阅读说明：本技术 一种即用型维生素b12检测载体及其制备方法 (Ready-to-use vitamin B12 detection carrier and preparation method thereof ) 是由 王舒乐 齐延林 张恒 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种即用型维生素B12检测载体及其制备方法,该制备方法包括以下步骤：(1)将维生素B12测试培养基与塑形填充剂混合,装入载体容器后冻干；(2)将抗氧化阻隔液与塑形填充剂混合,装入载体容器后冻干,获得位于维生素B12测试培养基固定层上的抗氧化阻隔层；(3)将已活化的维生素B12测试菌种分散于凝胶保护剂中,取菌悬液黏附于载体容器中并固定化干燥。本发明将精准定量的维生素B12测试菌种及维生素B12测试培养基溶液重塑于容器中,确保菌种及培养基的稳定性,无需菌种复壮及培养基称量、溶解、灭菌、分装等繁琐的制备过程,检测时只需加入维生素B12标准溶液或样品提取液即可完成实验,减少实验操作步骤,提高检测效率。(The invention discloses a ready-to-use vitamin B12 detection carrier and a preparation method thereof, wherein the preparation method comprises the following steps: (1) mixing vitamin B12 test medium with shaping filler, placing into carrier container, and lyophilizing; (2) mixing the antioxidant barrier solution with a shaping filler, filling the mixture into a carrier container, and freeze-drying to obtain an antioxidant barrier layer on a vitamin B12 test medium fixing layer; (3) dispersing activated vitamin B12 test strain in gel protectant, taking the strain suspension, adhering to carrier container, immobilizing and drying. According to the invention, the vitamin B12 test strain and the vitamin B12 test culture medium solution which are accurately and quantitatively remodeled in the container, so that the stability of the strain and the culture medium is ensured, the complicated preparation processes of strain rejuvenation, culture medium weighing, dissolving, sterilizing, subpackaging and the like are not needed, the experiment can be completed only by adding the vitamin B12 standard solution or the sample extracting solution during detection, the operation steps of the experiment are reduced, and the detection efficiency is improved.)

1. A preparation method of a ready-to-use vitamin B12 detection carrier is characterized by comprising the following steps:

(1) mixing the vitamin B12 test culture medium with a shaping filler, filling the mixture into a carrier container, and freeze-drying to obtain a vitamin B12 test culture medium fixed layer;

(2) mixing the antioxidant barrier solution with a shaping filler, filling into a carrier container to cover the freeze-dried vitamin B12 test medium fixing layer, and freeze-drying to obtain an antioxidant barrier layer on the vitamin B12 test medium fixing layer;

(3) dispersing the activated vitamin B12 test strain in a gel protective agent, taking the strain suspension to adhere to a carrier container, immobilizing and drying to obtain the ready-to-use vitamin B12 detection carrier.

2. The method for preparing a ready-to-use vitamin B12 detection vector, according to claim 1, wherein in step (1) (2), the shaping filler comprises: 0.5-5 wt% dextran 5000-7500, 1-10 wt% polyethylene glycol 4000-8000, 0.5-2 wt% alpha-cyclodextrin, and the balance water.

3. The method for preparing a ready-to-use vitamin B12 assay carrier according to claim 1 or 2, wherein the mixing volume ratio of the vitamin B12 test medium and the shaping filler in step (1) is (0.5-4): 1.

4. the preparation method of the ready-to-use vitamin B12 detection carrier, wherein in the step (2), the components of the antioxidant barrier liquid comprise: 10k to 40k of polyvinylpyrrolidone (PVP), 0.2 to 2wt% of vitamin E, 0.5 to 3wt% of sodium ascorbate, 0.001 to 0.05wt% of glutathione, and the balance of water.

5. The preparation method of the ready-to-use vitamin B12 detection carrier according to claim 1 or 4, wherein the volume ratio of the antioxidant barrier fluid and the shaping filler in the step (2) is (0.5-2): 1.

6. the method for preparing a ready-to-use vitamin B12 test carrier according to claim 1, wherein the gel protectant components of step (3) comprise: 1-10 wt% of mannitol, 0.05-0.5M of magnesium chloride, 0.2-5 wt% of milk casein, 0.05-0.2 wt% of guar gum/carrageenan/xanthan gum and the balance of water.

7. The method for preparing a ready-to-use vitamin B12 assay vector as claimed in claim 1, wherein the activated vitamin B12 test strain of step (3) is prepared by adding vitamin B12 test strain powder or beads to 0.5-5 mL of vitamin B12 test medium, adding 0.05-0.2 ng/mL of vitamin B12 standard solution 0.5-5 mL, and activating, wherein the vitamin B12 test strain comprises at least one of Lactobacillus reuteri and its variant strain, Escherichia coli and its variant strain.

8. The method for preparing a ready-to-use vitamin B12 assay carrier, according to claim 1, wherein the immobilization drying procedure of step (3) is: the pre-freezing stage is to control the temperature to be minus 80 ℃ to minus 60 ℃ and keep the temperature for 1 to 3 hours; in the primary sublimation drying stage, the temperature is controlled to be minus 60 ℃ to minus 48 ℃, the vacuum degree is reduced to 0.18 mbar to 0.38mbar, and the time for reaching the final vacuum degree is 7 hours to 12 hours; in the secondary sublimation drying stage, the temperature is controlled at 0 ℃, the vacuum degree is controlled at 0.18-0.38 mbar, and the time for reaching the final temperature is 6-12 hours; in the analysis and drying stage, the temperature is controlled at 27 ℃, the vacuum degree is controlled at 0.010-0.020 mbar, and the time for reaching the final temperature is 6-12 hours.

9. The preparation method of the ready-to-use vitamin B12 detection carrier, according to claim 1, wherein the carrier container is a microplate or a centrifuge tube or a culture tube.

10. A ready-to-use vitamin B12 test vector prepared by the method of any one of claims 1 to 9, wherein the ready-to-use vitamin B12 test vector is prepared.

Technical Field

The invention belongs to the technical field of preparation of water-soluble vitamin detection carriers, and particularly relates to a ready-to-use vitamin B12 detection carrier and a preparation method thereof.

Background

The microbiological method is the first method for measuring vitamins such as vitamin B12, folic acid, pantothenic acid, biotin, vitamin B6, nicotinic acid and the like in food according to the national standard. The preparation before the microbiological method experiment mainly comprises three steps: preparation of test strains, preparation of samples/standards and preparation of culture medium. Wherein, the preparation of the test strain needs passage for 2-3 generations to enhance activity, the consumed time is long, and the contamination probability is high. The correct preparation of the culture medium is the basis of the detection work of the microorganism laboratory, and the accuracy and the reliability of the detection work are concerned. The culture system for measuring the water-soluble vitamins by the national standard microbiological method is 10mL, the reagent dosage is large, and the detection cost is high.

Because of the wide nutrition requirement range of the microorganism, the nutrient components in the vitamin B12 test culture medium can reach more than 28, the content of partial reagent components is very small, and the weighing and preparing work is heavy. The commercialized dry powder synthetic culture medium solves the complicated preparation process of the culture medium to a certain extent, but the dry powder culture medium has higher storage condition, the dry powder is easy to absorb moisture, agglomerate and deteriorate after being unsealed, and the homogeneity and the repeatability of different batches of dry powder materials are difficult to ensure by the domestic existing dry powder culture medium production technology. No matter the laboratory self-prepared culture medium or the commercial dry powder synthetic culture medium is purchased, the preparation processes of weighing, dissolving, sterilizing and subpackaging are required, and the pollution risk brought by the substances to be detected is also frequently generated in the preparation process of the culture medium, so that the detection effect of the experimental method cannot be achieved.

Chinese invention patent CN201611094901 discloses a microplate for quantitatively detecting vitamin B12 by microbiological method, its kit and its preparation method, US invention patent US8357504B2 discloses a method for quantitatively detecting vitamin in mixture by microorganism and its kit, the above invention patent schemes all provide preparation methods of strain, culture medium, sterile water and standard, but still have the following problems: (1) the test strains are prepared by culturing the strains to be in a milk state by using skim milk powder, and the strains are not easy to separate; (2) drying the strains in a boiling mode at low vacuum room temperature easily causes partial bacteria liquid to be sprayed out of the container, so that the dried bacteria amount in the container is inconsistent; (3) the microporous plate only contains dry strains, a culture medium needs to be additionally prepared when the microporous plate is used, and the strain contamination risk exists in the preparation and subpackage process of the culture medium.

Disclosure of Invention

The invention provides a ready-to-use vitamin B12 detection carrier and a preparation method thereof, aiming at the defects of the prior art, the invention remolds precisely and quantitatively vitamin B12 test strains and vitamin B12 test culture medium solution in a container, ensures the stability of the strains and the culture medium, does not need the complex preparation processes of strain rejuvenation, culture medium weighing, dissolving, sterilizing, subpackaging and the like, the carrier container contains a vitamin B12 test culture medium layer, an antioxidant barrier layer and dry vitamin B12 test strains, and the experiment can be completed only by adding a vitamin B12 standard solution or a sample extracting solution during detection, thereby reducing the operation steps of the experiment and improving the detection efficiency.

The above object of the present invention is achieved by the following technical solutions:

a preparation method of a ready-to-use vitamin B12 detection carrier comprises the following steps:

(1) fully mixing the vitamin B12 test culture medium with a shaping filler, filling the mixture into a carrier container, and freeze-drying to obtain a vitamin B12 test culture medium fixed layer;

(2) fully mixing the antioxidant barrier solution with a shaping filler, filling the mixture into a carrier container to cover the freeze-dried vitamin B12 test medium fixing layer, and freeze-drying to obtain an antioxidant barrier layer positioned on the vitamin B12 test medium fixing layer;

(3) dispersing the activated vitamin B12 test strain in a gel protective agent, taking the strain suspension to adhere to a carrier container, immobilizing and drying to obtain the ready-to-use vitamin B12 detection carrier.

Preferably, but not limited to, a method for preparing a ready-to-use vitamin B12 test carrier, which comprises the following steps:

(1) fully mixing a vitamin B12 test culture medium with a shaping filler, packaging 50-200 mu L of the mixture into micropores, ensuring that the volume of the solution is full of the bottoms of the micropores, and quickly freezing and fixing the mixture for 1-3 hours in an aseptic freezing device at-80 to-60 ℃ to obtain a vitamin B12 test culture medium fixing layer;

(2) fully mixing an antioxidant barrier solution and a shaping filler, covering 30-60 mu L of the mixture on the surface of a frozen and fixed B12 test culture medium to ensure that the volume of the solution is enough to completely cover a culture medium fixing layer, and then quickly freezing and fixing the mixture in an aseptic freezing device at-80 to-60 ℃ for 1-3 hours to obtain a vitamin B12 test culture medium fixing layer and an antioxidant barrier layer;

(3) dispersing activated vitamin B12 test strains in a gel protective agent, controlling the light transmittance of the bacterial suspension within the range of 40-80%, adhering 1-10 mu L of bacterial suspension in the micropores, immobilizing and drying;

(4) and (5) packaging and storing the vitamin B12 detection micro-porous plate.

As an embodiment of the preparation method of the ready-to-use vitamin B12 detection carrier of the present invention, in the step (1) (2), the shaping filler component comprises: 0.5-5 wt% dextran 5000-7500, 1-10 wt% polyethylene glycol 4000-8000, 0.5-2 wt% alpha-cyclodextrin, and the balance water.

The shaping filler used in the invention can ensure that the culture medium solution provides a 'skeleton' after freeze-drying, ensures the loose and stable structure of the dried culture medium, and is beneficial to rapid and complete dissolution during rehydration.

In the concrete implementation, the shaping filler is sterilized by an autoclave at 121 +/-3 ℃ for 5-15 min and then rapidly cooled in a water bath for later use.

As an embodiment of the preparation method of the ready-to-use vitamin B12 detection carrier, the mixing volume ratio of the vitamin B12 test medium and the shaping filler in the step (1) is (0.5-4): 1.

as an embodiment of the preparation method of the ready-to-use vitamin B12 detection carrier, in the step (2), the components of the antioxidant barrier liquid include: 10k to 40k of polyvinylpyrrolidone (PVP), 0.2 to 2wt% of vitamin E, 0.5 to 3wt% of sodium ascorbate, 0.001 to 0.05wt% of glutathione, and the balance of water.

In the concrete implementation, the anti-oxidation barrier liquid is filtered and sterilized by using a sterile filter membrane with the aperture of 0.22-0.45 mu m for standby, and in order to achieve effective filtration, the filter membrane is wetted by sterile water in advance. Because the components of the culture medium contain a large amount of sugar and reducing components and have the problems of easy moisture absorption and easy oxidation, aiming at the two problems, the invention uses an anti-oxidation barrier solution and an isolation protection method of layered freeze-drying to ensure the stability of the immobilized culture medium.

As an embodiment of the preparation method of the ready-to-use vitamin B12 detection carrier, the volume ratio of the antioxidant barrier liquid and the shaping filler in the step (2) is (0.5-2): 1.

as an embodiment of the preparation method of the ready-to-use vitamin B12 detection carrier of the present invention, the gel protectant component of step (3) comprises: 1-10 wt% of mannitol, 0.05-0.5M of magnesium chloride, 0.2-5 wt% of milk casein, 0.05-0.2 wt% of guar gum/carrageenan/xanthan gum and the balance of water. The gel protective agent used in the invention can protect the freeze-dried strain from being stressed by freezing and drying, still keep higher survival rate, and ensure the stability of the long-term storage activity of the strain.

In the concrete implementation, the gel protective agent is sterilized by an autoclave at 121 +/-3 ℃ for 5-15 min and then rapidly cooled in a water bath for later use.

In one embodiment of the method for preparing the ready-to-use vitamin B12 assay vector according to the present invention, the activated vitamin B12 test strain in step (3) is obtained by adding vitamin B12 test strain powder or beads to 0.5-5 mL of a vitamin B12 test medium, and then adding 0.05-0.2 ng/mL of a vitamin B12 standard solution 0.5-5 mL for activation, wherein the vitamin B12 test strain comprises at least one of lactobacillus reuteri and its variant strain, escherichia coli and its variant strain.

Preferably, but not limited to, the activation temperature is controlled to be 37-42 ℃ and the activation time is 16-24 h in the activation process.

As an embodiment of the method for preparing the ready-to-use vitamin B12 detection carrier of the present invention, the immobilization drying procedure in step (3) is: the pre-freezing stage is to control the temperature to be minus 80 ℃ to minus 60 ℃ and keep the temperature for 1 to 3 hours; in the primary sublimation drying stage, the temperature is controlled to be minus 60 ℃ to minus 48 ℃, the vacuum degree is reduced to 0.18 mbar to 0.38mbar, and the time for reaching the final vacuum degree is 7 hours to 12 hours; the secondary sublimation drying stage is-60 ℃ to-48 → 0 ℃, the vacuum degree is controlled to be 0.18-0.38 mbar, and the time for reaching the final temperature is 6-12 hours; in the analysis and drying stage, the temperature is controlled at 27 ℃, the vacuum degree is controlled at 0.010-0.020 mbar, and the time for reaching the final temperature is 6-12 hours.

Because of the higher concentration of the immobilized culture medium and the large proportion of saccharides and proteins, the rapid heating and the vacuum pumping are not beneficial to the immobilization of the culture medium, so that the vacuum degree is controlled by one-time sublimation, the heating speed is controlled by two-time sublimation, and the analytic use is close to normal temperature and lower vacuum degree, thereby being beneficial to the sublimation of residual moisture and ensuring the dryness of strains and the culture medium.

As an embodiment of the preparation method of the ready-to-use vitamin B12 test carrier, the packaging and storage in the step (4) are that the carrier container with the fixed vitamin B12 culture medium and the fixed vitamin B12 test strain is sealed and stored by an aluminum foil bag, and a sterile drying agent and an antioxidant are placed in the carrier container and stored at the temperature of less than or equal to 8 ℃.

Preferably, but not limitatively, the types of carriers include: a micro-porous plate (200-450 mu L), a centrifuge tube (300-5000 mu L) and a culture test tube (1-10 mL).

It should be noted that the vitamin B12 test medium described in step (1) was prepared at a concentration of 2-fold concentration. The culture medium may be appropriately concentrated or diluted for further immobilization depending on the volume of the vessel and the culture volume required for the detection method. In the present invention, the test medium is not particularly limited, and the test medium may include a test medium provided in the national standard law, and a commercially available test medium, but is not limited thereto, and may be used as long as the test medium does not contain the component to be tested and the test medium can satisfy the requirement that the test bacterial species has a correlation with the growth of the concentration of the component to be tested.

A ready-to-use vitamin B12 detection vector is prepared by any one of the preparation methods of the ready-to-use vitamin B12 detection vector.

Compared with the prior art, the invention has the beneficial effects that:

the ready-to-use vitamin B12 detection carrier provided by the invention is suitable for quantitatively detecting vitamin B12 by a microbiological method, a vitamin B12 test strain and a culture medium for vitamin B12 determination are pre-immobilized in a microporous plate, when the vitamin B12 is detected, the carrier container comprises a vitamin B12 test culture base layer, an antioxidant barrier layer and a dry vitamin B12 test strain, and only a vitamin B12 standard solution or a sample extracting solution needs to be added, so that an experiment can be completed, the complicated process of strain and culture medium preparation is omitted, the experimental operation steps of adding the strain and the culture medium are also reduced, and the detection efficiency is improved.

The ready-to-use vitamin B12 detection carrier comprises a vitamin B12 test culture medium layer, an antioxidant barrier layer and dry vitamin B12 test strains. The dry strain and the solid culture medium have the advantages of high structural stability and good rehydration, can realize the immobilization of mass culture media and strains, can complete an experiment only by adding a vitamin B12 standard solution or a sample extracting solution when detecting the vitamin B12, does not need the complicated process of preparing the strain and the culture medium, also saves the experimental step of adding the strain and the culture medium, and improves the experimental efficiency of detecting the vitamin B12 by a microplate method.

According to the ready-to-use vitamin B12 detection carrier prepared by the technical scheme, the survival rate of dry strains is more than or equal to 92%, and the variation coefficient of a microporous plate in a batch is less than 5%; and the antioxidant blocking liquid is adopted to cover the culture medium for layered immobilization, so that the problems of moisture absorption, oxidative browning and the like of the culture medium are effectively prevented, the dry solid test culture medium is dissolved immediately after rehydration, and the culture effect is basically the same as that of a liquid culture medium.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.