CN113846003A - Research, preparation method and application of bifidobacterium colony counting test piece - Google Patents

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

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CN113846003A
CN113846003A CN202111262433.0A CN202111262433A CN113846003A CN 113846003 A CN113846003 A CN 113846003A CN 202111262433 A CN202111262433 A CN 202111262433A CN 113846003 A CN113846003 A CN 113846003A
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bifidobacterium
test piece
colony
colony counting
powder
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陈萍
王熙钰
何湃
王鑫
孟锋
康艳萍
叶登羿
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Jilin Agricultural University
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Abstract

A study and preparation method of a bifidobacterium colony count test piece and application of the bifidobacterium colony count test piece in detecting bifidobacterium count in dairy products 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 viscous lower surface of the transparent plastic upper layer film is coated with compound cold water soluble gel, and the hollow area is filled with a bifidobacterium colony counting chromogenic medium and compound cold water soluble gel powder which are prepared according to a certain proportion. The invention uses the test piece to count and measure the bifidobacteria in the dairy products, and the detection procedure is convenient and quick. The color development characteristic of the bifidobacterium counting test piece is systematically researched under the condition of the optimal cold water soluble gel proportion, and isopropyl-beta-D-thiogalactoside is added into the culture medium components, so that the color development definition of bacterial colonies can be improved, the color development time of the bacterial colonies is accelerated, and the accuracy rate of bacterial colony counting of the test piece is improved.

Description

Research, preparation method and application of bifidobacterium colony counting test piece
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a bifidobacterium counting test sheet, a preparation method and application thereof in detecting bifidobacterium counting in dairy products.
Background
Bifidobacterium genus(Bifidobacterium) Is a gram-positive bacterium, is an important beneficial microorganism in intestinal tracts, has been paid more attention to the efficacy and the prospect of bifidobacterium in food, health care and medical treatment in recent years, and is also widely applied to various dairy products. The bifidobacterium is an important index for evaluating the quality and the nutritional and health functions of dairy products, the bifidobacterium count in milk in China is measured according to GB 4789.34-2016 (national food safety standard food microbiology inspection bifidobacterium inspection), the national standard flat plate counting method has high accuracy, but the traditional detection method mainly depends on microorganism enrichment culture, selective separation and biochemical identification, has the defects of complicated operation steps, long detection period and the like, and is not suitable for the requirement of convenient detection of a basic layer. Aiming at the detection requirement of modern dairy product industry which is developed at a high speed at present, the technology for detecting one bifidobacterium which is lactobacillus is developed conveniently, quickly, standardizedly and highly sensitively is very important.
In recent years, with the intensive research on probiotics, the detection of bifidobacteria at home and abroad is increasing day by day, including Flow Cytometry (FCM), fluorescence quantitative PCR (polymerase chain reaction), PMA/EMA-QPCR (polymerase chain reaction-quantitative polymerase chain reaction) method and the like, but most of methods for detecting bifidobacteria have the disadvantages of more complicated operation steps, higher detection cost and higher requirement on technical personnel, so that the application range of the methods is limited. The microbial test strip method is a very ideal detection method at present, the bacterial colony growth is good, the culture period is short, several domestic microbial companies have bacterial colony counting test strip products using cold water soluble gel as a carrier, although the test strips are simple and convenient to operate, the technical problem of influencing the growth and counting of the bacterial colony still exists, and at present, no test strip product aiming at bifidobacterium counting exists in China, so that the invention provides a simple, quick and accurate bifidobacterium bacterial colony counting test strip which has very important application value.
Aiming at the problems, the invention carries out systematic research on the color development characteristic of the bifidobacterium counting test piece, optimizes the color development agent of the bifidobacterium test piece culture medium according to the optimal cold water soluble gel proportion which provides a proper growth environment for culturing the bifidobacterium, determines the composition of a color development system according to the growth condition and the color development effect of bacterial colonies, and ensures that the bacterial colonies are clear in color development and convenient to identify and count. According to the requirements of GB 4789.34-2016 national standard on bifidobacterium counting, a bifidobacterium colony counting test piece is prepared, and the total number of the bifidobacterium can be counted. According to the invention, a bifidobacterium colony counting test sheet product which is convenient to operate and accurate in counting is developed through the research on the color development characteristic.
Disclosure of Invention
One of the purposes of the invention is to provide a bifidobacterium counting test piece which is simple in operation, low in price and accurate in detection, and consists of a transparent plastic upper layer film with a sticky lower surface (such as a PET silica gel film which is high in transparency, non-toxic, free of pungent smell, waterproof and breathable and has a thickness of 0.05-0.10 mm, and the lower surface of the PET silica gel film has stickiness), a plastic middle layer plate with a hollow area (such as a polypropylene (PP) plastic plate or a PET plastic plate with good hardness and a thickness of 0.15-0.30 mm), and a bottom plate printed with grids (such as waterproof and anti-seepage white coated paper with a thickness of 0.10-0.20 mm and a size of 1cm multiplied by 1cm) from top to bottom in sequence; the high temperature resistant temperature range of the materials is 130-150 ℃; the viscous lower surface of the transparent plastic upper layer film is coated with compound cold water (the temperature range is 20-45 ℃) soluble gel, the hollow area of the plastic middle layer plate is of a circular, oval or square structure, and the proportion range of the area of the hollow area to the area of the plastic middle layer plate is 1: 1.6-2.7, filling a bifidobacterium colony counting chromogenic medium and compound cold water soluble gel powder which are prepared according to a certain proportion in the hollow area, wherein the mass ratio of the compound cold water soluble gel powder to the bifidobacterium colony counting chromogenic medium is 3: 7-9.
The compound cold water soluble gel comprises the following components: mixing the guar gum, gellan gum, sodium polyacrylate and carrageenan which are ground and pass through a 100-150-mesh standard sieve, wherein the guar gum comprises the following components in percentage by weight: gellan gum: sodium polyacrylate: the mass ratio of the carrageenan is 5: 1-2.
The bifidobacterium colony counting chromogenic medium comprises the following components in parts by weight: 8-12 g of peptone, 4-6 g of beef powder, 3-5 g of yeast powder, 18-22 g of glucose, 800.5-2 mL of Tween, 1.5-2.5 g of dipotassium phosphate, 1.5-2.5 g of diammonium hydrogen citrate, 4-6 g of sodium acetate, 0.1-0.4 g of magnesium sulfate, 0.01-0.1 g of manganese sulfate, 0.01-0.1 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.1-1 g of an inducer isopropyl-beta-D-thiogalactoside, 0.01-0.1 g of a bacteriostatic agent mupirocin lithium salt and 1000mL of distilled water.
The invention also aims to provide a preparation method of the bifidobacterium colony counting test piece, which comprises the following steps:
(1) preparation of bifidobacterium colony counting chromogenic detection culture medium
Weighing 8-12 g of peptone, 4-6 g of beef powder, 3-5 g of yeast powder, 18-22 g of glucose, 800.5-2 mL of tween, 1.5-2.5 g of dipotassium 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, filtering 0.01-0.1 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.1-1 g of inducer isopropyl-beta-D-thiogalactoside and 0.01-0.1 g of bacteriostatic agent mupirocin lithium salt by using a needle filter with the diameter of 25 mm and the pore diameter of 0.22 mu m, and uniformly mixing to obtain bifidobacterium colony counting chromogenic culture medium powder;
(2) preparation of compound cold water soluble gel
Mixing the guar gum, gellan gum, sodium polyacrylate and carrageenan which are ground and pass through a 100-150-mesh standard sieve, wherein the guar gum comprises the following components in percentage by weight: gellan gum: sodium polyacrylate: preparing carrageenan with the mass ratio of 5: 1-2;
(3) preparation of bifidobacterium colony counting test piece
Cutting a transparent plastic upper layer film, a plastic middle layer plate with a hollow area and a bottom plate printed with squares into the same size, wherein the length is 9-12 cm, the width is 8-10 cm, and polyacrylic resin pressure-sensitive adhesive (transparent, nontoxic, tasteless and sterile) is utilized; sealing and bonding the three sides with the labels, 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 a polyacrylic resin pressure-sensitive adhesive on the bottom plate exposed out of the hollow area of the plastic middle-layer plate, wherein the thickness of the pressure-sensitive adhesive is 0.05-0.10 mm, and mixing the compound cold water soluble gel powder with the bifidobacterium colony counting chromogenic medium powder according to the mass ratio of 3: 7-9, uniformly coating the obtained mixed powder in a hollow area of a plastic middle layer plate and compacting;
and finally, carrying out vacuum packaging, and sterilizing by adopting an ethylene oxide method, thereby obtaining the bifidobacterium colony counting and developing test piece.
(4) Operation steps for measuring bifidobacterium count by test strip method
Sucking 25mL of sample (the sample should be dairy products) by using 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 using a beating type homogenizer for 1-2 min to prepare a product 1:10 of the diluted homogenate.
1mL of the above 1:10, adding the diluted solution into a test tube containing 9 mL 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 content of lactic acid bacteria in a dairy product sample, selecting 2-3 diluted uniform solutions with proper dilution degrees, opening the upper film of the transparent plastic of the bifidobacterium colony counting test piece, taking 1mL of the diluted uniform solution of the sample, adding the diluted uniform solution of the sample to the area with the bifidobacterium colony counting chromogenic medium in the middle layer, and uniformly distributing the diluted uniform solution of the sample in the whole culture area; slowly dropping the plastic upper layer film, avoiding extrusion, standing for 30 s-1 min until the compound cold water soluble gel is solidified with water, putting the plastic upper layer film into a constant temperature incubator to be cultured for 48-72 h at 36 +/-1 ℃, and counting according to GB 4789.34-2016 (national food safety standard for food microbiology inspection) Bifidobactirium inspection). Meanwhile, 1mL of physiological saline was used as a blank control.
The invention relates to a bifidobacterium colony counting test piece, wherein a counting chromogenic medium of the bifidobacterium colony counting test piece consists of compound cold water soluble gel (prepared from guar gum, gellan gum, sodium polyacrylate and carrageenan in a mass ratio of 5: 1-2), a basic medium (peptone, beef powder, yeast powder, glucose, Tween 80, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate and manganese sulfate), a specific chromogenic substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside, an inducer isopropyl-beta-D-thiogalactoside and a bacteriostatic agent mupirocin lithium salt. Compared with similar products, the bifidobacterium colony counting test piece prepared by the invention has the advantages of good colony growth, clear color development, high sensitivity, strong specificity, convenient operation and accurate counting. Mainly comprising 2 important technical features.
Important technical features 1: the invention adopts 5-bromo-4-chloro-3-indole-beta-D-galactoside as a chromogenic substrate, adds an inducer isopropyl-beta-D-thiogalactoside, and designs a bifidobacterium enzyme specific chromogenic system, so that the bacterial colony of the test piece develops color clearly. The bifidobacterium can produce galactosidase in the growth process, so that the substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside is decomposed to release indole, thereby enabling the colony to develop color. On the basis, an inducer isopropyl-beta-D-thiogalactoside is added, the isopropyl-beta-D-thiogalactoside can induce the synthesis of beta-galactosidase, and the color can be developed well after culturing for 48 hours, so that the colony number and the color development effect of bifidobacterium growth are effectively improved, and the observation and counting are more convenient.
Important technical features 2: the invention develops a bacterial colony counting test piece for detecting bifidobacteria in dairy products for the first time, the test piece can accurately and rapidly quantitatively detect the bacterial colony number of the bifidobacteria in the dairy products, and has the advantages of simple and convenient procedure, clear color of the bacterial colony, accurate counting, simple operation and low cost.
The invention carries out systematic research on the color development characteristic of the bifidobacterium colony counting test piece, adds the inducer isopropyl-beta-D-thiogalactoside and the bacteriostatic agent mupirocin lithium salt according to the optimal cold water soluble gel proportion, colony growth condition and color development effect, establishes a color development system suitable for the bifidobacterium test piece, ensures that the colony of the test piece develops color clearly and the colony counting is accurate.
According to the requirements of GB 4789.34-2016 national standard on bifidobacterium counting, a bifidobacterium colony counting test piece is prepared, and the total number of the bifidobacterium can be counted. According to the invention, the bifidobacterium in the milk is counted and tested, the color development of bacterial colonies is clear, the operation is convenient and fast, the counting is accurate, the accuracy rate reaches 99%, and the lowest detection limit reaches 3 CFU/mL. The test piece showed good growth of Bifidobacterium and blue color, while the other bacteria could not be detected. In the dairy product sample inspection, the bifidobacterium colony counting test piece method has no difference significance (P is more than 0.05) with the detection result of the national standard method, accords with the national standard of the bifidobacterium inspection in China, and can be applied in large scale in detection institutions and food enterprises.
Drawings
FIG. 1: the structure of the bifidobacterium colony 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 bifidobacterium colony counting test piece added with the color developing agent with different concentrations has the color developing effect.
FIG. 3: the bifidobacterium colony counting test piece added with the inducer with different concentrations has the color development effect.
FIG. 4: the invention discloses a bifidobacterium colony test piece method and a national standard method detection result linear correlation curve.
FIG. 5: the bifidobacterium colony test piece method of the invention is compared with the national standard method detection picture.
Detailed Description
Example 1 preparation of culture Medium for Bifidobacterium colony count assay
The original weight components of the bifidobacterium colony counting chromogenic medium are as follows: 10 g of peptone, 5 g of beef powder, 4g of yeast powder, 20 g of glucose, 801 mL of tween, 2g of dipotassium phosphate, 2g of diammonium hydrogen citrate, 5 g of sodium acetate, 0.2g of magnesium sulfate, 0.05 g of manganese sulfate, 0.06 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.6 g of inducer isopropyl-beta-D-thiogalactoside, 0.06 g of bacteriostatic agent mupirocin lithium salt and 1000mL of distilled water.
The preparation method of the bifidobacterium colony counting chromogenic medium comprises the following steps: weighing 10 g of peptone, 5 g of beef powder, 4g of yeast powder, 20 g of glucose, 801 mL of tween, 2g of dipotassium phosphate, 2g of diammonium hydrogen citrate, 5 g of sodium acetate, 02 g of magnesium sulfate and 0.05 g of manganese sulfate, filtering 0.06 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.6 g of inducer isopropyl-beta-D-thiogalactoside and 0.06 g of bacteriostatic agent mupirocin lithium salt by using a needle filter with the diameter of 25 mm and the pore diameter of 0.22 mu m, uniformly mixing, freeze-drying into powder, and uniformly mixing with other components to obtain chromogenic culture medium powder for later use.
The preparation method of the compound cold water soluble gel comprises the following steps: mixing the guar gum, gellan gum, sodium polyacrylate and carrageenan which are ground and pass through a 120-mesh standard sieve, and mixing the guar gum, the gellan gum, the sodium polyacrylate and the carrageenan according to the weight ratio of the guar gum: gellan gum: sodium polyacrylate: fully and uniformly mixing carrageenan in a mass ratio of 5:2:2:1 for later use.
EXAMPLE 2 preparation of Bifidobacterium colony counting test piece
1) The test piece consists of three layers, namely an upper PET silica gel film, a middle polypropylene plastic plate with a hollow area and a bottom layer printed with faint yellow 1cm multiplied by 1cm square white square copper plate paper. Cutting the three parts into the same size, length 9cm and width 8cm, and sealing and bonding the side with label with polyacrylic resin pressure sensitive adhesive (transparent, nontoxic, odorless, and sterile) with bonding width of 5 mm.
2) The compounded cold water soluble gel powder is evenly sprayed on the inner side of the film of the test piece, the thickness is 0.15mm, and the mass of the cold hydrogel of each test piece is 0.2 g. 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) And (3) uniformly coating 0.2g of transparent polyacrylic resin pressure-sensitive adhesive on the bottom plate corresponding to the hollow area of the middle layer, preparing the compound cold water soluble gel powder and the bifidobacterium colony counting chromogenic medium powder according to the mass ratio of 3:7, uniformly coating the mixed powder in the hollow area of the plastic middle layer plate, and compacting.
4) The test pieces were vacuum packed.
5) The test piece was sterilized by ethylene oxide sterilization.
EXAMPLE 3 investigation of the chromogenic System of Bifidobacterium colony counting test piece
1) Influence of 5-bromo-4-chloro-3-indole-beta-D-galactoside as color developing agent on color developing effect of test piece
As the bifidobacteria can generate galactosidase in the growth process, the substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside is decomposed to release indole, blue precipitate is generated, and the existence of bacterial colony can be judged. In the manufacturing process of the test piece, as shown in fig. 2, when the concentrations of the chromogenic substrates are respectively 0.04 g/L, 0.05 g/L, 0.06 g/L, 0.07 g/L and 0.08 g/L, when the concentration of the chromogenic substrates is 0.06 g/L, the bacterial colony is blue, the chromogenic effect is obvious, and the counting is convenient. In combination with the consideration of FIG. 2, the substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside with a concentration of 0.06 g/L was selected as the optimum concentration to be added to the culture medium to prepare the test piece.
2) Influence of inducer isopropyl-beta-D-thiogalactoside on color development effect of test piece
The isopropyl-beta-D-thiogalactoside can induce the synthesis of enzyme, is similar to natural beta-galactosidase and cannot be decomposed by enzyme, thereby improving the colony number and the color development effect of bifidobacterium growth and being more convenient to observe and count. The optimal color developing agent 5-bromo-4-chloro-3-indole-beta-D-galactoside dosage is added into the sterilized modified MRS culture medium, and then 0.4 g/L, 0.6 g/L and 0.8 g/L inducer isopropyl-beta-D-thiogalactoside are respectively added, and the colony color developing effect is shown in table 1 and figure 3. When the concentration of the inducer reaches 0.6 g/L, the growth condition of the bifidobacteria is optimal, the colony is clearly developed, and the colony growth is inhibited along with the increase of the dosage.
Table 1: effect of color-developing agent and inducer concentration on color development of bacterial colonies
Figure DEST_PATH_IMAGE002
Example 4 method for determining the colony count of Bifidobacterium in a milk product Using a test piece
1) Weighing 25mL of a dairy product sample to be detected by aseptic operation, adding the dairy product sample to be detected into an aseptic homogenizer bag of 225mL diluent, beating for 1-2 min by using a homogenizer, and preparing into a product 1: sample aliquot of 10. And performing 10-fold serial gradient dilution on the sample homogeneous solution, and selecting 2-3 dilutions according to the estimation of the content of lactic acid bacteria in the dairy product sample, wherein each dilution is used for preparing two parallel samples.
2) Taking 1mL of sample diluent to be detected, lifting the upper membrane of the test piece, adding the sample 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 after the proliferation culture is placed in a sterile operating table, the presence or absence of blue colonies is observed, and the blue colonies are counted. The counting method is carried out according to GB 4789.34-2016 (national food safety Standard food microbiology inspection) for detecting bifidobacterium.
If the colony number of the test piece of all the dilutions is more than 300 CFU, 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 30 CFU, the colony number of the test strip at the lowest dilution is calculated by multiplying the dilution factor by the average colony number at the lowest dilution.
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 30 CFU and 300 CFU, and a part of the test piece is less than 30 CFU or more than 300 CFU, the colony number is calculated by multiplying the average colony number closest to 30 CFU or 300 CFU by the dilution factor.
Example 5: evaluation of Effect by Using Bifidobacterium colony count test piece
1) A test piece for colony count of Bifidobacterium was prepared as in examples 1 and 2 and was used.
2) Determination of detection limit and sensitivity of test strip
The experimental strains are bifidobacterium lactis HN019, bifidobacterium longum CICC 24632, bifidobacterium bifidum CICC 10395, bifidobacterium infantis CICC 6069, bifidobacterium breve CICC 22948, bifidobacterium adolescentis CICC 2473, lactobacillus paracasei CICC 20241, streptococcus thermophilus IFFI 6038, lactobacillus acidophilus CICC 20244 and lactobacillus lactis CICC 20090.
And respectively inoculating single colonies into 100 mL national standard MRS liquid culture medium, and culturing in a constant temperature incubator at 36 +/-1 ℃ for 48 h. Adding 25mL of bacterial liquid into 225mL of sterile normal saline, and mixing uniformly to prepare a diluent with a ratio of 1: 10. The above procedure was repeated to dilute the bacterial solution to a dilution (concentration of 10) suitable for counting0~102 CFU/mL), the number of colonies of bifidobacterium was measured using the test piece using 1mL of the bacterial solution according to the method in example 4. Meanwhile, the bifidobacterium colony count is measured according to GB 4789.34-2016 (national food safety Standard food microbiology inspection for bifidobacterium inspection), two parallel samples are made for each dilution, and the average value of the detection results is taken.
And taking the detection result of the lowest dilution as the lowest detection limit, and taking the lowest detection limit as the detection standard of the sensitivity of the test strip. The detection result is positive when the detection result is larger than the minimum detection limit, the detection result is negative when the detection result is smaller than the minimum detection limit, the sensitivity = positive/(positive + negative), and the experimental result shows that the two methods can reach the detection limit of the same level, the detection limit is 3 CFU/mL, and the sensitivity is 100%. The colony number between 30 and 300 CFU/mL is analyzed, t =0.095, and P =0.5> 0.05. The detection result of the bifidobacterium colony counting test piece developed by the invention has no difference significance with the national standard flat plate method. See table 2, fig. 4 and fig. 5.
Table 2: sensitivity detection result of bifidobacterium test piece
Figure DEST_PATH_IMAGE004
2) Test strip specificity detection
Bacterial suspensions of strains such as bifidobacterium lactis, bifidobacterium longum, bifidobacterium bifidum, bifidobacterium infantis, bifidobacterium breve, bifidobacterium adolescentis, lactobacillus paracasei, streptococcus thermophilus, lactobacillus acidophilus, lactococcus lactis and the like are selected, 10-fold gradient dilution is carried out on the bacterial suspensions by using normal saline, and 1mL of bacterial solution is taken to carry out colony counting measurement on 10 strains of bacteria according to the method in example 4 and GB 4789.34-2016 (national standard food safety food microbiology inspection bifidobacterium inspection).
The experimental result shows that the colony of the bifidobacterium on the bifidobacterium counting test piece grows well and shows blue after being cultured for 48 hours, and other bacteria can not be detected. The bifidobacterium counting test piece has no difference significance (P is more than 0.05) with the counting result of GB 4789.34-2016 (national food safety standard food microbiology inspection) bifidobacterium inspection, and the counting result is accurate. The results are shown in Table 3.
Table 3: test piece specificity test data
Colony name Test strip counting method (log)10CFU/mL) National Standard plate count (log)10CFU/mL)
Bifidobacterium lactis 7.492 7.544
Bifidobacterium longum 7.361 7.432
Bifidobacterium bifidum 6.740 6.807
Bifidobacterium infantis 7.286 7.373
Bifidobacterium breve 6.895 6.975
Bifidobacterium adolescentis 7.633 7.681
Lactobacillus paracasei — —
Streptococcus thermophilus — —
Lactobacillus acidophilus — —
Lactococcus lactis — —
3) Application of test piece in dairy product sample detection
Purchasing actual samples such as live lactobacillus beverages of different brands from supermarket, diluting the samples to concentration of 100~102CFU/mL was reserved, and the number of colonies of Bifidobacterium was determined using the test piece from 1mL of the bacterial suspension by the method described in example 4. Simultaneously, according to GB 4789.34-2016 (national food safety Standard for food microbiology inspection) for detecting bifidobacteriaAnd (4) carrying out bifidobacterium colony counting measurement, and comparing the conformity of the measurement of the test piece and the measurement result of the national standard method.
Table 4: result comparison and coincidence rate of dairy product sample detected by test piece method and national standard method
Figure DEST_PATH_IMAGE006
120 dairy product samples are respectively detected by using a bifidobacterium counting test piece method and a national standard method, the detection results of the samples are shown in table 4, the data show that the highest coincidence rate of the two methods can reach 98.8 percent, and the detection results of the two methods have no difference significance (P is more than 0.05). The results show that: the bifidobacterium counting test piece is suitable for detecting the total number of the bifidobacterium in the dairy product.

Claims (7)

1. A bifidobacterium colony 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 viscous lower surface of the transparent plastic upper layer film is coated with compound cold water soluble gel, and the hollow area is filled with a bifidobacterium colony counting chromogenic medium and compound cold water soluble gel powder which are prepared according to a certain proportion; the compound cold water soluble gel is prepared by grinding guar gum, gellan gum, sodium polyacrylate and carrageenan which pass through a 120-mesh standard sieve in a ratio of 5:2:2: 1; the bifidobacterium colony counting chromogenic medium comprises 10 g of peptone, 5 g of beef powder, 4g of yeast powder, 20 g of glucose, 801 mL of tween, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5 g of sodium acetate, 02 g of magnesium sulfate, 0.05 g of manganese sulfate, 0.06 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.6 g of an inducer isopropyl-beta-D-thiogalactoside, 0.06 g of a bacteriostatic agent mupirocin lithium salt and 1000mL of distilled water.
2. A bifidobacterium colony counting test strip as claimed in 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 bifidobacterium colony counting test strip as claimed in 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 bifidobacterium colony counting test strip as claimed in 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 bifidobacterium colony counting test strip as claimed in 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.
6. a method for preparing a Bifidobacterium colony count test piece as claimed in claim 1, which comprises the following steps:
(1) preparation of bifidobacterium colony counting chromogenic detection culture medium
Weighing 10 g of peptone, 5 g of beef powder, 4g of yeast powder, 20 g of glucose, 801 mL of tween, 2g of dipotassium phosphate, 2g of diammonium hydrogen citrate, 5 g of sodium acetate, 02 g of magnesium sulfate and 0.05 g of manganese sulfate, filtering 0.06 g of 5-bromo-4-chloro-3-indole-beta-D-galactoside, 0.6 g of inducer isopropyl-beta-D-thiogalactoside and 0.06 g of bacteriostatic agent mupirocin lithium salt by using a needle filter with the diameter of 25 mm and the pore diameter of 0.22 mu m, uniformly mixing, freeze-drying into powder, and uniformly mixing with other components to obtain bifidobacterium colony counting chromogenic culture medium powder;
(2) preparation of compound cold water soluble gel
Mixing the guar gum, gellan gum, sodium polyacrylate and carrageenan which are ground and pass through a 120-mesh standard sieve, and mixing the guar gum, the gellan gum, the sodium polyacrylate and the carrageenan according to the weight ratio of the guar gum: gellan gum: sodium polyacrylate: fully and uniformly mixing carrageenan with the mass ratio of 5:2:2: 1;
(3) preparation of bifidobacterium colony 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, preparing the compound cold water soluble gel powder and bifidobacterium colony counting chromogenic medium powder according to the mass ratio of 3:7, uniformly coating the mixed powder in the hollow area of the plastic middle-layer plate and compacting;
vacuum packaging, and sterilizing by ethylene oxide sterilization method to obtain Bifidobacterium colony count test piece.
7. Use of a test piece for the colony count of bifidobacteria according to claim 1 in the colony count of bifidobacteria in a dairy product.
CN202111262433.0A 2021-10-28 2021-10-28 Research, preparation method and application of bifidobacterium colony counting test piece Pending CN113846003A (en)

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