

[0086] Table 4

[0087] Grouping Ct value CFU / g 1 group 4.32 2.09E+04 2 groups 4.49 3.09E+04 3 groups 4.40 2.51E+04 4 groups 5.11 1.29E+05 Control group 4.31 2.04E+04

[0088] This patent provides a method for determining the number of viable bacteria, which is closer to the culture method for determining the number of viable bacteria than the group without Nison, PMA, or the group without both.

[0089] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate a method for rapid quantitative detection of the number of viable fungi in wheat products and its application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0090] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0091] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for rapid quantitative detection of the number of viable fungi in wheat products, characterized in that: The method comprises: (1) Mixing wheat with sterile water, filtering and collecting the supernatant to obtain a primary spore suspension; (2) mixing the primary spore suspension with nisin to obtain a secondary spore suspension; (3) The secondary spore suspension was mixed with propidium azidobromide, incubated in the dark and then exposed to light. After centrifugation, the precipitate was collected and DNA was extracted for real-time fluorescence quantitative PCR detection. The number of viable fungi was calculated according to the standard curve.

2. The method for rapid quantitative detection of the number of viable fungi in wheat products according to claim 1, characterized in that: The ratio of wheat to sterile water in step (1) is 1 g: (10-100) mL.

3. The method for rapid quantitative detection of the number of viable fungi in wheat products according to claim 1 or 2, characterized in that: In step (2), the mass ratio of nisin to the primary spore suspension is (0.01-0.1) g:1 kg; Preferably, the potency of nisin is 900-1000 IU / mg.

4. The method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 3, characterized in that: The mixing temperature in step (2) is 35-40° C. and the mixing time is 1-4 h; Preferably, before the secondary spore suspension is mixed with propidium azidobromide, the concentration of spores in the secondary spore suspension is adjusted to 1×10 3 -1×10 6 Pieces / mL.

5. The method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 4, characterized in that: The final concentration of propidium bromide azide in step (3) is 5-50 μg / mL.

6. The method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 5, characterized in that: The incubation time in the dark is 10-30 minutes.

7. The method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 6, characterized in that: The exposure time is 5-10 minutes.

8. The method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 7, characterized in that: The method for establishing a standard curve comprises: preparing a fungal spore suspension of known concentration, performing gradient dilution using sterile water, performing fluorescent quantitative PCR, and establishing a standard curve with the number of live fungal spores as the horizontal axis and the threshold cycle number Ct value as the vertical axis; Preferably, the fungi in the fungal spore suspension of known concentration include Aspergillus flavus.

9. The method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 8, characterized in that: The nucleotide sequences of the primers for real-time fluorescence quantitative PCR detection include the sequences shown in SEQ ID NO:1 and SEQ ID NO:

2.

10. Use of the method for rapid quantitative detection of the number of viable fungi in wheat products according to any one of claims 1 to 9 in fungal detection in wheat products.

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