Research, preparation method and application of a bifidobacterium colony counting test piece

Abstract

This invention relates to the research, preparation method, and application of a Bifidobacterium colony counting test strip in the detection of Bifidobacterium counts in dairy products, belonging to the field of food testing technology. The test strip consists of, from top to bottom, a transparent plastic upper film with an adhesive lower surface, a plastic middle layer with perforated areas, and a base plate printed with squares. All materials are heat-resistant within a temperature range of 130℃ to 150℃. The adhesive lower surface of the transparent plastic upper film is coated with a compound cold-water soluble gel. The perforated areas are filled with a Bifidobacterium colony counting colorimetric culture medium and the compound cold-water soluble gel powder prepared in a specific ratio. This invention uses this test strip to determine the Bifidobacterium count in dairy products, providing a convenient and rapid detection procedure. The colorimetric characteristics of the Bifidobacterium counting test strip were systematically studied under optimal cold-water soluble gel formulation conditions. The addition of isopropyl-β-D-thiogalactoside to the culture medium components improves the clarity of colony color development and accelerates the color development time, thereby improving the accuracy of colony counting on the test strip.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a bifidobacterium counting test piece, a preparation method and an application thereof in detecting bifidobacterium counting in dairy products. Background Art

[0002] Bifidobacterium (Bifidobacterium ) is a Gram-positive bacterium and an important beneficial intestinal microorganism. In recent years, the efficacy and prospects of bifidobacteria in food, health care and medical treatment have been valued by more people, and bifidobacteria have also been widely used in various dairy products. Bifidobacteria are an important indicator for evaluating the quality and nutritional health of dairy products. At present, the count of bifidobacteria in milk in my country is determined in accordance with GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test". The national standard plate count method has high accuracy, but the traditional detection method mainly relies on microbial enrichment culture, selective separation, and biochemical identification. There are shortcomings such as complicated operating steps and long detection cycle, which are not suitable for the requirements of convenient detection at the grassroots level. In view of the detection needs of today's rapidly developing modern dairy industry, it is particularly important to develop convenient, fast, standardized, and highly sensitive detection technology for bifidobacteria, one of the lactic acid bacteria.

[0003] In recent years, with the deepening of research on probiotics, the detection of bifidobacteria has increased both at home and abroad, including flow cytometry (FCM), fluorescence quantitative PCR, PMA / EMA-QPCR, etc. However, most of the detection methods for bifidobacteria have complicated operation steps, expensive detection costs, and high requirements for technical personnel, which limits their scope of application. The microbial test piece method is currently a very ideal detection method, with good colony growth and short culture cycle. Several domestic microbiology companies have colony counting test pieces with cold water soluble gel as carrier. Although these test pieces are easy to operate, there are still technical problems that affect the growth and counting of colonies. At present, there are still no test pieces for bifidobacterium counting in China. Inventing a simple, fast and accurate test piece for bifidobacterium colony counting has very important application value.

[0004] In view of the above-mentioned problems, the present invention systematically studies the color development characteristics of the bifidobacterium counting test piece, optimizes the color developer of the bifidobacterium test piece culture medium according to the optimal cold water soluble gel ratio that provides a suitable growth environment for culturing bifidobacteria, determines the composition of the color development system according to the colony growth status and the color development effect, and the colony color development is clear and convenient for identification and counting. According to the requirements of the national standard bifidobacterium counting GB 4789.34-2016, a bifidobacterium colony counting test piece is prepared, and the total number of bifidobacteria can be counted. Through the study of the color development characteristics, the present invention develops a bifidobacterium colony counting test piece product that is easy to operate and accurately count. Summary of the invention

[0005] One of the purposes of the present invention is to provide a bifidobacterium counting test piece with simple operation, low price and accurate detection. The test piece is composed of a transparent plastic upper layer film with a sticky lower surface (such as a PET silicone film with high transparency, non-toxic and non-irritating odor, waterproof and breathable, with a thickness of 0.05mm-0.10mm, and the lower surface of the PET silicone film itself is sticky), a plastic middle layer plate with a hollow area (such as a polypropylene (PP) plastic plate or a PET plastic plate with good hardness, with a thickness of 0.15mm-0.30mm), a bottom plate printed with squares (such as waterproof and impermeable white coated paper, with a thickness of 0 .10mm~0.20mm; the size of the square is 1cm×1cm); the temperature range of the above materials being resistant to high temperatures is 130℃~150℃; the sticky lower surface of the transparent plastic upper film is coated with a compound cold water (temperature range is 20℃~45℃) soluble gel, the hollow area of ​​the plastic middle layer plate is a circular, oval or square structure, the ratio of the hollow area area to the plastic middle layer plate area is in the range of 1:1.6~2.7, the hollow area is filled with a bifidobacterium colony counting chromogenic medium and a compound cold water soluble gel powder prepared in a certain ratio, and the compound cold water soluble gel powder and the bifidobacterium colony counting chromogenic medium are prepared in a mass ratio of 3:7~9.

[0006] The cold water soluble gel component is prepared by mixing guar gum, gellan gum, sodium polyacrylate and carrageenan which are ground and passed through a standard sieve of 100-150 meshes, and preparing the mixture in a mass ratio of guar gum: gellan gum: sodium polyacrylate: carrageenan of 5:1-2:1-2:1-2.

[0007] The weight components of the bifidobacterium colony counting chromogenic medium are: peptone 8~12 g, beef powder 4~6 g, yeast powder 3~5 g, glucose 18~22 g, Tween 80 0.5~2 mL, dipotassium hydrogen phosphate 1.5~2.5 g, diammonium hydrogen citrate 1.5~2.5 g, sodium acetate 4~6 g, magnesium sulfate 0.1~0.4 g, manganese sulfate 0.01~0.1 g, 5-bromo-4-chloro-3-indole-β-D-galactoside 0.01~0.1 g, inducer isopropyl-β-D-thiogalactoside 0.1~1 g, antibacterial agent mupirocin lithium salt 0.01~0.1 g and distilled water 1000 mL.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned Bifidobacterium colony count test piece, the steps of which are as follows:

[0009] (1) Preparation of culture medium for colorimetric detection of bifidobacterium colony count

[0010] Weigh 8-12 g of peptone, 4-6 g of beef powder, 3-5 g of yeast powder, 18-22 g of glucose, 0.5-2 mL of Tween 80, 1.5-2.5 g of dipotassium hydrogen phosphate, 1.5-2.5 g of diammonium hydrogen citrate, 4-6 g of sodium acetate, 0.1-0.4 g of magnesium sulfate, and 0.01-0.1 g of manganese sulfate, and filter 0.01-0.1 g of 5-bromo-4-chloro-3-indole-β-D-galactoside, 0.1-1 g of inducer isopropyl-β-D-thiogalactoside, and 0.01-0.1 g of antibacterial agent mupirocin lithium salt through a needle filter with a diameter of 25 mm and a pore size of 0.22 μm, and mix them to obtain a colorimetric medium powder for counting bifidobacterium colonies;

[0011] (2) Preparation of composite cold water soluble gel

[0012] The guar gum, gellan gum, sodium polyacrylate and carrageenan that have been ground and passed through a 100-150 mesh standard sieve are mixed to prepare a mixture with a mass ratio of guar gum: gellan gum: sodium polyacrylate: carrageenan of 5:1-2:1-2:1-2;

[0013] (3) Preparation of Bifidobacterium colony count test piece

[0014] The transparent plastic upper film, the plastic middle plate with a hollow area and the bottom plate printed with squares are cut into the same size, with a length of 9 to 12 cm and a width of 8 to 10 cm, and polyacrylic resin pressure-sensitive adhesive (transparent, non-toxic, odorless, and sterile) is used to seal and bond the three on the side with the label, with a bonding width of 4 to 6 mm;

[0015] The above-mentioned composite cold water soluble gel powder is evenly coated on the sticky lower surface of the transparent plastic upper film with a thickness of 0.10 to 0.20 mm;

[0016] The exposed bottom plate of the hollowed-out area of ​​the plastic middle layer plate is evenly coated with a polyacrylic resin pressure-sensitive adhesive with a thickness of 0.05 to 0.10 mm, and a compound cold water-soluble gel powder and a bifidobacterium colony counting colorimetric medium powder are prepared in a mass ratio of 3:7 to 9, and the obtained mixed powder is evenly applied to the hollowed-out area of ​​the plastic middle layer plate and compacted;

[0017] Finally, vacuum packaging is performed and sterilization is performed using the ethylene oxide method, thereby obtaining the bifidobacterium colony counting colorimetric test piece of the present invention.

[0018] (4) Operation steps of the test strip method for determining the number of bifidobacteria

[0019] Use a sterile pipette to draw 25 mL of sample (the sample should be dairy product) and place it in a container or sterile homogenizing bag containing 225 mL of sterile diluent (distilled water, saline or phosphate buffer). Shake it thoroughly or beat it with a slapping homogenizer for 1 to 2 minutes to make a 1:10 diluted solution.

[0020] Take 1 mL of the above 1:10 dilution solution and add it to a test tube containing 9 mL of sterile diluent, mix well on a vortex mixer to obtain a 1:100 dilution solution; prepare 10-fold gradient dilution solutions in sequence and set aside.

[0021] According to the estimation of lactic acid bacteria content in dairy samples, select 2 to 3 dilutions with appropriate dilutions, open the transparent plastic upper film of the bifidobacterium colony counting test piece, take 1 mL of the diluted homogenate of the above sample and add it to the area with bifidobacterium colony counting chromogenic medium in the middle layer, so that the sample diluted homogenate is evenly distributed in the entire culture area; slowly drop the plastic upper film to avoid squeezing, let it stand for 30 s to 1 min to allow the compound cold water soluble gel and water to solidify, put it in a constant temperature incubator at 36±1℃ for 48~72 h, and count according to GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test". At the same time, take 1 mL of physiological saline as a blank control.

[0022] The present invention develops a bifidobacterium colony counting test piece, and the counting color development medium is composed of a composite cold water soluble gel (prepared with a mass ratio of guar gum: gellan gum: sodium polyacrylate: carrageenan of 5:1~2:1~2:1~2), a basic culture medium (peptone, beef powder, yeast powder, glucose, Tween 80, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate, manganese sulfate), a specific color development substrate 5-bromo-4-chloro-3-indole-β-D-galactoside, an inducer isopropyl-β-D-thiogalactoside and an antibacterial agent mupirocin lithium salt. Compared with similar products, the bifidobacterium colony counting test piece prepared by the present invention has good colony growth, clear color development, high sensitivity, strong specificity, convenient operation and accurate counting. It mainly includes two important technical features.

[0023] Important technical feature 1: The present invention uses 5-bromo-4-chloro-3-indole-β-D-galactoside as a chromogenic substrate, adds an inducer isopropyl-β-D-thiogalactoside, and designs a bifidobacterium enzyme-specific color development system, so that the colony color of the test piece is clear. Bifidobacteria can produce β-galactosidase during growth, which decomposes the substrate 5-bromo-4-chloro-3-indole-β-D-galactoside to release indole, thereby causing the colony to develop color. On this basis, the inducer isopropyl-β-D-thiogalactoside is added, and isopropyl-β-D-thiogalactoside can induce the synthesis of β-galactosidase. The color development is good after 48 hours of cultivation, which effectively increases the number of colonies and the color development effect of bifidobacterium growth, making it easier to observe and count.

[0024] Important technical feature 2: The present invention is the first to develop a colony counting test piece for detecting bifidobacteria in dairy products. This test piece can accurately and quickly quantitatively detect the number of bifidobacterium colonies in dairy products. The procedure is simple, the colony color is clear, the counting is accurate, the operation is simple, and the cost is low.

[0025] The present invention systematically studies the color development characteristics of a bifidobacterium colony counting test piece, and according to the optimal cold water-soluble gel ratio, colony growth conditions and color development effect, an inducer isopropyl-β-D-thiogalactoside and an antibacterial agent mupirocin lithium salt are added to establish a color development system suitable for a bifidobacterium test piece, so that the colony color of the test piece is clear and the colony count is accurate.

[0026] According to the requirement of GB 4789.34-2016 national standard bifidobacterium counting, prepare bifidobacterium colony counting test piece, can count the total number of bifidobacteria. The present invention counts and tests bifidobacteria in milk, and colony color is clear, easy to operate, and counting is accurate, with an accuracy rate of 99%, and the minimum detection limit reaches 3 CFU / mL. The specific test result of the test piece is that bifidobacterium grows well and is blue, while other bacteria can not be detected. In the inspection of dairy product samples, there is no significant difference (P>0.05) between the bifidobacterium colony counting test piece method of the present invention and the national standard method detection result, which meets the national standard of my country's bifidobacterium inspection and can be widely used in testing institutions and food companies. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Schematic diagram of the structure of the Bifidobacterium colony counting test piece;

[0028] like Figure 1 As shown, the various parts are: label 1, upper transparent PET silicone film 2, transparent polypropylene plastic middle plate with a hollow area in the middle 3, culture area 4, white coated paper 5;

[0029] Figure 2: The color development effect of the Bifidobacterium colony counting test piece with different concentrations of color developer added;

[0030] Figure 3 : The color development effect of the Bifidobacterium colony counting test piece with different concentrations of inducers added;

[0031] Figure 4 : Linear correlation curve between the test results of the bifidobacterium colony test piece method of the present invention and the national standard method;

[0032] Figure 5 : Comparison of the detection pictures of the bifidobacterium colony test piece method of the present invention and the national standard method. DETAILED DESCRIPTION

[0033] Example 1 Preparation of Bifidobacterium Colony Count Determination Medium

[0034] The original weight components of the chromogenic medium for counting bifidobacterium colonies were as follows: peptone 10 g, beef powder 5 g, yeast powder 4 g, glucose 20 g, Tween 80 1 mL, dipotassium hydrogen phosphate 2 g, diammonium hydrogen citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, 5-bromo-4-chloro-3-indole-β-D-galactoside 0.06 g, inducer isopropyl-β-D-thiogalactoside 0.6 g, antibacterial agent mupirocin lithium salt 0.06 g and distilled water 1000 mL.

[0035] The preparation method of the bifidobacterium colony counting chromogenic medium is as follows: weigh 10 g of peptone, 5 g of beef powder, 4 g of yeast powder, 20 g of glucose, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, and 0.05 g of manganese sulfate; use a needle filter with a diameter of 25 mm and a pore size of 0.22 μm to filter 0.06 g of 5-bromo-4-chloro-3-indole-β-D-galactoside, 0.6 g of inducer isopropyl-β-D-thiogalactoside, and 0.06 g of antibacterial agent mupirocin lithium salt; mix well and then freeze-dry into powder; then mix well with other ingredients to obtain chromogenic medium powder for later use.

[0036] The preparation method of the composite cold water soluble gel is as follows: guar gum, gellan gum, sodium polyacrylate and carrageenan which have been ground and passed through a 120-mesh standard sieve are mixed, and the mass ratio of guar gum: gellan gum: sodium polyacrylate: carrageenan is 5:2:2:1, and the mixture is fully mixed and set aside.

[0037] Example 2 Preparation of Bifidobacterium Colony Count Test Plate

[0038] 1) The test piece consists of three layers, namely the upper PET silicone film, the middle polypropylene plastic plate with a hollow area, and the bottom layer printed with a white checkered copperplate paper with a light yellow 1cm×1cm checkered pattern. The three parts are cut into the same size, 9cm in length and 8cm in width, and the side with the label is sealed and bonded with a polyacrylic resin pressure-sensitive adhesive (transparent, non-toxic, odorless, and sterile), with a bonding width of 5mm.

[0039] 2) Spray the compound cold water soluble gel powder evenly on the inner side of the upper film of the test piece with a thickness of 0.15mm. The mass of cold water gel for each test piece is 0.2g. Spray the compound cold water soluble gel powder evenly on the inner side of the upper film of the test piece with a thickness of 0.15mm. The mass of cold water gel for each test piece is 0.2g.

[0040] 3) First, evenly apply 0.2 g of transparent polyacrylic resin pressure-sensitive adhesive on the bottom plate corresponding to the hollow area of ​​the middle layer, prepare the compound cold water-soluble gel powder and the bifidobacterium colony count colorimetric medium powder in a mass ratio of 3:7, and then evenly apply the mixed powder on the hollow area of ​​the plastic middle layer plate and compact it.

[0041] 4) Vacuum pack the test piece.

[0042] 5) Use ethylene oxide sterilization method to sterilize the test piece.

[0043] Example 3 Study on the color development system of the Bifidobacterium colony counting test piece

[0044] 1) The effect of the color developer 5-bromo-4-chloro-3-indole-β-D-galactoside on the color development of the test piece

[0045] Since bifidobacteria can produce β-galactosidase during their growth, the substrate 5-bromo-4-chloro-3-indole-β-D-galactoside is decomposed to release indole, producing a blue precipitate, which can be used to determine the presence of colonies. Figure 2 As shown in the figure, when the concentrations of the color developing substrate added to the sterilized modified MRS medium were 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, and 0.08 g / L, respectively, when the concentration of the color developing substrate reached 0.06 g / L, the colonies were blue, the coloring effect was obvious, and it was easy to count. Figure 2 After comprehensive consideration, the substrate 5-bromo-4-chloro-3-indole-β-D-galactoside with a concentration of 0.06 g / L was selected as the optimal concentration and added to the culture medium to prepare the test piece.

[0046] 2) Effect of the inducer isopropyl-β-D-thiogalactoside on the color development of the test piece

[0047] Isopropyl-β-D-thiogalactoside can induce the synthesis of enzymes, which is similar to natural β-galactosidase and cannot be decomposed by enzymes, increasing the number of colonies and color development effect of bifidobacterium growth, making it easier to observe and count. The optimal color developer 5-bromo-4-chloro-3-indole-β-D-galactoside was added to the sterilized modified MRS medium, and then 0.4 g / L, 0.6 g / L, and 0.8 g / L of the inducer isopropyl-β-D-thiogalactoside were added respectively. The color development effect of the colonies is shown in Table 1 and Figure 3 As shown. When the inducer concentration reaches 0.6 g / L, the growth of bifidobacteria is optimal, and the colony color is clear. As the dose increases, the colony growth is inhibited.

[0048]

[0049] Example 4 Method for determining the colony count of bifidobacteria in dairy products using a test piece

[0050] 1) Aseptically weigh 25 mL of the dairy sample to be tested and add it to a sterile homogenizing bag containing 225 mL of diluent. Beat with a homogenizer for 1 to 2 minutes to make a 1:10 sample homogenate. Make a 10-fold series gradient dilution of the above sample homogenate. According to the estimation of the lactic acid bacteria content in the dairy sample, select 2 to 3 dilutions, and make two parallel samples for each dilution.

[0051] 2) Take 1mL of the sample dilution to be tested, open the upper film of the test piece, add it to the culture medium area, and evenly distribute the sample liquid in the entire culture area. Slowly drop the upper film to avoid squeezing with fingers, let it stand for 30 seconds until the gel solidifies, and place it in an incubator at 28℃ for 48 hours. Make two parallel samples for each dilution, and take 1mL of physiological saline as a blank control.

[0052] 3) Place the test piece that has completed the proliferation culture on a sterile operating table, observe whether there are blue colonies, and count the blue colonies. The counting method is in accordance with GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test".

[0053] If the colony counts on all dilution test pieces are greater than 300 CFU, the test piece with the highest dilution shall be counted, and the other test pieces may be recorded as uncountable. The result is calculated by multiplying the average colony count by the highest dilution factor.

[0054] If the colony counts of the test pieces at all dilutions are less than 30 CFU, the calculation should be based on the average colony count of the lowest dilution multiplied by the dilution factor.

[0055] If no colonies grow on the test pieces at all dilutions (including the original liquid sample solution), the value is calculated as less than 1 multiplied by the lowest dilution factor.

[0056] If the colony counts of all dilution test pieces are not between 30 CFU and 300 CFU, and some of them are less than 30 CFU or greater than 300 CFU, the average colony count closest to 30 CFU or 300 CFU shall be multiplied by the dilution factor for calculation.

[0057] Example 5: Evaluation of the effect of using the Bifidobacterium colony count test piece

[0058] 1) Prepare a Bifidobacterium colony count test piece according to Example 1 and Example 2 and set aside.

[0059] 2) Determination of detection limit and sensitivity of test strips

[0060] The experimental strains were Bifidobacterium lactis HN019, Bifidobacterium longum CICC 24632, Bifidobacterium bifidum CICC10395, Bifidobacterium infantis CICC 6069, Bifidobacterium breve CICC 22948, Bifidobacterium adolescentis CICC 24573, Lactobacillus paracasei CICC 20241, Streptococcus thermophilus IFFI 6038, Lactobacillus acidophilus CICC 20244, and Lactococcus lactis CICC20090.

[0061] Take a single colony and inoculate it into 100 mL of national standard MRS liquid medium, and place it in a constant temperature incubator at 36±1℃ for 48 h. Take 25 mL of bacterial solution and add 225 mL of sterile saline to mix and make a 1:10 dilution. Repeat the above steps to dilute the bacterial solution to a dilution suitable for counting (concentration of 10 0 ~10 2 CFU / mL), take 1mL of bacterial solution and use the test piece to determine the number of bifidobacterium colonies according to the method in Example 4. At the same time, the bifidobacterium colony count was determined according to GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test", and two parallel samples were made for each dilution, and the test results were averaged.

[0062] The detection result of the lowest dilution is used as the lowest detection limit, and the lowest detection limit is used as the detection standard for the sensitivity of the test piece. The test result greater than the lowest detection limit is positive, and the result less than the lowest detection limit is negative. Sensitivity = positive / (positive + negative). The experimental results show that the two methods can achieve the same level of detection limit, with a detection limit of 3 CFU / mL and a sensitivity of 100%. At the same time, the colony count between 30 and 300 CFU / mL was analyzed, t=0.095, P=0.5>0.05. There is no significant difference between the detection results of the bifidobacterium colony count test piece developed by the present invention and the national standard plate method. See Table 2, Figure 4 and Figure 5 ;

[0063]

[0064] 2) Test piece specificity detection

[0065] A bacterial suspension of Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus paracasei, Streptococcus thermophilus, Lactobacillus acidophilus, Lactococcus lactis and other bacterial species was selected, and a 10-fold gradient dilution was performed with normal saline. 1 mL of the bacterial solution was taken and the colony count of 10 strains of bacteria was determined according to the method in Example 4 and GB 4789.34-2016 "National Food Safety Standard Food Microbiology Examination Bifidobacterium Examination".

[0066] The experimental results showed that the colonies of Bifidobacterium genus on the Bifidobacterium Count Test Plate grew well and showed blue color after 48 hours of culture, while other bacteria could not be detected. There was no significant difference between the counting results of the Bifidobacterium Count Test Plate and GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test" (P>0.05), and the counting results were accurate. The experimental results are shown in Table 3;

[0067]

[0068] 3) Test strips are used for testing dairy samples

[0069] Purchase actual samples of live lactic acid bacteria drinks of different brands from supermarkets and dilute the samples to a concentration of 10 0 ~10 2 CFU / mL for later use, take 1mL of bacterial solution and use the test piece to determine the number of bifidobacterium colonies according to the method in Example 4. At the same time, the bifidobacterium colony count was determined according to GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test", and the conformity of the test piece determination results with the national standard method determination results was compared;

[0070]

[0071] 120 dairy samples were tested using the bifidobacterium count test piece method and the national standard method. The sample test results are shown in Table 4. The data show that the highest compliance rate of the two methods can reach 98.8%, and there is no significant difference in the test results of the two methods (P>0.05). The results show that the bifidobacterium count test piece is suitable for the detection of the total number of bifidobacteria in dairy products.

Claims

1. A test piece for counting bifidobacterium colonies, characterized in that: The test piece is composed of a transparent plastic upper film with a sticky lower surface, a plastic middle plate with a hollow area, and a bottom plate printed with squares from top to bottom; the above materials are resistant to high temperatures in the range of 130°C to 150°C; the sticky lower surface of the transparent plastic upper film is coated with a compound cold water soluble gel, and the hollow area is filled with a bifidobacterium colony counting color development medium and a compound cold water soluble gel powder prepared in a certain proportion; and the preparation method of the bifidobacterium colony counting test piece is obtained by the following steps: (1) Preparation of chromogenic medium for counting bifidobacterium colonies Weigh 10 g of peptone, 5 g of beef powder, 4 g of yeast powder, 20 g of glucose, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, and 0.05 g of manganese sulfate, and use a needle filter with a diameter of 25 mm and a pore size of 0.22 μm to filter 0.06 g of 5-bromo-4-chloro-3-indole-β-D-galactoside, 0.6 g of inducer isopropyl-β-D-thiogalactoside, and 0.06 g of mupirocin lithium salt, an antibacterial agent, mix well, and then freeze-dry into powder, and then mix well with other ingredients to obtain a colorimetric medium powder for counting bifidobacterium colonies; (2) Preparation of compound cold water soluble gel powder Mix the guar gum, gellan gum, sodium polyacrylate and carrageenan that have been ground and passed through a 120-mesh standard sieve, and mix them thoroughly in a mass ratio of guar gum: gellan gum: sodium polyacrylate: carrageenan of 5:2:2:1; (3) Preparation of Bifidobacterium colony count test piece Cut the transparent plastic upper film, the plastic middle plate and the bottom plate printed with squares into the same size, and seal and bond the side with the label with polyacrylic resin pressure-sensitive adhesive; The above-mentioned composite cold water soluble gel powder is evenly coated on the sticky lower surface of the transparent plastic upper film; The polyacrylic acid resin pressure-sensitive adhesive is evenly coated on the bottom plate exposed in the hollow area of ​​the plastic middle layer plate, and the compound cold water-soluble gel powder and the bifidobacterium colony counting colorimetric medium powder are prepared in a mass ratio of 3:7, and then the mixed powder is evenly applied to the hollow area of ​​the plastic middle layer plate and compacted; The mixture is vacuum packed and then sterilized by ethylene oxide sterilization method to obtain a bifidobacterium colony counting test piece.

2. A test piece for counting bifidobacteria colonies as claimed in claim 1, characterized in that: The transparent plastic upper film is a PET silicone film with a thickness of 0.05 mm to 0.10 mm, and the lower surface of the PET silicone film itself is sticky.

3. A test piece for counting bifidobacterium colonies as claimed in claim 1, characterized in that: The plastic middle layer board is a polypropylene plastic board or a PET plastic board with a thickness of 0.15 mm to 0.30 mm.

4. A test piece for counting bifidobacterium colonies as claimed in claim 1, characterized in that: The base is white coated paper with a thickness of 0.10 mm~0.20 mm.

5. A test piece for counting bifidobacterium colonies as claimed in claim 1, characterized in that: The hollow area of ​​the plastic middle layer board is a circular, oval or square structure, and the ratio of the hollow area to the area of ​​the middle layer board is in the range of 1:1.6~1:2.

7.

6. Use of the bifidobacterium colony count test piece according to claim 1 in counting bifidobacterium colonies in dairy products.

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