CN115651962A - Detection method for rapidly determining vitamin B12 in food and dairy products - Google Patents
Detection method for rapidly determining vitamin B12 in food and dairy products Download PDFInfo
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Abstract
The invention belongs to the technical field of food rapid detection, and particularly relates to a detection method for rapidly determining vitamin B12 in food and dairy products, which comprises the following steps: s1, preparing a strain stock solution; s2, preparing a flat plate for determination; s3, preparing a standard solution; s4, preparing a sample solution; s5, preparing a standard curve and measuring the content of the vitamin B12. The invention adopts a cup and dish method to establish a rapid determination method of vitamin B12, overcomes the detection defects of the traditional microbiological method, has simple operation, high efficiency, low cost, good reproducibility and stable result, and is suitable for the detection requirements of mass samples.
Description
Technical Field
The invention belongs to the technical field of rapid detection of foods, and particularly relates to a detection method for rapidly detecting vitamin B12 in foods and dairy products.
Background
At present, the detection methods of vitamin B12 at home and abroad mainly comprise: microbiological method, high performance liquid chromatography, ultra performance liquid chromatography-tandem mass spectrometry, enzyme-linked immunosorbent assay and the like.
Compared with other methods, the method has the advantages that the microbial hair has higher sensitivity, and the vitamin B12 with biological activity can be detected. The national food safety standard of China also uses a microbiological method as a preferred method and an arbitration method of vitamin B12. However, the existing microbiological method has the following problems:
1. in terms of strain use, lactobacillus reuteri (Lactobacillus leichmannii ATCC 7830) is transferred to an agar culture medium to be activated and stored as a stock strain, and then the strain is inoculated every 15 days. Before the test, the stock strain is transferred to a broth culture medium for 24h to be reactivated, and then the stock strain is centrifugally eluted for a plurality of times to adjust the concentration of the strain to prepare inoculation liquid. The test process is easy to cause strain pollution, and has large workload and poor timeliness.
Although the method controls the concentration of the bacteria and the culture time within a certain range, the method is not suitable for the growth of strains in different generations by using the same concentration of the bacteria and the same culture time due to different strains in different generations. The growth rule of the bacteria has certain deviation, and the stability of the test result cannot be effectively controlled.
2. In the preparation of the sample solution, the sample is sterilized, pH adjusted, diluted, and 1.0mL, 2.0mL, 3.0mL, and 4.0mL of the sample extract are added to the test tube, and 5.0mL of the vitamin B12 assay culture solution is added after 5.0mL of water is added.
A large amount of glassware is used in the preparation process, and the requirement on cleaning the glassware is high. The test operation is complicated, the consumed time is long, and the detection requirement of a large batch of samples cannot be met. And the test process is easy to have human errors, so that the test result is unstable.
3. The culture of a single sample needs at least 60mL of culture medium, and the culture medium is divided into 12 glass test tubes. High cost and requires sufficient space for cultivation.
4. In the aspect of determination, in the existing method, the culture solution is uniformly mixed and then transferred to a cuvette, and a spectrophotometer is used for reading. 12 data are required to be measured for each sample, and the bacteria in the culture solution are in an active state, so that the data are easy to be unstable, the operation is complicated, and the measurement time is long.
5. In the aspect of test cost, the existing method uses two testers to calculate by taking 100 samples, the time is more than 35 days, and the consumable material is about 1.5 ten thousand yuan.
In conclusion, the existing microbiological method for detecting vitamin B12 has the problems of complex operation, long measuring time, high requirement on personnel, difficulty in mastering, easiness in pollution, poor reproducibility, incapability of meeting the requirement of rapid detection of mass samples and the like.
Although the chinese patent CN201010277447.5 discloses a biological detection method for measuring vitamin H, a method for rapidly and simply detecting the content of vitamin H is established by the linear relationship between the logarithmic value of the concentration of vitamin H and the diameter of the growth circle. However, the following problems still remain:
1. before a test is carried out, a bacterial suspension needs to be prepared, the preparation time of the bacterial suspension is long, generally 3 days, and the detection efficiency is low;
2. the adopted stock bacterial liquid needs to be repeatedly activated and passaged, so that the experimental result is influenced;
3. the culture medium content in the culture dish is 25mL, the thickness of the culture medium is large, growth and diffusion of bacteria are not facilitated, and a growth ring is not easy to observe and measure; simultaneously, a covering method is adopted: the thickness of the culture medium is large, the light transmission is not strong, and the measurement is difficult due to fuzziness;
4. the culture time is 2 days, and the culture time is longer.
Disclosure of Invention
In view of the above, the present invention provides a method for rapidly detecting vitamin B12 in food and dairy products.
The technical scheme of the invention is as follows:
a detection method for rapidly determining vitamin B12 in food and dairy products is characterized by comprising the following steps:
s1, preparing a strain stock solution; inoculating the lyophilized auxotrophic strain to lactobacillus broth culture medium, and culturing at 33-39 deg.C for 16-24 hr; then 3 generations of seeds are transferred to enhance vitality; centrifuging the activated bacterial liquid for 5min at 8000 r/min, discarding the supernatant, adding 10mL sterile water, and mixing; the elution was repeated 3 times to ensure no residual medium; adding culture medium containing glycerol for vitamin B12 determination, mixing to obtain strain stock solution, subpackaging in strain storage tube, and freezing at-70 deg.C;
s2, preparing a flat plate for determination; preparing a certain amount of culture medium for vitamin B12 determination, adding agar powder, sterilizing at 121 deg.C for 5min, cooling to 50-60 deg.C, adding appropriate amount of stock solution of strain to make the concentration of bacteria in the culture medium at the set OD 600 A value; after fully and uniformly mixing, subpackaging in a sterile culture dish; gently shaking the plate to uniformly spread the bacteria liquid, solidifying to obtain a plate for measurement, and storing at 2-4 deg.C for use, wherein the bacteria liquid is taken according to the amount during the test;
s3, preparing a standard solution;
s4, preparing a sample solution;
s5, preparing a standard curve and measuring the content of vitamin B12;
preparing a standard curve: placing the sterile oxford cups into a flat plate for vitamin B12 determination, transferring a standard curve solution into each oxford cup, and preparing 3 copies of a blank control group of sterile distilled water; culturing the flat plate in a constant temperature incubator at 33-39 ℃ for 16-24h; measuring the diameter of the strain growth circle of each solution; drawing a standard curve by taking the logarithmic value of the concentration of the vitamin B12 standard substance as an abscissa and the diameter of a growth ring as an ordinate;
determination of vitamin B12 content: transferring the sample solution into each Oxford cup, and performing 3 copies of a sterile distilled water blank control group; culturing the flat plate in a constant temperature incubator at 33-39 ℃ for 16-24h; the diameter of the circle of growth of the strain for each solution was measured, and then the content of vitamin B12 in the sample was calculated based on the standard curve.
The invention uses the specificity of the microorganism, gives certain nutrition condition or changes the living environment of the microorganism, and completes the determination of the single vitamin component through the growth, the reproduction and the metabolic expression of the microorganism. Although the microbiological method in the prior art is mature, the advantages of low detection limit, high sensitivity and reliable result of the microbiological method are determined by utilizing the extremely sensitivity of microorganisms to the storage environment and the specific expression of the microorganisms, the defects of long culture period, poor reproducibility and the like are caused by the easy contamination and strong specificity of the microorganisms. Especially, the activation culture and separation, no matter the traditional method or the modern detection technology, are limited by the detection sensitivity, and after pretreatment, the activation culture and the selective separation are mostly needed to be carried out, so that the method can be used for subsequent detection and analysis. The two steps in the traditional microorganism detection take as long as 24-96 hours, and are key speed-limiting steps in the whole detection process.
According to the method, the freeze-dried strain of the Lactobacillus reuteri is activated and passaged, and the same batch of reserved strains are subpackaged and frozen in the strain storage tube by using the glycerol, so that the activity of the strains can be maintained for a long time, the strain preparation time is saved, the experimental operation is simplified, and the pollution probability is effectively reduced; moreover, because the activity of the strains subpackaged in the same batch is basically consistent, repeated measurement is not needed when the plate is prepared. The activity of the test strains is effectively controlled, the stability of the test is ensured, the experiment operation is simplified, and the cross contamination is avoided. In addition, the prepared flat plate is equivalent to a specificity detection kit, has long storage time, can be taken before the test, saves the test time, effectively improves the detection efficiency, solves the problems of long test period and complex operation of the traditional microbiological method, and greatly shortens the detection time.
Further, in step S1, the auxotrophic strain is lactobacillus gasseri. In the invention, the vitamin B12 is a nutrient necessary for the growth of lactobacillus leishmanii, and according to the principle of molecular diffusion, the cup and dish method is utilized to ensure that the vitamin B12 is diffused in a spherical shape in a specific culture medium containing lactobacillus leishmaniasis to form a spherical area containing a certain concentration of the vitamin B12. Lactobacillus leishmanii cannot grow and propagate in a medium area without vitamin B12, and grows and propagates in a medium area with vitamin B12 to form a turbid growth circle. The size of the growth circle of the Lactobacillus leishmanii is in a linear relation with the concentration of the vitamin B12 in a certain concentration range.
Further, in step S1, a vitamin B12 assay medium containing 10 to 30% glycerol is added.
According to the invention, 10-30% of glycerol is added, and the glycerol is used for protecting the strains, so that the strain preparation time is saved, the test operation is simplified, the strain activity is more stable, and the test effectiveness is ensured.
Further, in step S2, the concentration of agar in the medium for vitamin B12 determination is 0.5% to 2%.
In the invention, by controlling the specific proportion of the agar in the flat plate for determination, a proper growth condition is provided for lactobacillus leishmaniae, the effective growth of bacteria in the flat plate for determination is ensured, and the effectiveness of the test is ensured; meanwhile, the agar can be used as a coagulant to convert a liquid culture medium into a solidified culture medium, so that subsequent tests are facilitated.
Further, in step S2, the concentration OD of bacteria in the culture medium is adjusted 600 The value is 0.015-0.035.
In the invention, by controlling the specific ratio of the culture medium to the strain stock solution, OD can be passed 600 The accurate bacterium concentration in the culture medium is obtained through numerical values, the bacterium concentration can be accurately quantified, and therefore the detection result error of each flat plate can be controlled. The OD 600 The values refer to the absorbance values at a wavelength of 600 nm.
According to the invention, a large number of creative tests show that the actual value of the concentration of the strain stock solution in the culture medium can be obtained more quickly and accurately under the wavelength of 600nm light, so that experimental errors are avoided, and the accuracy of experimental results can be ensured.
Further, in step S2, the cells are subpackaged in 10-15mL sterile culture dishes. After the mixture is solidified, a plate for determination is prepared and stored at the temperature of 2-4 ℃ for later use.
According to the invention, the flat plate for determination is prepared in advance, stored in the environment of 2-4 ℃, taken according to the quantity before the test, so that the test operation is simplified, the test time is saved, and the detection efficiency is effectively improved.
Further, step S3, precisely weighing a vitamin B12 standard substance, diluting the vitamin B12 standard substance into 1ng/mL standard working solution by using distilled water, sterilizing the working solution at 121 ℃ for 5min, and rapidly cooling the working solution to room temperature; diluting with sterile water to obtain standard solution of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100ng/L for use.
Further, step S4 includes taking 1g of a sample to be detected, adding 40mL of distilled water, and fully and uniformly mixing; sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature; diluting the sample to be within the standard curve according to the concentration of the vitamin B12 in the sample for later use.
Further, in step S5, the sample adding amount of the oxford cup is 100-300uL.
Further, in step S5, the same batch of the plate prepared in step S2 is used for preparing the standard curve and measuring the content of vitamin B12, and the standard curve and the vitamin B12 are simultaneously detected.
It is emphasized that although lactobacillus leishmaniae used in the present application is disclosed in the international microbiological method, the test principle is completely different from the present application. Although the prior art discloses the principle of growth circle testing, no solution for growth circle testing with different fungi is disclosed in the art. It is emphasized that the technical solution of the present invention, which is not randomly selected, is obtained by a great number of creative experiments and the technical effect obtained by the inventor is unexpected.
In particular, the detection method of the present application is also applicable to detection of other bacteria (auxotrophic strains) having specificity for vitamin B12, and has a wide range of applications. The screening method for other specific fungi (auxotrophic strains) is as follows: taking 1mL of different sample solutions, respectively coating the different sample solutions into an MRS culture medium and a supplementary culture medium (the MRS culture medium added with a vitamin B12 standard solution in advance), culturing for a certain time, observing the difference of the growth rate and the morphology of bacteria on the two culture media, selecting single bacterial colonies with obvious difference in a vitamin B12 determination culture medium containing vitamin B12 for verification, and selecting effective bacteria for verification.
Meanwhile, aiming at different types of detection samples, the auxotrophic strain can be obtained through natural pollution samples such as agricultural products and milk powder rich in vitamin components, so that the specificity and sensitivity of detection can be further improved, and a detection plate has a lower detection limit of concentration for corresponding specific products.
In addition, compare with the luminousness that detects needs repeated confirmation under spectrophotometer, the detection experiment requirement of this application is lower, and experimental error is controllable, and detection efficiency obviously improves, can not receive the influence of spectrophotometer equipment stability in the testing process simultaneously, and detectivity and reproducibility are better.
In addition, the research and development in this field is different from other fields, and the research and development is not purely pioneering research, and often needs to be based on the prior art, and the corresponding exploration and development are performed with the help of the guidance of the existing results of the prior art, and for those skilled in the art, the technical solutions of the present application cannot be obtained through limited experiments.
The invention has the beneficial effects that:
1. after the freeze-dried strain of the lactobacillus gasseri is activated and passaged, the same batch of stored strains are separately packaged and frozen into the strain storage tube by using the glycerol, so that the activity of the strains can be maintained for a long time, the time for preparing the strains is saved, the experimental operation is simplified, and the pollution probability is effectively reduced.
2. And (4) taking one tube of the stock strains for measurement, and determining the addition amount required by the preparation of the plate of the batch of the stock solution of the strains. Because the activity of the strains packed in the same batch is basically consistent, repeated measurement is not needed when preparing the plate. The activity of the test strain is effectively controlled, the stability of the test is ensured, the experiment operation is simplified, and the cross contamination is avoided.
3. Adding agar powder into culture medium for vitamin B12 determination, sterilizing at 121 deg.C for 5min, cooling, adding appropriate amount of strain stock solution, and making into plate, and storing at 2-4 deg.C for 30 days. The test tool is used before the test, so that the test time is saved, and the detection efficiency is effectively improved.
4. The samples were sterilized after initial dilution. Diluting the mixture by a certain time into a sterile centrifuge tube by using sterile water, and adding the diluted mixture into an Oxford cup. A large number of utensils are not needed, the centrifuge tube and the oxford cup are disposable, cross interference in testing is avoided, the preparation process is simple and rapid, and the method is suitable for detection of large-batch samples.
5. A single sample only needs 15mL of culture medium at most, and is cultured in one culture dish, so that the cost is low and the occupied space is small.
6. In the measurement aspect, the diameter of the growth ring is measured by using a vernier caliper or a bacteriostatic ring measuring instrument. The measuring speed is high and the data is stable.
7. In terms of test cost, the time is about 2 days and the consumable is about 1000 yuan for 100 samples and 1 experimenter.
The invention adopts a cup and dish method to establish a rapid determination method of vitamin B12, overcomes the detection defects of the traditional microbiological method, has simple operation, high efficiency, low cost, good reproducibility and stable result, and is suitable for the detection requirements of mass samples.
Detailed Description
The technical solutions will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A detection method for rapidly determining vitamin B12 in food and dairy products adopts instruments, equipment and reagents which comprise:
1. the instrument equipment comprises: an inhibition zone measuring instrument, a sterilization pot, a disposable culture dish (sterile), an ultra-clean bench, an incubator at 36 ℃, a strain preservation tube (1.5 mL), pipette tips at 20-200 muL and 100-1000 muL (sterilized), a 50mL centrifuge tube and a 1.5mL centrifuge tube (sterilized).
2. Reagent: (1) Lactobacillus mansoni (Lactobacillus leichmannii ATCC 8014), commercially available; (2) vitamin B12 standard, commercially available; (3) A medium for vitamin B12 assay, which contains, in addition to vitamin B12, other nutrients necessary for the growth of Lactobacillus gasseri, and is commercially available; and (4) sterile water is prepared by self.
The method comprises the following steps:
1. preparation of stock solution of the strain:
inoculating lyophilized strain of Lactobacillus reuteri to lactobacillus broth, and culturing at 36 deg.C for 20h. And then 3 generations of seeds are transplanted to enhance the vitality. Centrifuging the activated bacterial liquid at 8000 r/min for 5min, discarding the supernatant, adding 10mL sterile water, and mixing. Elution was repeated 3 times to ensure no residual medium. Adding culture medium containing 20% glycerol for vitamin B12 determination, mixing to obtain stock solution, and subpackaging in strain storage tube at-70 deg.C for freezing storage.
2. Preparation of assay plate:
preparing a certain amount of culture medium for vitamin B12 determination, adding 1% agar powder, sterilizing at 121 deg.C for 5min, cooling to 55 deg.C, adding appropriate amount of strain stock solution to make the concentration OD of Lactobacillus reuteri in the culture medium 600 The value was 0.025. After being fully mixed, the mixture is subpackaged in sterile culture dishes with 12mL each. And (4) shaking the plate gently to uniformly spread the bacterial liquid, preparing a plate for measurement after the bacterial liquid is solidified, and storing at 2-4 ℃ for later use.
3. Preparation of a standard curve:
accurately weighing appropriate amount of vitamin B12 standard, diluting with distilled water to obtain 1ng/mL standard working solution, sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature. Diluting with sterile water to obtain standard solution of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100ng/L for use.
4. Preparation of sample solution:
1g of a sample to be detected is taken, added with 40mL of distilled water and fully mixed. Sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature. Diluting the sample to be within the standard curve according to the concentration of the vitamin B12 in the sample for later use.
5. Determination of vitamin B12 content:
the sterile Oxford cup is placed into a flat plate for vitamin B12 determination, 200uL of standard curve solution and sample solution are respectively transferred into each Oxford cup, and 200uL of sterile distilled water blank control group is transferred. In 3 replicates. The plate was incubated for 20h in an incubator at 36 ℃. The diameter of the circle of strain growth of each solution was measured.
And drawing a standard curve by taking the logarithmic value of the concentration of the vitamin B12 standard substance as an abscissa and the diameter of the growth ring as an ordinate. The vitamin B12 content of the sample was calculated according to the standard curve.
Example 2
A detection method for rapidly determining vitamin B12 in food and dairy products is characterized by comprising the following steps:
s1, preparing a strain stock solution; inoculating the freeze-dried auxotrophic strain to a lactobacillus broth culture medium, and culturing at 39 ℃ for 24h; then 3 generations of seeds are transferred to enhance the vitality; centrifuging the activated bacterial liquid for 5min at 8000 r/min, discarding the supernatant, adding 10mL sterile water, and mixing; the elution was repeated 3 times to ensure no residual medium; adding culture medium containing 30% glycerol for vitamin B12 determination, mixing to obtain strain stock solution, subpackaging in strain storage tube, and freezing at-70 deg.C;
s2, preparing a flat plate for determination; preparing a certain amount of culture medium for vitamin B12 determination, adding 2% agar powder, sterilizing at 121 deg.C for 5min, cooling to 60 deg.C, adding appropriate amount of stock solution of strain to make the strain concentration OD in the culture medium 600 0.035; after fully and uniformly mixing, subpackaging in a sterile culture dish; gently shaking the plate to uniformly spread the bacteria liquid, solidifying to obtain a plate for determination, and storing at 2-4 deg.C;
s3, preparing a standard solution;
s4, preparing a sample solution;
s5, preparing a standard curve and measuring the content of vitamin B12;
preparing a standard curve: placing the sterile oxford cups into a flat plate for vitamin B12 determination, transferring a standard curve solution into each oxford cup, and preparing 3 copies of a blank control group of sterile distilled water; the flat plate is placed in a constant temperature incubator at 39 ℃ for 24 hours; measuring the diameter of the strain growth circle of each solution; drawing a standard curve by taking the logarithmic value of the concentration of the vitamin B12 standard substance as an abscissa and the diameter of a growth ring as an ordinate;
determination of vitamin B12 content: transferring the sample solution into each Oxford cup, and performing 3 copies of a sterile distilled water blank control group; the flat plate is placed in a constant temperature incubator at 39 ℃ for 24 hours; the diameter of the growth circle of the strain of each solution was measured, and then the vitamin B12 content in the sample was calculated according to the standard curve.
Further, in step S1, the auxotrophic strain is lactobacillus gasseri.
Further, in step S2, the cells were dispensed into sterile petri dishes at 15 mL/dish.
Further, step S3, precisely weighing a vitamin B12 standard substance, diluting the vitamin B12 standard substance into 1ng/mL standard working solution by using distilled water, sterilizing the working solution at 121 ℃ for 5min, and rapidly cooling the working solution to room temperature; diluting with sterile water to obtain standard solution of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100ng/L for use.
Further, step S4 includes taking 1g of a sample to be detected, adding 40mL of distilled water, and fully and uniformly mixing; sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature; diluting the sample to be within the standard curve according to the concentration of the vitamin B12 in the sample for later use.
Further, in step S5, the amount of the oxford cup added was 300uL.
Further, in step S5, the same batch of the flat plate prepared in step S2 is used for preparing the standard curve and measuring the vitamin B12 content, and the detection is carried out simultaneously.
Example 3
A detection method for rapidly determining vitamin B12 in food and dairy products is characterized by comprising the following steps:
s1, preparing a strain stock solution; inoculating the lyophilized auxotrophic strain to a lactobacillus broth culture medium, and culturing at 33 ℃ for 16h; then 3 generations of seeds are transferred to enhance vitality; centrifuging the activated bacteria liquid at 8000 r/min for 5min, discarding the supernatant, adding 10mL sterile water, and mixing; the elution was repeated 3 times to ensure no residual medium; adding culture medium containing 10% glycerol for vitamin B12 determination, mixing to obtain strain stock solution, subpackaging in strain storage tube, and freezing at-70 deg.C;
s2, preparing a flat plate for determination; preparing a certain amount of culture medium for vitamin B12 determination, adding 0.5% agar powder, sterilizing at 121 deg.C for 5min, cooling to 50 deg.C, adding appropriate amount of stock solution of strain to make the strain concentration OD in the culture medium 600 Is 0.015; after fully and uniformly mixing, subpackaging in sterile culture dishes; gently shaking the plate to uniformly spread the bacteria liquid, solidifying to obtain a plate for determination, and storing at 2-4 deg.C;
s3, preparing a standard solution;
s4, preparing a sample solution;
s5, preparing a standard curve and measuring the content of the vitamin B12;
preparing a standard curve: placing the sterile oxford cups into a flat plate for vitamin B12 determination, transferring a standard curve solution into each oxford cup, and preparing 3 copies of a blank control group of sterile distilled water; the flat plate is placed in a constant temperature incubator at 33 ℃ for culturing for 16h; measuring the diameter of the strain growth circle of each solution; drawing a standard curve by taking the logarithmic value of the concentration of the vitamin B12 standard substance as an abscissa and the diameter of a growth ring as an ordinate;
determination of vitamin B12 content: transferring the sample solution into each oxford cup, and performing sterile distilled water blank control in 3 copies; the flat plate is placed in a constant temperature incubator at 33 ℃ for 16h; the diameter of the circle of growth of the strain for each solution was measured, and then the content of vitamin B12 in the sample was calculated based on the standard curve.
Further, in step S1, the auxotrophic strain is lactobacillus gasseri.
Further, in step S2, the cells were dispensed into 10mL sterile petri dishes.
Further, step S3 comprises accurately weighing vitamin B12 standard, diluting with distilled water to obtain 1ng/mL standard working solution, sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature; diluting with sterile water to obtain standard solution of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100ng/L for use.
Further, step S4 includes taking 1g of a sample to be detected, adding 40mL of distilled water, and fully and uniformly mixing; sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature; diluting the sample to be within the standard curve according to the concentration of the vitamin B12 in the sample for later use.
Further, in step S5, the amount of the oxford cup added was 100uL.
Further, in step S5, the same batch of the plate prepared in step S2 is used for preparing the standard curve and measuring the content of vitamin B12, and the standard curve and the vitamin B12 are simultaneously detected.
The experimental effect is verified:
1. a standard curve; the logarithmic values of the concentrations of the vitamin B12 standard and the diameters of the growth circles obtained by the method of example 1 are shown in Table 1 below, and based thereon, a standard curve (shown in the following table) was drawn:
TABLE 1 vitamin B 12 Standard curve
The concentration of vitamin B12 is in the range of 10-100ng/L, the logarithm value of the concentration of vitamin B12 has good linear relation with the diameter of the growth ring, the linear regression equation is Y = 7.9468X +4.8405, and the correlation coefficient R 2 = 0.9985. The deviation of each point of the standard curve is small, and the result is stable.
2. Performing precision test;
taking the vitamin B12 content of an SRM 1869a quality control sample as a reference, and performing parallel tests for 6 times; and compared with the measurement results of the national standard method (GB 5413.14-2010 national standard for food safety of infant food and vitamin B12 in milk), the results are shown in the following table 2
TABLE 2 comparison of the cup and dish method with the national standard method for determining the vitamin B12 content in SRM 1869a
As can be known from the specification of SRM 1869a, the content of the vitamin B12 in the sample is (4.47 +/-0.49) ug/100g, and the results (shown in the table 2) measured by 2 methods are all consistent with the labeled values, thereby meeting the requirements and having good method accuracy. Compared with the national Standard method, the cup and dish method has more accurate results, and the Relative Standard Deviation (RSD) of the parallel test is smaller.
3. Repeatability test
Randomly, 5 different samples were taken and 6 replicates were run for each sample. As can be seen from Table 3, the RSD of the vitamin B12 content in 5 different samples is less than 5%, and the method has good repeatability.
TABLE 3 repeatability test results of the cup and dish method
4. Strain viability assay with same batch split
By adopting the method, ten strains are randomly taken from the strain freezing tubes which are separately packed in the same batch, 1mL of bacterial liquid is respectively absorbed, diluted by a certain multiple, and the bacterial colonies on the plate are counted after the strains are cultured under a certain condition by using an MRS plate culture medium. The results are shown in the following table:
TABLE 4 test results of strain viability test
The results show that the total number of bacterial colonies of the frozen and stored strains subpackaged in the same batch is basically consistent after the culture, and the activities of the strains subpackaged in the same batch are basically consistent.
5. Flat panel test validation
The method is adopted to prepare the flat plate for determination, the flat plate is stored at the temperature of 2-4 ℃, and the batch flat plate is used to determine the content of the vitamin B12 in the SRM 1869a quality control sample in 0, 5, 15, 20, 25 and 30 days respectively. The results are shown in the following table:
TABLE 5 test results of the plate validity test
The results show that the deviation of the vitamin B12 measurement results can be controlled during the 30-day storage of the measurement plate, and the measurement plate is effective in the time, so that the storage life can reach 30 days.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein. It should be noted that the technical features not described in detail in the present invention can be implemented by any prior art.
Claims (10)
1. A detection method for rapidly determining vitamin B12 in food and dairy products is characterized by comprising the following steps:
s1, preparing a strain stock solution; inoculating the lyophilized auxotrophic strain to a lactobacillus broth culture medium, and culturing at 33-39 deg.C for 16-24h; then 3 generations of seeds are transferred to enhance vitality; centrifuging the activated bacterial liquid for 5min at 8000 r/min, discarding the supernatant, adding 10mL sterile water, and mixing; the elution was repeated 3 times to ensure no residual medium; adding culture medium containing glycerol for vitamin B12 determination, mixing to obtain stock solution of strain, and packaging into strain storage tube, and freezing at-70 deg.C;
s2, preparing a flat plate for determination; preparing a certain amount of vitamin B12 determination culture medium, adding agar powder, sterilizing at 121 deg.C for 5min, and cooling to 50-Adding appropriate amount of stock solution of strain at 60 deg.C to make the concentration of bacteria in the culture medium at the set OD 600 A value; after fully and uniformly mixing, subpackaging in sterile culture dishes; gently shaking the plate to uniformly spread the bacteria liquid, solidifying to obtain a plate for measurement, and storing at 2-4 deg.C for use, wherein the bacteria liquid is taken according to the amount during the test;
s3, preparing a standard solution;
s4, preparing a sample solution;
s5, preparing a standard curve and measuring the content of the vitamin B12;
preparing a standard curve: placing the sterile oxford cups into a flat plate for vitamin B12 determination, transferring a standard curve solution into each oxford cup, and preparing 3 copies of a blank control group of sterile distilled water; culturing the flat plate in a constant temperature incubator at 33-39 ℃ for 16-24h; measuring the diameter of the strain growth circle of each solution; drawing a standard curve by taking the logarithmic value of the concentration of the vitamin B12 standard substance as an abscissa and the diameter of a growth ring as an ordinate;
determination of vitamin B12 content: transferring the sample solution into each oxford cup, and performing sterile distilled water blank control in 3 copies; culturing the flat plate in a constant temperature incubator at 33-39 ℃ for 16-24h; the diameter of the circle of growth of the strain for each solution was measured, and then the content of vitamin B12 in the sample was calculated based on the standard curve.
2. The assay for the rapid determination of vitamin B12 in food and dairy products according to claim 1, wherein in step S1 the auxotrophic strain is Lactobacillus gasseri.
3. The assay method for the rapid determination of vitamin B12 in food and dairy products according to claim 1, wherein in step S1, a vitamin B12 assay medium containing 10-30% glycerol is added.
4. The assay for the rapid determination of vitamin B12 in food and dairy products according to claim 1, wherein in step S2 the concentration of agar in the medium for vitamin B12 determination is between 0.5% and 2%.
5. The assay for the rapid determination of vitamin B12 in food and dairy products according to claim 1 wherein in step S2 the bacterial concentration OD in the culture medium is adjusted 600 The value is 0.015-0.035.
6. The assay method for the rapid determination of vitamin B12 in foods and dairy products according to claim 1, wherein in step S2, the sample is dispensed into sterile culture dishes at a volume of 10-15 mL/dish.
7. The detection method for rapidly determining vitamin B12 in food and dairy products according to claim 1, wherein step S3 comprises accurately weighing vitamin B12 standard, diluting with distilled water to obtain 1ng/mL standard working solution, sterilizing at 121 ℃ for 5min, and rapidly cooling to room temperature; diluting with sterile water to obtain standard solution of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100ng/L for use.
8. The detection method for rapidly determining vitamin B12 in food and dairy products according to claim 1, wherein step S4 comprises taking 1g of a sample to be determined, adding 40mL of distilled water, and fully mixing; sterilizing at 121 deg.C for 5min, and rapidly cooling to room temperature; diluting the sample to be within the standard curve according to the concentration of the vitamin B12 in the sample for later use.
9. The detection method for rapidly detecting vitamin B12 in food and dairy products as claimed in claim 1, wherein the amount of the Oxford cup added in step S5 is 100-300uL.
10. The assay method for the rapid determination of vitamin B12 in food and dairy products according to claim 1, wherein in step S5, the preparation of the standard curve and the determination of the vitamin B12 content are performed simultaneously using the same batch of plates prepared in step S2.
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