Preparation method of cell imprinting electrochemical sensor and method for rapid quantitative detection of lactic acid bacteria in dairy products using the same

Abstract

The present invention relates to the field of microbial detection technology, and specifically to a method for preparing a cell-imprinted electrochemical sensor and a method for rapidly and quantitatively detecting lactic acid bacteria in dairy products using the same. The method comprises: 1) immersing the cell-imprinted electrochemical sensor in a lactic acid bacteria solution of known concentration, then washing it with PBS and using differential pulse voltammetry to detect the concentration of lactic acid bacteria in a solution containing [Fe(CN)6] 3‑ / 4‑ , KCl solution; draw a standard curve with the logarithm of the lactic acid bacteria solution concentration as the horizontal coordinate and the current value as the vertical coordinate; 2) detect lactic acid bacteria in dairy products. The cell imprinting electrochemical sensor of the present invention can realize rapid, sensitive and selective detection of lactic acid bacteria, and has simple sample pretreatment, short analysis time, simple experimental operation and strong specific recognition ability. The sensor is used in dairy product production, and can quickly and accurately quantitatively detect lactic acid bacteria in dairy products, effectively reducing production costs, and is also suitable for the detection of lactic acid bacteria in various dairy products.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a method for preparing a cell imprinting electrochemical sensor and a method for rapidly and quantitatively detecting lactic acid bacteria in dairy products by using the cell imprinting electrochemical sensor. Background Art

[0002] Yogurt is a fermented dairy product made by inoculating one or more probiotic lactic acid bacteria into sterilized cow's milk or goat's milk. During the fermentation process, the lactic acid bacteria can produce a large amount of organic acids, amino acids, B vitamins, enzymes and other nutrients. The lactic acid bacteria in yogurt also have the functions of improving intestinal flora, regulating gastrointestinal motility, and promoting digestion, making yogurt very popular among consumers.

[0003] In yogurt, the number of lactic acid bacteria is an important indicator of yogurt quality, and the quantitative detection of lactic acid bacteria has become an important indicator for product quality and functional evaluation. At present, the traditional culture method is a common method for detecting lactic acid bacteria in yogurt. This method has low operational sensitivity, and the selective culture medium used cannot count bacterial cells that are difficult to culture despite being metabolically active. Moreover, this method is time-consuming and easily interfered by miscellaneous bacteria. In addition, yogurt is usually co-fermented by a variety of different lactic acid bacteria, and it is necessary to clarify the number of live bacteria of each lactic acid bacteria in yogurt. Although molecular biological techniques such as polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), and high-throughput sequencing technology can achieve quantitative detection of different lactic acid bacteria, they are time-consuming, require expensive instruments, and have complex data analysis. Therefore, it is of great significance to develop a simple, rapid, and highly specific detection technology to dynamically monitor the number of lactic acid bacteria during yogurt fermentation for the quality control of dairy products.

[0004] Biosensors are sensors that use biological materials such as microorganisms, enzymes, antibodies, DNA, and RNA as molecular recognition elements to quantitatively analyze the matrix content in the sample liquid. They have the advantages of high sensitivity, fast analysis speed, and strong anti-interference ability, and have great application prospects in food microbial detection. At present, the application of immunosensors in microbial detection is relatively mature. This type of sensor uses specific modifications of antibodies, aptamers, or bacteriophages to selectively identify target bacteria. However, the synthesis process of biorecognition elements is complex, the pretreatment steps are many, and there are strict requirements on environmental pH and temperature, which to a certain extent limits the further development of immunosensors. Molecular imprinting technology has been widely used as a substitute for natural receptors. During its preparation process, a mixture of functional monomers and crosslinkers polymerizes around the template cells and can specifically bind to the cells to form cell imprinted polymers. After the polymerization reaction is completed, the template is eluted from the imprinted polymer to obtain a cell imprinted sensor. The cell imprinting sensor has holes (i.e., imprinting sites) that are complementary to the shape, size, and chemical functional groups of the template molecule. These imprinting sites can specifically recognize and bind to cells in the sample to be tested that are similar to the template cells. By detecting the changes in the electrical signals before and after the cell imprinting sensor adsorbs the cells, the target microorganisms can be selectively and quantitatively detected. Currently, the technology of molecular imprinting sensors for dairy microbial detection is still immature. Summary of the invention

[0005] To solve the above problems, the purpose of the present invention is to provide a method for preparing a cell imprinting electrochemical sensor and a method for rapidly and quantitatively detecting lactic acid bacteria in dairy products using the same. The method is a cell imprinting electrochemical sensor method for detecting lactic acid bacteria in dairy products. In the present invention, the cell imprinting electrochemical sensor can detect lactic acid bacteria rapidly, sensitively and selectively; in addition, it has simple sample pretreatment, high detection sensitivity, short analysis time, simple experimental operation and strong specific recognition ability.

[0006] In order to achieve the above invention object, the specific technical scheme of the present invention is as follows:

[0007] A method for preparing a cell imprinting electrochemical sensor comprises the following steps:

[0008] 1) Preparation of activated biochar: heating the biomass to obtain biochar; then activating the biochar to obtain activated biochar;

[0009] 2) Modified electrode: add PBS solution to the activated biochar, stir until dissolved, and prepare a suspension with a biochar concentration of 1 mg / mL; then polish the screen-printed electrode, rinse with ethanol and ultrapure water respectively, and dry at room temperature; take the activated biochar suspension and drop it on the surface of the above electrode, and obtain the modified electrode after natural drying;

[0010] 3) Preparation of an electrode with lactic acid bacteria-polypyrrole polymer: placing the modified electrode in a PBS solution containing polypyrrole, KCl and lactic acid bacteria template, and performing electrochemical polymerization by cyclic voltammetry to obtain an electrode with lactic acid bacteria-polypyrrole polymer;

[0011] 4) Preparation of cell imprinted electrochemical sensor: The electrode with lactic acid bacteria-polypyrrole polymer was rinsed with PBS and then immersed in lysozyme. Then NaOH was added and voltage was applied to remove the lactic acid bacteria encapsulated in the polymer, leaving the imprinted cavity, thereby obtaining a cell imprinted electrochemical sensor with specific binding ability.

[0012] Furthermore, 1) the specific preparation steps of activated biochar are: heating the biomass at 600-800°C for 1-2 h (more preferably 700°C for 2 h) to obtain biochar.

[0013] Furthermore, the biomass includes all plants, microorganisms and animals and their waste products (specifically bamboo leaves, straws, distiller's grains, etc.).

[0014] Furthermore, the biomass carbon is mixed with potassium carbonate and heated, and the entire pyrolysis process is carried out in nitrogen to obtain activated biochar.

[0015] Furthermore, the weight ratio of biomass carbon to potassium carbonate is 1:2, the heating temperature is 800-900°C, and the heating time is 1-2 h.

[0016] Furthermore, the specific steps of 2) modifying the electrode are as follows: polishing the screen-printed electrode (SPE) to a mirror surface with 0.05 μm alumina powder, rinsing with ethanol and ultrapure water, and drying at room temperature. The biochar suspension is dripped on the electrode surface and dried naturally to obtain the modified electrode.

[0017] Furthermore, the screen-printed electrode includes a working electrode, a reference electrode and an auxiliary electrode; the working electrode is made of a bare glassy carbon electrode with a diameter of 3 mm, the auxiliary electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.

[0018] Furthermore, the volume of the biochar suspension dripped on the electrode surface is 6-10 μL.

[0019] Furthermore, 3) the specific preparation steps of the electrode with lactic acid bacteria-polypyrrole polymer are as follows: put the modified electrode into 10 mL of 0.1-0.2 mol / L polypyrrole, 0.05-0.1 mol / L KCl and 10 7 ~10 9Electrochemical polymerization was carried out in a PBS solution containing CFU / mL lactic acid bacteria template by cyclic voltammetry to obtain an electrode with lactic acid bacteria-polypyrrole polymer.

[0020] The lactic acid bacteria template includes but is not limited to Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophilic. The bacterial template can be selected accordingly according to the different types of detected microorganisms.

[0021] The PBS solution contains NaH2PO4 and Na2HPO4, both with a concentration of 0.1 mol / L and a pH of 7.4, the same below.

[0022] The cyclic voltammetry method is used for electrochemical polymerization, with a scanning number of 9 to 11, a voltage of 0 to 1.2 V, and a scanning rate of 40 to 60 mV / s.

[0023] Furthermore, 4) the specific preparation steps of the cell imprinted electrochemical sensor are:

[0024] The electrode with lactic acid bacteria-polypyrrole polymer was rinsed with PBS and then immersed in 10-15 mg / mL lysozyme for 1-2 hours. Subsequently, 0.1 mol / L NaOH was added and a voltage of 0.98 V was applied to remove the lactic acid bacteria encapsulated in the polymer, leaving the imprinted cavity, thereby obtaining a cell-imprinted electrochemical sensor with specific binding ability.

[0025] Another aspect of the present invention provides the use of a cell imprinting electrochemical sensor in the detection of lactic acid bacteria in dairy products.

[0026] A method for rapid quantitative detection of lactic acid bacteria in dairy products comprises the following steps:

[0027] 1) Preparation of standard curve for detection of lactic acid bacteria by cell imprinting electrochemical sensor:

[0028] The cell-imprinted electrochemical sensor was immersed in a known concentration of lactic acid bacteria solution and then rinsed with PBS. Differential pulse voltammetry was used to measure the concentration of [Fe(CN)6] 3- / 4- , KCl solution to detect the current on the electrode surface; draw a standard curve with the logarithm of the lactic acid bacteria solution concentration as the abscissa and the current value as the ordinate;

[0029] 2) Detection of lactic acid bacteria in dairy products:

[0030] The cell-imprinted electrochemical sensor was immersed in the sample to be tested and then rinsed with PBS. Differential pulse voltammetry was used to measure the concentration of [Fe(CN)6] 3- / 4- , KCl solution; the measured current value is used to find the corresponding bacterial concentration on the standard curve.

[0031] Furthermore, 1) the preparation of the standard curve of the cell-imprinted electrochemical sensor for detecting lactic acid bacteria is as follows: immerse the cell-imprinted electrochemical sensor in 100 μL of lactic acid bacteria solution of known concentration and keep it at room temperature for 1 hour. Then rinse it with PBS and use differential pulse voltammetry in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , 0.1 mol / L KCl solution to detect the current on the electrode surface. Draw a standard curve with the logarithm of the lactic acid bacteria solution concentration as the horizontal axis and the current value as the vertical axis.

[0032] The voltage range of the differential pulse voltammetry is 0-0.6 V, the pulse amplitude is 50 mV, and the pulse width is 0.2 s, the same below.

[0033] The lactic acid bacteria liquid includes but is not limited to Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophilic, and the concentration of the bacterial liquid is 1×10 1 CFU / mL, 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL, the standard method for determining the concentration of bacterial solution is the plate count method.

[0034] Furthermore, 2) the specific steps for detecting lactic acid bacteria in dairy products are:

[0035] The cell-imprinted electrochemical sensor was immersed in 100 μL of the sample to be tested and kept at room temperature for 1 hour. After being rinsed with PBS, differential pulse voltammetry was used in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , 0.1 mol / L KCl solution to detect the current on the electrode surface. The measured current value is found on the standard curve to determine the corresponding bacterial concentration.

[0036] The samples to be tested include but are not limited to yogurt, fresh milk, sterilized milk, milk powder, etc. Solid samples can be prepared into liquid samples by adding pretreatment steps as needed.

[0037] The working principle of the present invention is as follows: under the modification of biochar, the specific surface area of ​​the electrode surface increases, the conductivity increases, and a higher peak current appears. Under further electropolymerization, the polymer of the functional monomer and the cross-linking agent can specifically bind to the living cells to form a polymer on the electrode surface. The accumulation of living cells will hinder the current transport and cause the current to decrease. After the template cells are removed, an imprinted cavity is left. The imprinted cavity promotes the current transport as a channel for electron transfer, and the current increases again. The imprinted cavity can specifically identify and re-bind to the cell according to the cell size, shape, and chemical group structure, so that the imprinted cavity is blocked again, hindering the electron transfer, and the current is reduced again. Therefore, the bacterial concentration can be calculated based on the current change value before and after the cell imprinted electrochemical sensor is combined with the cell.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The method of detecting lactic acid bacteria using the cell imprinting electrochemical sensor of the present invention has the advantages of high efficiency, sensitivity and high selectivity, and has low preparation cost, simple sample pretreatment, short analysis time and simple experimental operation. It has the potential to be applied to real-time monitoring of microbial indicators in fermented foods.

[0040] (2) The cell imprinting electrochemical sensor prepared in the present invention can be applied to the production of dairy products, and can achieve the purpose of rapid and accurate quantitative detection of lactic acid bacteria in dairy products, shortening the detection time of lactic acid bacteria from 1 to 2 days by traditional culture method to 4 to 6 hours, effectively reducing production costs, and is suitable for the detection of lactic acid bacteria in various dairy products.

[0041] (3) The present invention can achieve quantitative detection of different lactic acid bacteria in dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The cell imprinting electrochemical sensor provided by the present invention is used for the standard curve of detecting Lactobacillus delbrueckii subspecies bulgaricus.

[0043] Figure 2 The cell imprinting electrochemical sensor provided by the present invention is used for the standard curve of detecting thermophilic subspecies of Streptococcus salivarius. DETAILED DESCRIPTION

[0044] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0045] Any feature disclosed in this specification (including claims and abstract), unless otherwise stated, may be replaced by other alternative features that are equivalent or have similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0046] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0047] The experimental methods and detection methods in the following embodiments are all conventional methods unless otherwise specified; the agents and materials described are all commercially available unless otherwise specified; the indicator data described are all conventional measurement methods unless otherwise specified.

[0048] Embodiment 1:

[0049] A method for preparing a cell-imprinted electrochemical sensor based on Lactobacillus delbrueckii subsp. bulgaricus.

[0050] (1) Preparation of activated biochar: Wash the bamboo leaves with water, dry them in an oven at 60°C for 12 h, and crush them after drying. Pour an appropriate amount of bamboo leaf powder into a crucible and heat it in a tube furnace at 700°C for 2 h to obtain biochar. Wash the biochar with 1 mol / L hydrochloric acid and ultrapure water until its pH is neutral, and then dry it in an oven for 12 h. Mix the dried material with potassium carbonate in a weight ratio of 1:2, and then heat it at 850°C in a tube furnace for 2 h. The entire pyrolysis process is carried out in nitrogen. After the pyrolysis is completed, wash it with 1 mol / L hydrochloric acid and ultrapure water until its pH is neutral, then dry it in an oven for 12 h, and resuspend it in PBS solution to obtain a 1 mg / mL biochar suspension;

[0051] (2) Electrode pretreatment: The screen-printed electrode was polished to a mirror surface with 0.05 μm alumina powder, rinsed with ethanol and ultrapure water, and dried at room temperature. 6 μL of the biochar suspension obtained in (1) was dropped on the electrode surface and dried naturally to obtain the modified electrode;

[0052] (3) Preparation of lactic acid bacteria-polypyrrole polymer: The product obtained in step (2) was placed in 10 mL of 0.2 mol / L polypyrrole, 0.1 mol / L KCl and 10 9 CFU / mL of Lactobacillus delbrueckii subsp. bulgaricus template in a PBS solution (the PBS contains NaH2PO4 and Na2HPO4, both at a concentration of 0.1 mol / L, and a pH of 7.4, the same below), electrochemical polymerization was performed by cyclic voltammetry, with a scan number of 9, a voltage of 1.1 V, and a scan rate of 50 mV / s;

[0053] (4) Preparation of cell imprinted electrochemical sensor: The sample obtained in step (3) was rinsed with PBS solution and then immersed in 10 mg / mL lysozyme for 2 h. Subsequently, 0.1 mol / L NaOH was added and an overvoltage of 0.98 V was applied to remove the Lactobacillus delbrueckii subsp. bulgaricus embedded in the polymer, thereby obtaining a cell imprinted electrochemical sensor with specific binding ability.

[0054] Embodiment 2:

[0055] A method for preparing a cell-imprinted electrochemical sensor based on Streptococcus salivarius thermophilus subsp.

[0056] The preparation of this example is the same as that of Example 1, except that the bacterial template used in step (3) is 10 9 CFU / mL Streptococcus salivarius subsp. thermophilus

[0057] Example 3

[0058] A method for preparing a standard curve for detecting Lactobacillus delbrueckii subspecies bulgaricus using a cell-imprinted electrochemical sensor.

[0059] (1) Preparation of different concentrations of Lactobacillus delbrueckii subspecies bulgaricus culture liquid: Lactobacillus delbrueckii subspecies bulgaricus lb31 was inoculated into sterile MRS liquid culture medium at a rate of 2% (v / v), and cultured at 37°C for 24 h to obtain the first generation of activated liquid. The first generation of activated liquid was then inoculated into sterile MRS liquid culture medium at a rate of 2% (v / v), and cultured at 37°C for 24 h to obtain the second generation of activated liquid. The second generation of activated liquid was centrifuged at 9000 r / min for 10 min, and the precipitate was retained. The remaining precipitate was resuspended with sterile purified water and the bacterial concentration was adjusted to 1.0×10 9 CFU / mL, and then the resuspension was diluted to 1×10 1 CFU / mL, 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL of bacterial solution;

[0060] (2) Detection of bacterial solution: The cell-imprinted electrochemical sensor obtained in Example 1 was immersed in the solution obtained in step (1) and kept at room temperature for 1 h. After rinsing with PBS, differential pulse voltammetry was used in 10 mL of 5 mmol / L [Fe(CN)6]3- / 4- , the current on the electrode surface was detected in a 0.1 mol / L KCl solution, with a voltage range of 0-0.6 V, a pulse amplitude of 50 mV, and a pulse width of 0.2 s;

[0061] (3) Drawing of standard curve: Draw a standard curve with the logarithm of the concentration of Lactobacillus delbrueckii subsp. bulgaricus as the horizontal axis and the corresponding current value as the vertical axis.

[0062] Example 4

[0063] A method for preparing a standard curve for detecting thermophilic Streptococcus salivarius subspecies using a cell-imprinted electrochemical sensor.

[0064] (1) Preparation of different concentrations of Streptococcus salivarius thermophilus subspecies: Streptococcus salivarius thermophilus subspecies st195 was inoculated into sterile MRS liquid culture medium at a rate of 2% (v / v), and cultured at 37°C for 24 h to obtain the first generation of activated liquid. The first generation of activated liquid was then inoculated into sterile MRS liquid culture medium at a rate of 2% (v / v) and cultured at 37°C for 24 h to obtain the second generation of activated liquid. The second generation of activated liquid was centrifuged at 9000 r / min for 10 min, and the precipitate was retained. The remaining precipitate was resuspended with sterile purified water and the bacterial concentration was adjusted to 1.0×10 9 CFU / mL, and then the resuspension was diluted to 1×10 1 CFU / mL, 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL of bacterial solution;

[0065] (2) Detection of bacterial solution: The cell-imprinted electrochemical sensor obtained in Example 2 was immersed in the solution obtained in step (1) and kept at room temperature for 1 h. After rinsing with PBS, differential pulse voltammetry was used in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , the current on the electrode surface was detected in a 0.1 mol / L KCl solution, with a voltage range of 0-0.6 V, a pulse amplitude of 50 mV, and a pulse width of 0.2 s;

[0066] (3) Drawing of the standard curve: Draw the standard curve with the logarithm of the concentration of Streptococcus salivarius thermophilus subspecies as the horizontal axis and the corresponding current value as the vertical axis.

[0067] Example 5

[0068] A cell-imprinted electrochemical sensor was used to detect Lactobacillus delbrueckii subsp. bulgaricus in yogurt.

[0069] (1) Preparation of standard curve: The method for preparing the standard curve is the same as that in Example 3;

[0070] (2) Sample detection: The cell imprinted electrochemical sensor obtained in Example 1 was immersed in 100 μL of yogurt (purchased from a supermarket, with a live bacterial content of Lactobacillus delbrueckii subspecies bulgaricus ≥ 2×10 8 CFU / 100 g) sample and kept it at room temperature for 1 hour. After washing with PBS, differential pulse voltammetry was performed in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , the current on the electrode surface was detected in a 0.1 mol / L KCl solution, with a voltage range of 0-0.6 V, a pulse amplitude of 50 mV, and a pulse width of 0.2 s;

[0071] (3) Calculation of results: Use the current value measured in step (2) to find the corresponding bacterial concentration on the standard curve.

[0072] Example 6

[0073] A method for detecting Lactobacillus delbrueckii subsp. bulgaricus in whole-fat sterilized milk using a cell-imprinted electrochemical sensor.

[0074] The preparation of this example is the same as that of Example 5, except that the sample used in step (2) is sterilized milk (purchased from a supermarket).

[0075] A method for detecting Streptococcus salivarius subsp. thermophilus in flavored yogurt using a cell-imprinted electrochemical sensor.

[0076] (1) Preparation of standard curve: The method for preparing the standard curve is the same as that in Example 4;

[0077] (2) Sample detection: The cell imprinted electrochemical sensor obtained in Example 2 was immersed in 100 μL of flavored yogurt (purchased from a supermarket, with Lactobacillus delbrueckii subspecies bulgaricus added in an amount of ≥ 2.3 × 10 8 CFU / 100 g, Streptococcus salivarius thermophilus subspecies added ≥3.2×10 8 / 100g) sample and kept at room temperature for 1 hour. After washing with PBS, differential pulse voltammetry was used in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , the current on the electrode surface was detected in a 0.1 mol / L KCl solution, with a voltage range of 0-0.6 V, a pulse amplitude of 50 mV, and a pulse width of 0.2 s;

[0078] (3) Calculation of results: Use the current value measured in step (2) to find the corresponding bacterial concentration on the standard curve.

[0079] Comparative Example 1

[0080] The method of Example 1 was used to prepare the sensor, except that no Lactobacillus delbrueckii subsp. bulgaricus template was added in step (3). The method of Example 3 was used to detect bacterial solutions of Lactobacillus delbrueckii subsp. bulgaricus of different concentrations using the sensor prepared in Comparative Example 1.

[0081] Comparative Example 2

[0082] The sensor was prepared by the method of Example 2, except that polypyrrole was not added in step (3), and the template bacteria used was 10 9 CFU / mL of Streptococcus salivarius thermophilus subsp. The method of Example 4 was used to detect Streptococcus salivarius thermophilus subsp. bacterial liquids of different concentrations using the sensor prepared in Comparative Example 2.

[0083] Comparative Example 3

[0084] The method of Example 5 was used to prepare yogurt (purchased from a supermarket, with a live bacterial content of Lactobacillus delbrueckii subspecies bulgaricus ≥ 2×10 8 The detection of Lactobacillus delbrueckii subspecies bulgaricus in 1000 μg of Lactobacillus delbrueckii CFU / 100 g was performed, the only difference being that the sensor used in step (2) was prepared by the method of Comparative Example 1.

[0085] Comparative Example 4

[0086] The method of Example 7 was used to prepare flavored yogurt (purchased from a supermarket, with Lactobacillus delbrueckii subspecies bulgaricus added in an amount of ≥ 2.3 × 10 8 CFU / 100 g, Streptococcus salivarius thermophilus subspecies added ≥3.2×10 8 CFU / 100g), the only difference is that the sensor used in step (2) is prepared by the method of Comparative Example 2.

[0087] Comparative Example 5

[0088] The method of GB 4789.35-2023 National Food Safety Standard Microbiological Test for Lactobacillus was used to test yogurt (purchased from a supermarket, with a live bacterial content of Lactobacillus delbrueckii subspecies bulgaricus ≥ 2 × 10 8 CFU / 100 g).

[0089] experiment

[0090] Experimental Example 1

[0091] This experimental example provides the standard curves prepared in Example 3, Experimental Example 4, Comparative Example 1, and Comparative Example 2.

[0092] Figure 1 is the standard curve obtained in Example 3. When the concentration of Lactobacillus delbrueckii subspecies bulgaricus is 1×10 1 ~1×10 9 CFU / mL, its linear regression equation is y=-2.4473x+46.789, R 2 =0.9986, and the detection limit was 10 CFU / mL.

[0093] Figure 2 is the standard curve prepared in Example 4. When the concentration of Streptococcus salivarius thermophilus subsp. 1 ~1×10 9 CFU / mL, its linear regression equation is y=-2.6283x+48.697, R 2 =0.9988, and the detection limit was 10 CFU / mL.

[0094] Comparative Example 1 cannot specifically bind to the target bacteria due to the lack of imprinting sites of the template bacteria. The current value does not change significantly with the increase of bacterial concentration, indicating that lactic acid bacteria cannot be detected.

[0095] Comparative Example 2 lacks the functional monomer polypyrrole, so the lactic acid bacteria cannot be effectively polymerized into the polymer to form imprinting sites, and therefore the current value does not change much with the increase in bacterial concentration.

[0096] Compared with the comparative example, the current value on the surface of the cell imprinted electrochemical sensor decreases with the increase of bacterial concentration, indicating that the cell imprinted electrochemical sensor can successfully identify and bind to the target bacteria, and the accumulation of cells in the polymer hinders the current transport. From the above experimental results, it can be seen that the current value of the cell imprinted electrochemical sensor prepared by the present invention can be linearly related to the bacterial concentration when detecting lactic acid bacteria, indicating that the cell imprinted electrochemical sensor prepared by the present invention is a highly sensitive and stable lactic acid bacteria detection tool.

[0097] Experimental Example 2

[0098] This experimental example provides the number of Lactobacillus delbrueckii subspecies bulgaricus in yogurt measured in Example 5, Example 6, Comparative Example 3, and Comparative Example 5. The results are shown in Table 1.

[0099] The number of Lactobacillus delbrueckii subspecies bulgaricus measured in yogurt using a cell-imprinted electrochemical sensor was 3.45×10 8CFU / mL, and the number of Lactobacillus delbrueckii subspecies bulgaricus detected from sterilized milk was less than the detection limit of 10 CFU / mL. In Comparative Example 3, the sensor lacked imprinting sites and could not identify the target bacteria, and the result was less than the detection limit of 10 CFU / mL. In Comparative Example 5, the number of Lactobacillus delbrueckii subspecies bulgaricus measured by the plate count method was 3.3×10 8 The results measured by the cell imprinting electrochemical sensor were not much different from those measured by the plate counting method, and the former took 4 hours to detect, while the latter took 2 days to detect, indicating that the cell imprinting electrochemical sensor prepared by the present invention can quickly and effectively detect the number of lactic acid bacteria in yogurt.

[0100] Table 1

[0101]

[0102] Experimental Example 3

[0103] This experimental example provides the number of thermophilic Streptococcus salivarius subspecies in yogurt measured in Example 7 and Comparative Example 4.

[0104] The number of Streptococcus salivarius subsp. thermophilus measured in flavored yogurt using a cell-imprinted electrochemical sensor was 1.42×10 7 CFU / mL, and the cell imprinting electrochemical sensor for comparative example 4 could not be successfully prepared because the sensor lacked the functional monomer polypyrrole, and no lactic acid bacteria were detected, and the result was less than the detection limit of 10 CFU / mL. In the flavored yogurt sample containing multiple lactic acid bacteria, when the template bacteria in the cell imprinting sensor was Streptococcus salivarius thermophilus subsp., the number of Streptococcus salivarius thermophilus subsp. in the sample could be detected, indicating that the cell imprinting electrochemical sensor prepared by the present invention has specificity and can quantitatively detect lactic acid bacteria of different cell morphologies.

[0105] Table 2

[0106]

[0107] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

[0108] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither explicitly nor implicitly admitted to be prior art to the present invention.

Claims

1. A method for preparing a cell-imprinted electrochemical sensor, characterized in that The following steps are involved: 1) Preparation of activated biochar: heating the biomass to obtain biochar; then activating the biochar to obtain activated biochar; 2) Modified electrode: add PBS solution to the activated biochar, stir until dissolved, and prepare a suspension with a biochar concentration of 1 mg / mL; then polish the screen-printed electrode, rinse with ethanol and ultrapure water respectively, and dry at room temperature; take the activated biochar suspension and drop it on the surface of the above electrode, and obtain the modified electrode after natural drying; 3) Preparation of an electrode with a lactic acid bacteria-polypyrrole polymer: placing the modified electrode in a PBS solution containing polypyrrole, KCl and a lactic acid bacteria template, and performing electrochemical polymerization by cyclic voltammetry to obtain an electrode with a lactic acid bacteria-polypyrrole polymer; the lactic acid bacteria template includes but is not limited to Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus; 4) Preparation of cell imprinted electrochemical sensor: The electrode with lactic acid bacteria-polypyrrole polymer was rinsed with PBS and then immersed in lysozyme. NaOH was then added and voltage was applied to remove the lactic acid bacteria encapsulated in the polymer, leaving the imprinted cavity, thereby obtaining a cell imprinted electrochemical sensor with specific binding ability. The cell imprinted electrochemical sensor was used for rapid quantitative detection of lactic acid bacteria in dairy products.

2. The method for preparing a cell imprinted electrochemical sensor according to claim 1, characterized in that: Step 1) The specific preparation steps of the activated biochar are: heating the biomass at 600-800°C for 1-2 h to obtain the biochar; then mixing the biochar with potassium carbonate and heating it, and the entire pyrolysis process is carried out in nitrogen to obtain the activated biochar; the weight ratio of the biomass carbon to potassium carbonate is 1:2, the heating temperature after mixing is 800~900°C, and the heating time is 1~2 h.

3. The method for preparing a cell imprinted electrochemical sensor according to claim 1 or 2, characterized in that: Step 2) The specific steps of modifying the electrode are as follows: add PBS solution to the activated biochar, stir until dissolved, and prepare a suspension with a biochar concentration of 1 mg / mL; polish the screen-printed electrode to a mirror surface with 0.05 μm alumina powder, rinse with ethanol and ultrapure water, and dry at room temperature; drip the activated biochar suspension on the surface of the dried screen-printed electrode, and obtain the modified electrode after natural drying; the drip volume is 6~10 μL; the screen-printed electrode includes a working electrode, a reference electrode, and an auxiliary electrode; the working electrode is made of a bare glassy carbon electrode with a diameter of 3 mm; the auxiliary electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.

4. The method for preparing a cell imprinted electrochemical sensor according to claim 3, characterized in that: Step 3) The specific preparation steps of the electrode with lactic acid bacteria-polypyrrole polymer are as follows: put the modified electrode into 10 mL of 0.1-0.2 mol / L polypyrrole, 0.05-0.1 mol / L KCl and 10 7 ~10 9 Electrochemical polymerization was carried out in a PBS solution containing CFU / mL lactic acid bacteria template by cyclic voltammetry to obtain an electrode with lactic acid bacteria-polypyrrole polymer.

5. The method for preparing a cell imprinted electrochemical sensor according to claim 4, characterized in that: Step 4) The specific preparation steps of the cell imprinted electrochemical sensor are as follows: the lactic acid bacteria-polypyrrole polymer electrode is rinsed with PBS and then immersed in 10-15 mg / mL lysozyme for 1-2 hours. Subsequently, 0.1 mol / L NaOH is added and a voltage of 0.98 V is applied to remove the lactic acid bacteria encapsulated in the polymer, leaving the imprinted cavity to obtain the cell imprinted electrochemical sensor.

6. The method for preparing a cell imprinted electrochemical sensor according to claim 5, characterized in that: The lactic acid bacteria template includes but is not limited to Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus. The bacterial template is selected according to the different types of detected microorganisms; the PBS contains NaH2PO4 and Na2HPO4, both with a concentration of 0.1 mol / L and a pH of 7.4; the scanning number of the cyclic voltammetry electrochemical polymerization is 9-11, the voltage is 0-1.2V, and the scanning rate is 40-60 mV / s.

7. A method for rapid quantitative detection of lactic acid bacteria in dairy products, characterized in that A cell imprinting electrochemical sensor prepared by the method according to claim 6, wherein the method comprises the following steps: 1) Preparation of standard curve for detection of lactic acid bacteria by cell imprinting electrochemical sensor: The cell-imprinted electrochemical sensor was immersed in a known concentration of lactic acid bacteria solution and then rinsed with PBS. Differential pulse voltammetry was used to measure the concentration of [Fe(CN)6] 3- / 4- , KCl solution to detect the current on the electrode surface; draw a standard curve with the logarithm of the lactic acid bacteria solution concentration as the abscissa and the current value as the ordinate; 2) Detection of lactic acid bacteria in dairy products: The cell-imprinted electrochemical sensor was immersed in the sample to be tested and then rinsed with PBS. Differential pulse voltammetry was used to measure the concentration of [Fe(CN)6] 3- / 4- , KCl solution; the measured current value is used to find the corresponding bacterial concentration on the standard curve.

8. The method for rapid quantitative detection of lactic acid bacteria in dairy products according to claim 7, characterized in that: Step 1) The specific preparation steps of the standard curve of cell-imprinted electrochemical sensor for detecting lactic acid bacteria are as follows: immerse the cell-imprinted electrochemical sensor in 100 μL of lactic acid bacteria solution of known concentration and keep it at room temperature for 1 hour; then rinse it with PBS several times and use differential pulse voltammetry in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , 0.1 mol / L KCl solution to detect the current on the electrode surface; then draw a standard curve with the logarithm of the lactic acid bacteria solution concentration as the horizontal axis and the current value as the vertical axis; the lactic acid bacteria solution includes but is not limited to Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophilic; the concentrations of the bacteria solutions are 1×10 1 CFU / mL, 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL, the standard method for determining the concentration of bacterial solution is the plate count method.

9. The method for rapid quantitative detection of lactic acid bacteria in dairy products according to claim 7 or 8, characterized in that: Step 2) The specific steps for detecting lactic acid bacteria in dairy products are as follows: immerse the cell-imprinted electrochemical sensor in 100 μL of the sample to be tested and keep it at room temperature for 1 hour; then rinse it with PBS and use differential pulse voltammetry in 10 mL of 5 mmol / L [Fe(CN)6] 3- / 4- , 0.1 mol / L KCl solution; the measured current value is used to find the corresponding bacterial concentration on the standard curve.

10. The method for rapid quantitative detection of lactic acid bacteria in dairy products according to claim 9, characterized in that: The voltage range of the differential pulse voltammetry is 0-0.6V, the pulse amplitude is 50 mV, and the pulse width is 0.2 s; the samples to be tested include but are not limited to yogurt, fresh milk, sterilized milk, and milk powder.

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