CN112683625A - Method for detecting content of free biotin in infant formula food - Google Patents
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Abstract
The invention discloses a method for detecting the content of free biotin in infant formula food, which comprises the following steps: preparing a standard solution; preparing inoculation liquid; processing the sample; preparing a solution to be detected; sterilizing; inoculating; culturing; and (5) analyzing and testing. The preparation method of the biotin standard solution is optimized, so that the linearity of the obtained standard curve is better, and the preparation difficulty of the standard curve is reduced; meanwhile, the dosage of glucose and urea in the culture solution for biotin determination is optimized, so that the strains are basically not limited by the substrate concentration of a carbon source and a nitrogen source in the culture and detection processes, and the initial dosage is ensured not to generate obvious inhibition effect on the growth of the strains, thereby improving the maximum growth quantity of the strains in a biotin linear range, relatively reducing the error in the detection process and improving the detection accuracy. After optimization, the method has better reproducibility and sensitivity, simplifies the experimental steps, reduces the detection time and reduces the detection cost.
Description
Technical Field
The invention relates to the technical field of microbial food detection, in particular to a method for detecting the content of free biotin in infant formula food.
Background
Biotin, also known as vitamin H, belongs to the B group of vitamins, and is a sulfur-containing vitamin. Biotin is one of important nutrients of dairy products, and the vitamin is involved in three major nutritional metabolisms of organisms, is a nutrient essential for the growth and development of the organisms and is involved in various major metabolic activities of the organisms. Especially, the biological nutrient supplement has special importance for infants and pregnant and lying-in women, so that the biological nutrient supplement is necessary to be added into infant dairy products, pregnant and lying-in women dairy products and food nutrient supplements. The main uses of biotin are: form the photosensitive substance in the visual sediment cells, maintain the integrity and the soundness of the epithelial tissue structure, enhance the immune response and the resistance of the organism and maintain the normal growth and development.
In recent years, as the development of biotin is receiving more attention, detection methods are more and more, but the principle, the aging and the aimed product of each detection method are different, so that in practical application, it is also important to select an appropriate biotin detection method according to different samples. Many common biotin detection methods are available, and mainly include a microbiological method, a high performance liquid chromatography, an enzyme linked immunosorbent assay, a fluorescence method and the like. The method of the microorganism is a method which is widely applied at present, and is the only detection method in national standard detection methods of free biotin in infant food and dairy products, and the method is mainly used for detecting the conditions of low biotin content and complex components in samples.
However, many problems are always encountered in practical operation of the microbiological method, for example, the light absorption value after the culture of the test tube does not form a gradient or the test bacteria does not grow, the concentration of the test sample does not fall within the range of a standard curve, the preparation operation of inoculation liquid is complex, the microorganisms are cross-contaminated, and the like, and in addition, the detection error of biotin is remarkably increased due to the long activation period of the strain, the complicated sample treatment process and the like. Aiming at the problems, the invention aims to provide a method for detecting the content of free biotin in infant formula food, which optimizes the aspects of strain activation, strain suspension preparation, standard solution preparation, sample treatment and the like so as to improve the test quality and the accuracy of a detection result and reduce the detection cost.
Disclosure of Invention
In order to solve the above problems, the first aspect of the present invention provides a method for detecting the content of free biotin in an infant formula, comprising the following steps:
s1, preparing a standard solution, namely weighing a certain amount of biotin standard substance, and using an ethanol solution to prepare the standard solution with different concentrations;
s2, preparing an inoculation liquid, namely directly inoculating a reserved strain into lactobacillus broth to culture for 16-20h at 36 +/-1 ℃ to obtain an activated strain; centrifuging the activated strain, and removing the supernatant; adding a sodium chloride solution, uniformly mixing, centrifuging again, removing a supernatant, repeating the operation for 2-3 times, and then adding a certain amount of sodium chloride solution for dilution to obtain a inoculation solution;
s3, processing the sample, grinding and sieving the powdery sample; uniformly mixing the liquid sample before use;
s4, preparing a liquid to be detected, wherein the liquid to be detected comprises a standard series tube and a sample series tube;
s5, sterilizing, namely covering test tubes in the S4 with test tube caps, and placing the test tubes in a sterilization kettle for sterilization;
s6, inoculating, and under the aseptic operation condition, adding an inoculating liquid into all test tubes in the S5, wherein blank inoculation and blank sample are not performed in a standard curve tube except for blank inoculation and blank sample;
s7, culturing, namely putting all the test tubes obtained in the step S6 into an incubator, and culturing for 18-20 h at constant temperature;
s8, analyzing and testing, mixing the cultured test tubes uniformly by using a vortex mixer, and testing at 550nm by using a cuvette with the thickness of 1 cm.
As a preferable technical scheme, the method for detecting the content of the free biotin in the infant formula food comprises the following specific steps:
s1, preparing a standard solution, namely accurately weighing 101.41mg of a biotin standard substance, metering the volume to 1000ml by using an ethanol solution, and preparing a standard stock solution with the concentration of 101.41 mu g/ml; sucking 1mL of standard stock solution, and fixing the volume to 100mL by using an ethanol solution to prepare a standard intermediate solution with the concentration of 1.0141 mu g/mL; sucking 1ml of standard intermediate solution, using an ethanol solution to fix the volume to 100ml, preparing standard working solution with the concentration of 10.141ng/ml, and preparing the working solution before use; a certain amount of standard working solution is sucked and diluted into 0.20282ng/ml high-concentration standard solution and 0.10141ng/ml low-concentration standard solution by using ethanol solution, and the standard working solution is prepared before use;
s2, preparing an inoculation liquid, transferring a strain lactobacillus plantarum into a lactobacillus agar culture medium, culturing for 20-24 h in a constant-temperature incubator at 37 +/-1 ℃, taking out, and storing in a refrigerator at 2-4 ℃ to serve as a storage strain; inoculating the stock strain into lactobacillus broth, and culturing at 36 + -1 deg.C for 16-20h to obtain activated strain; centrifuging the activated strain at 2000r/min for 2-3min, and removing supernatant; adding 1mL of sterilized NaCl solution, uniformly mixing, centrifuging for 2-3min, removing supernatant, repeating the operation for 2-3 times, and then adding sodium chloride solution for dilution to obtain inoculation liquid;
s3, processing the sample, grinding and sieving the powdery sample; uniformly mixing the liquid sample before use; accurately weighing a proper amount of sample (about 0.2-0.5 mu g containing biotin), pouring the sample into a 250ml conical flask, adding 100ml of sulfuric acid solution, hydrolyzing at 80-100 ℃ for 1-2 h, cooling, adjusting the pH to 4.5 +/-0.2 by using an alkaline carbonate solution, transferring into a 250ml volumetric flask, fixing the volume by using water, and uniformly mixing; filtering with filter paper to obtain filtrate, discarding the first several mL, sucking 5mL of filtrate, adding 20mL of water, adjusting pH to 6.8 + -0.2 with alkali carbonate solution, transferring into 100mL volumetric flask, and fixing volume to obtain sample extractive solution;
s4, preparing a liquid to be detected, wherein the liquid to be detected comprises a standard series tube and a sample series tube;
standard series of tubes: taking test tubes C1-C10, respectively adding 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL of low-concentration standard solution and 3.0mL, 4.0mL and 5.0mL of high-concentration standard solution, supplementing water to 5.00mL, adding 5.0mL of culture solution for biotin determination, uniformly mixing, and making 3 parallel samples per gradient; wherein C1 is a non-inoculated blank and C2 is an inoculated blank;
sample series tube: taking test tubes D1-D5, adding 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of sample extracting solution respectively, supplementing water to 5.0mL (except for a D5 tube), adding 5.0mL of culture solution for biotin determination, uniformly mixing, and preparing 3 parallel samples per gradient, wherein D5 is a sample blank tube;
s5, sterilizing, namely covering test tubes in the S4 with test tube caps, and placing the test tubes in a sterilization kettle for sterilization;
s6, inoculating, and under the aseptic operation condition, adding 50 mu L of inoculating liquid into all test tubes in the S5 by using a liquid transfer gun, wherein the non-inoculated blank tube C1 and the sample blank tube D5 are excluded;
s7, culturing, namely putting all the test tubes obtained in the step S6 into an incubator, and culturing for 18-20 h at the constant temperature of 36 +/-1 ℃;
and S8, analyzing and testing, namely uniformly mixing the cultured test tubes by using a vortex mixer, adjusting the light transmittance to 100% by using an inoculation blank tube C2 in a cuvette with the thickness of 1cm at 550nm, and then sequentially measuring the light absorption values of a standard series tube and a sample series tube.
As a preferable technical solution, in step S1, the volume fraction of the ethanol solution is 20%.
In a preferable embodiment, in step S2, the absorbance of the inoculation liquid is 0.5 to 0.65.
In a preferred embodiment, in step S3, the sieve plate has a sieve opening size of 0.3mm to 0.5 mm.
As a preferable mode, in step S4, the raw material of the culture solution for biotin action includes 10.0g of casamino acid, 30.0g of glucose, 16.0g of urea, 1.6g of aspartic acid, 0.8g of serine, 1.6g of glycine, 1.0g of threonine, 1.0g of alanine, 1.4g of arginine, 0.4g of tyrosine, 0.7g of valine, 0.3g of methionine, 1.0g of phenylalanine, 0.7g of isoleucine, 1.6g of leucine, 1.0g of dipotassium hydrogen phosphate, 1.0g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate, 20.0mg of sodium chloride, 20.0mg of ferrous sulfate, 20.0mg of leucine, and a predetermined amount of water.
As a preferable technical scheme, in the step S5, the sterilization temperature is 121 ℃, and the sterilization time is 5 min.
As a preferred technical solution, step S5 includes the following test procedures:
(1) and (3) immediately taking out the culture medium from the pressure cooker after the sterilization is finished, putting the culture medium into an ice water bath for timely cooling, wherein the obtained culture medium is light yellow, and if the color of the culture medium is dark after the sterilization, the culture medium cannot be used and needs to be prepared again.
As a preferred technical solution, step S8 includes the following test procedures:
(1) visual inspection is carried out on each test tube, the culture solution in the inoculation blank tube C2 is clear, and if turbidity occurs, the result is invalid;
(2) and (4) taking an inoculation blank tube C2 as a control, measuring the absorbance of the test tube with the highest concentration standard curve, re-measuring after two hours, and taking out all test tubes to measure the absorbance A if the difference of the light transmittance obtained by the two times is less than 2%.
The second aspect of the invention also provides the use of the method for detecting the content of free biotin in an infant formula.
Advantageous effects
The preparation method of the biotin standard solution is optimized, so that the linearity of the obtained standard curve is better, and the preparation difficulty of the standard curve is reduced; the lactobacillus plantarum is directly transferred into lactobacillus broth for activation, and the activity is better than that of the lactobacillus broth after being transferred into lactobacillus agar culture medium for activation, so that the activity of the strain is improved, and the experimental steps are simplified. The absorbance of the inoculation liquid is set to be 0.5-0.65, so that the experimental error caused by the concentration of the inoculation liquid can be effectively reduced. The invention optimizes the dosage of glucose and urea in the culture solution for biotin determination, so that the strain is not limited by the substrate concentration of carbon source and nitrogen source in the culture and detection processes, and the initial dosage can not generate obvious inhibition effect on the growth of the strain, thereby improving the maximum growth amount of the strain in the biotin linear range, relatively reducing the error in the detection process and improving the detection accuracy. After optimization, the method has better reproducibility and sensitivity, simplifies the experimental steps, reduces the detection time and reduces the detection cost.
Drawings
Fig. 1 is a standard graph.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
In order to solve the above problems, a first aspect of the present invention provides a method for detecting the content of free biotin in an infant formula, comprising the following steps:
s1, preparing a standard solution, namely weighing a certain amount of biotin standard substance, and using an ethanol solution to prepare the standard solution with different concentrations;
s2, preparing an inoculation liquid, namely directly inoculating a reserved strain into lactobacillus broth to culture for 16-20h at 36 +/-1 ℃ to obtain an activated strain; centrifuging the activated strain, and removing the supernatant; adding a sodium chloride solution, uniformly mixing, centrifuging again, removing a supernatant, repeating the operation for 2-3 times, and then adding a certain amount of sodium chloride solution for dilution to obtain a inoculation solution;
s3, processing the sample, grinding and sieving the powdery sample; uniformly mixing the liquid sample before use;
s4, preparing a liquid to be detected, wherein the liquid to be detected comprises a standard series tube and a sample series tube;
s5, sterilizing, namely covering test tubes in the S4 with test tube caps, and placing the test tubes in a sterilization kettle for sterilization;
s6, inoculating, and under the aseptic operation condition, adding an inoculating liquid into all test tubes in the S5, wherein blank inoculation and blank sample are not performed in a standard curve tube except for blank inoculation and blank sample;
s7, culturing, namely putting all the test tubes obtained in the step S6 into an incubator, and culturing for 18-20 h at constant temperature;
s8, analyzing and testing, mixing the cultured test tubes uniformly by using a vortex mixer, and testing at 550nm by using a cuvette with the thickness of 1 cm.
In some preferred embodiments, the method for detecting the content of free biotin in the infant formula comprises the following specific steps:
s1, preparing a standard solution, namely accurately weighing 101.41mg of a biotin standard substance, metering the volume to 1000ml by using an ethanol solution, and preparing a standard stock solution with the concentration of 101.41 mug/ml; sucking 1mL of standard stock solution, and fixing the volume to 100mL by using an ethanol solution to prepare a standard intermediate solution with the concentration of 1.0141 mu g/mL; sucking 1ml of standard intermediate solution, using an ethanol solution to fix the volume to 100ml, preparing standard working solution with the concentration of 10.141ng/ml, and preparing the working solution before use; a certain amount of standard working solution is sucked and diluted into 0.20282ng/ml high-concentration standard solution and 0.10141ng/ml low-concentration standard solution by using ethanol solution, and the standard working solution is prepared before use;
s2, preparing an inoculation liquid, transferring a strain lactobacillus plantarum into a lactobacillus agar culture medium, culturing for 20-24 h in a constant-temperature incubator at 37 +/-1 ℃, taking out, and storing in a refrigerator at 2-4 ℃ to serve as a storage strain; inoculating the stock strain into lactobacillus broth, and culturing at 36 + -1 deg.C for 16-20h to obtain activated strain; centrifuging the activated strain at 2000r/min for 2-3min, and removing supernatant; adding 1mL of sterilized NaCl solution, uniformly mixing, centrifuging for 2-3min, removing supernatant, repeating the operation for 2-3 times, and then adding sodium chloride solution for dilution to obtain inoculation liquid;
s3, processing the sample, grinding and sieving the powdery sample; uniformly mixing the liquid sample before use; accurately weighing a proper amount of sample (about 0.2-0.5 mu g containing biotin), pouring the sample into a 250ml conical flask, adding 100ml of sulfuric acid solution, hydrolyzing at 80-100 ℃ for 1-2 h, cooling, adjusting the pH to 4.5 +/-0.2 by using an alkaline carbonate solution, transferring into a 250ml volumetric flask, fixing the volume by using water, and uniformly mixing; filtering with filter paper to obtain filtrate, discarding the first several mL, sucking 5mL of filtrate, adding 20mL of water, adjusting pH to 6.8 + -0.2 with alkali carbonate solution, transferring into 100mL volumetric flask, and fixing volume to obtain sample extractive solution;
s4, preparing a liquid to be detected, wherein the liquid to be detected comprises a standard series tube and a sample series tube;
standard series of tubes: taking test tubes C1-C10, respectively adding 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL of low-concentration standard solution and 3.0mL, 4.0mL and 5.0mL of high-concentration standard solution, supplementing water to 5.00mL, adding 5.0mL of culture solution for biotin determination, uniformly mixing, and making 3 parallel samples per gradient; wherein C1 is a non-inoculated blank and C2 is an inoculated blank;
sample series tube: taking test tubes D1-D5, adding 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of sample extracting solution respectively, supplementing water to 5.0mL (except for a D5 tube), adding 5.0mL of culture solution for biotin determination, uniformly mixing, and preparing 3 parallel samples per gradient, wherein D5 is a sample blank tube;
s5, sterilizing, namely covering test tubes in the S4 with test tube caps, and placing the test tubes in a sterilization kettle for sterilization;
s6, inoculating, and under the aseptic operation condition, adding 50 mu L of inoculating liquid into all test tubes in the S5 by using a liquid transfer gun, wherein the non-inoculated blank tube C1 and the sample blank tube D5 are excluded;
s7, culturing, namely putting all the test tubes obtained in the step S6 into an incubator, and culturing for 18-20 h at the constant temperature of 36 +/-1 ℃;
and S8, analyzing and testing, namely uniformly mixing the cultured test tubes by using a vortex mixer, adjusting the light transmittance to 100% by using an inoculation blank tube C2 in a cuvette with the thickness of 1cm at 550nm, and then sequentially measuring the light absorption values of a standard series tube and a sample series tube.
In some preferred embodiments, in step S1, the ethanol solution has a volume fraction of 20%. In the invention, the volume fraction of the ethanol solution is 20%, the linearity of the standard curve is greatly improved, the standard working solution is prepared by using the 50% ethanol solution, and the obtained standard curve hardly meets the linearity requirement, because the ethanol volatilization amount is large due to the problems of high temperature and test tube airtightness in the sterilization process when the ethanol concentration is large, the volume of the test tube is greatly changed, and the curve linearity is poor; meanwhile, the growth of the lactobacillus plantarum can be inhibited when the ethanol concentration is higher, and the growth amount of the lactobacillus plantarum is influenced, so that the linearity of a standard curve is poor or not good. When the volume fraction of the ethanol is reduced to 20%, the influence factors are greatly reduced, a standard curve with good linearity is easier to obtain, and the detection cost is reduced.
In some preferred embodiments, in step S2, the absorbance of the inoculation liquid is 0.5 to 0.65. In the invention, the absorbance of the inoculation liquid is 0.5-0.65, the absorbance value of the inoculation liquid reflects the concentration value of the inoculation liquid, and too low concentration of the inoculation liquid can cause the time for the test tube to exhaust biotin to be prolonged, thereby increasing the probability of bacterial contamination; too high concentration of the inoculation liquid can cause too large growth amount of the test tube, and the concentration of the inoculation liquid is only in a linear relation with absorbance within a certain range, so that the concentration is not in a linear relation with the absorbance when being too high; the absorbance of the inoculation liquid is set to be 0.5-0.65, and experimental errors caused by the concentration of the inoculation liquid can be effectively reduced.
In some preferred embodiments, the inoculation liquid is centrifuged and washed 3-4 times before use. According to the method, the seed culture solution is subjected to centrifugal washing for 3-4 times before detection, so that the components of the seed culture medium can be effectively removed, the removal rate is over 99.9%, and the influence of residual amino nitrogen and trace growth factors on the detection process can be ignored, so that the nutrient components of the seed culture medium are prevented from being brought into the detection culture medium to cause errors on detection.
In some preferred embodiments, in step S3, the sieve plate has a pore size of 0.3mm to 0.5mm when sieved.
In some preferred embodiments, in step S3, the basic carbonate solution is a mixed solution of sodium carbonate and sodium bicarbonate, and the volume ratio of the two is 1: 2; in GB 5009.259-2016, sodium hydroxide is used to adjust the pH value, although the content of sodium hydroxide is low, the sodium hydroxide still affects the biotin when the sodium hydroxide is not completely reacted with acid in the sample, and a small amount of biotin may be decomposed, resulting in experimental errors.
In some preferred embodiments, in step S4, the raw material of the biotin-determination culture solution includes 10.0g of casamino acid, 30.0g of glucose, 16.0g of urea, 1.6g of aspartic acid, 0.8g of serine, 1.6g of glycine, 1.0g of threonine, 1.0g of alanine, 1.4g of arginine, 0.4g of tyrosine, 0.7g of valine, 0.3g of methionine, 1.0g of phenylalanine, 0.7g of isoleucine, 1.6g of leucine, 1.0g of dipotassium hydrogen phosphate, 1.0g of potassium dihydrogen phosphate, 0.4g of magnesium sulfate, 20.0mg of sodium chloride, 20.0mg of ferrous sulfate, 20.0mg of leucine, and an amount of water of manganese sulfate. The invention optimizes the components and content in the culture solution for biotin determination, glucose is the main source of carbon source required by thallus growth, urea can provide inorganic nitrogen source for thallus growth and has the function of adjusting pH value, and the influence of the urea and the inorganic nitrogen source on thallus growth is larger than that of other components, so the invention optimizes the dosage of the glucose and the urea in the culture solution for biotin determination, ensures that the bacterial strain is not limited by the substrate concentration of the carbon source and the nitrogen source in the culture and detection processes basically, and ensures that the initial dosage can not generate obvious inhibition effect on the growth of the bacterial strain, thereby improving the maximum growth amount of the bacterial strain in a biotin linear range, relatively reducing the error in the detection process and improving the detection accuracy.
In some preferred embodiments, in step S5, the sterilization temperature is 121 ℃ and the sterilization time is 5 min.
In some preferred embodiments, the following test procedure is included in step S5:
(1) and (3) immediately taking out the culture medium from the pressure cooker after the sterilization is finished, putting the culture medium into an ice water bath for timely cooling, wherein the obtained culture medium is light yellow, and if the color of the culture medium is dark after the sterilization, the culture medium cannot be used and needs to be prepared again.
In some preferred embodiments, the following test procedure is included in step S8:
(1) visual inspection is carried out on each test tube, the culture solution in the inoculation blank tube C2 is clear, and if turbidity occurs, the result is invalid;
(2) and (4) taking an inoculation blank tube C2 as a control, measuring the absorbance of the test tube with the highest concentration standard curve, re-measuring after two hours, and taking out all test tubes to measure the absorbance A if the difference of the light transmittance obtained by the two times is less than 2%.
The second aspect of the invention also provides the use of the method for detecting the content of free biotin in an infant formula.
Examples
The technical solution of the present invention is described in detail by the following examples, but the scope of the present invention is not limited to the examples. Unless otherwise specified, all the raw materials in the present invention are commercially available.
Example 1
1. Range of
The method is suitable for measuring the content of free biotin in infant formula food.
2. Principle of
The content of the substance to be detected in the sample is quantitatively determined by utilizing the specificity and the sensitivity of the Lactobacillus plantarum (Lactobacillus plantarum) to free biotin. In a culture medium containing all nutrient components except the substance to be detected, the growth of microorganisms and the content of the substance to be detected are in a linear relation, and the content of the substance to be detected in the sample can be calculated by comparing the light transmittance with a standard working curve.
3. Reagents and materials
Unless otherwise specified, the reagents used in the method are analytically pure, and the water is secondary water specified in GB/T6682.
3.1 reagents
3.1.1 Anhydrous ethanol (C)2H6O)。
3.1.2 sodium carbonate (Na)2CO3) Sodium bicarbonate (Na)2HCO3)。
3.1.3 hydrochloric acid (HCl).
3.1.4 citric acid salt.
3.1.5 α -amylase: more than or equal to 1.5U/mg.
3.1.6 papain: more than or equal to 5U/mg.
3.1.7 sulfuric acid (H)2SO4)。
3.2 reagent configuration
3.2.1 ethanol solution (20%): 200mL of absolute ethyl alcohol and 500mL of water are weighed and mixed uniformly.
3.2.2 sodium carbonate solution (0.5 mol/L): weighing 53g of sodium carbonate, dissolving in 1000mL of water, and uniformly mixing; sodium bicarbonate solution (0.5 mol/L): 53.5g of sodium bicarbonate was weighed, dissolved in 1000mL of water and mixed well.
3.2.3 sodium chloride solution (0.85%): 8.5g of sodium chloride is weighed, dissolved in water and diluted to 1000mL, and mixed evenly.
3.2.4 hydrochloric acid solution (1 mol/L): absorb 83mL of hydrochloric acid, dilute to 1000mL with water, and mix well.
3.2.5 citrate buffer (pH 4.5): 1.5g of citric acid was weighed into a 100mL beaker with magnetic stirrer, about 50mL of distilled water was added to dissolve, 12mL of NaOH (1mol/L) was added, the pH was adjusted to 4.5 (with 0.1mol/LHCl), the solution was transferred to a 100mL volumetric flask and the volume was fixed with distilled water. The buffer solution can be stored for 3 days at the temperature of 2-8 ℃.
3.2.6 protease-amylase solution: 200mg of papain and alpha-amylase are respectively weighed, 20mL of water is added to be ground into homogenate, and the homogenate is centrifuged at 3000r/min for 8 min. It is prepared as before use.
3.2.7 sulfuric acid solution (3%): 30mL of sulfuric acid was measured and added to 1000mL of water and mixed well.
3.3 Standard substance
The purity of Biotin (d-Biotin or Vitamin H) standard (C10H16N2O3S) is greater than or equal to 99%.
3.4 Standard solution preparation
Standard stock solution (101.41. mu.g/ml): accurately weighing 101.42mg biotin standard substance, adding ethanol solution to a constant volume of 1000ml, preparing into standard stock solution with concentration of 101.41 μ g/ml, storing in brown bottle, and storing in refrigerator at 3 deg.C for 12 months.
Standard intermediate (1.0141. mu.g/ml): accurately sucking 1mL of standard stock solution, diluting to 100mL with ethanol solution to obtain standard intermediate solution with concentration of 1.0141 μ g/mL, mixing, storing in 3 deg.C refrigerator for 6 months
Standard working solution (10.141 ng/ml): sucking 1ml of standard intermediate solution, using an ethanol solution to fix the volume to 100ml, preparing standard working solution with the concentration of 10.141ng/ml, and preparing the working solution before use;
the standard uses working liquids: dividing the solution into two concentrations, wherein the concentration of the high-concentration standard solution is 0.20282 ng/ml; the concentration of the low-concentration standard solution is 0.10141ng/ml, 5ml of the low-concentration standard solution is sucked from the standard working solution twice, and the volume is respectively determined to be 250ml and 500ml by water.
3.5 Medium
3.5.1 Lactobacillus agar Medium: can be formulated according to standard GB 5009.259-2016.
3.5.2 Lactobacillus broth: can be formulated according to standard GB 5009.259-2016.
3.5.3 culture medium for biotin measurement:
the components: 10.0g casamino acid, 30.0g glucose, 16.0g urea, 1.6g aspartic acid, 0.8g serine, 1.6g glycine, 1.0g threonine, 1.0g alanine, 1.4g arginine, 0.4g tyrosine, 0.7g valine, 0.3g methionine, 1.0g phenylalanine, 0.7g isoleucine, 1.6 leucine, 1.0g dipotassium hydrogen phosphate, 1.0g potassium dihydrogen phosphate, 0.4g magnesium sulfate, 20.0mg sodium chloride, 20.0mg ferrous sulfate, 20.0mg manganese sulfate, water to 1000ml, pH6.8 + -0.2 (25 deg.C + -5 deg.C).
The preparation method comprises the following steps: dissolving the above components in water, and adjusting pH.
Note that some commercial synthetic media have good results and are prepared according to the label instructions.
4 instruments and devices
4.1 balance, sensitive quantity 0.1 mg.
4.2 constant temperature incubator at 37 ℃. + -. 1 ℃.
4.3 steam sterilizer under pressure at 121 deg.C (0.10-0.12 mPa).
4.4 vortex oscillator.
4.5 the rotating speed of the centrifuge is more than or equal to 2000 r/min.
4.6 liquid separator, 0 mL-10 mL.
4.7 Adjustable electric stove.
4.8pH meter, accuracy is +/-0.01.
4.9 Spectrophotometer.
4.10 clean bench.
Before the glass instrument is used, a hard glass measuring tube and other necessary glassware are cleaned by an active agent (sodium lauryl sulfate or a household detergent is added into washing water), and then the hard glass measuring tube and other necessary glassware are dried and heated for 2 hours at 200 ℃.
5 preparation and preservation of Strain
5.1 strain: lactobacillus plantarum (ATCC 8014).
5.2 preparation of the inoculum
5.2.1 transferring the strain Lactobacillus plantarum into a Lactobacillus agar culture medium, culturing for 22h in a constant temperature incubator at 37 +/-1 ℃, taking out and storing in a refrigerator at 23 ℃. At least once a month, and stored as a stock strain.
5.2.2 inoculating the stock strain into lactobacillus broth by using an inoculating loop, and culturing for 18h at 36 +/-1 ℃ to obtain an activated strain; centrifuging the activated strain at 2000r/min for 3min, and removing the supernatant; adding 1mL of sterilized NaCl solution (0.85%), uniformly mixing, centrifuging for 3min, removing supernatant, repeating the operation for 3 times, then adding sodium chloride solution for dilution, and measuring the absorbance of the inoculation solution to be 0.5-0.55.
6 analytical procedure
6.1 treatment of the samples
Grinding and sieving the powdery sample (the aperture of a sieve plate is 0.3 mm-0.5 mm); uniformly mixing the liquid sample before use; accurately weighing a proper amount of sample (m) (the content of biotin is about 0.2-0.5 mu g), accurately weighing to 0.001g, pouring into a 250ml conical flask, adding 100ml of sulfuric acid solution, hydrolyzing at 100 ℃ for 1h, cooling, adjusting the pH to 4.5 +/-0.2 by using a mixed solution of sodium carbonate and sodium bicarbonate (the volume ratio of the sodium carbonate to the sodium bicarbonate is 1: 2), transferring into a 250ml volumetric flask, fixing the volume by using water, and uniformly mixing; filtering with filter paper to obtain filtrate, discarding the first few mL, sucking 5mL of filtrate, adding 20mL of water, adjusting pH to 6.8 + -0.2 with mixed solution of sodium carbonate and sodium bicarbonate (volume ratio of the two is 1: 2), transferring into 100mL volumetric flask, and metering to constant volume to obtain sample extractive solution.
6.2 preparation of the test solution
6.2.1 Standard series pipe
Taking test tubes C1-C10, respectively adding 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL of low-concentration standard solution and 3.0mL, 4.0mL and 5.0mL of high-concentration standard solution, supplementing water to 5.00mL, adding 5.0mL of culture solution for biotin determination, uniformly mixing, and making 3 parallel samples per gradient; wherein C1 is an unseeded blank and C2 is a seeded blank.
6.2.2 sample line tubes
Taking test tubes D1-D5, adding 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of sample extracting solution respectively, supplementing water to 5.0mL (except for a D5 tube), adding 5.0mL of culture solution for biotin determination, mixing uniformly, and preparing 3 parallel samples per gradient, wherein D5 is a sample blank tube.
6.3 Sterilization
And covering test tube caps on all the test tubes, and sterilizing in a sterilization kettle at 121 deg.C for 5 min. And (3) immediately taking out the culture medium from the pressure cooker after the sterilization is finished, putting the culture medium into an ice water bath for timely cooling, wherein the obtained culture medium is light yellow, and if the color of the culture medium is dark after the sterilization, the culture medium cannot be used and needs to be prepared again.
6.4 inoculation
Under sterile operating conditions, 50 μ L of inoculum was added to all tubes in S5 with a pipette gun, with the exception of non-inoculated blank tube C1 and sample blank tube D5.
6.5 cultivation
All the test tubes are put into an incubator and are incubated for 18 to 20 hours at the constant temperature of 36 plus or minus 1 ℃.
6.6 analytical testing
6.6.1 visually inspecting each test tube, wherein the culture solution in the inoculated blank tube C2 is clear, and if the test tube is turbid, the test needs to be redone;
6.6.2 the absorbance of the test tube with the highest concentration standard curve is measured by taking an inoculation blank tube C2 as a control, the absorbance is measured again after two hours, and if the difference of the light transmittance of the two results is less than 2%, the absorbance A can be measured.
6.6.3 the cultured test tubes are mixed uniformly by a vortex mixer, a cuvette with the thickness of 1cm is used at 550nm, the light transmittance is adjusted to 100% by an inoculation blank tube C2, and then the light absorption values of the standard series tube and the sample series tube are measured in sequence.
6.7 results of analysis
6.7.1 standard curve: and (5) drawing a standard curve by taking the biotin content of the standard series tube as an abscissa and the light absorption value as an ordinate.
6.7.2 calculation of results:
the biotin content in the sample was calculated according to formula (1):
in the formula:
x-biotin content in samples in micrograms per hundred grams or milliliters (μ g/100g or mL);
f is the dilution multiple of the sample solution, wherein f is 1;
m is the sample mass in grams (g);
the calculation results are expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, with the results remaining in three significant digits.
7. Precision degree
The absolute difference between two independent measurements obtained under repetitive conditions must not exceed the arithmetic mean by 10%.
8. Others
The standard detection limit is 20.0 mug/kg.
9. The standard curve obtained by the method of the invention is shown in FIG. 1.
Claims (10)
1. The method for detecting the content of free biotin in the infant formula food is characterized by comprising the following steps of:
s1, preparing a standard solution, namely weighing a certain amount of biotin standard substance, and using an ethanol solution to prepare the standard solution with different concentrations;
s2, preparing an inoculation liquid, namely directly inoculating a reserved strain into lactobacillus broth to culture for 16-20h at 36 +/-1 ℃ to obtain an activated strain; centrifuging the activated strain, and removing the supernatant; adding a sodium chloride solution, uniformly mixing, centrifuging again, removing a supernatant, repeating the operation for 2-3 times, and then adding a certain amount of sodium chloride solution for dilution to obtain a inoculation solution;
s3, processing the sample, grinding and sieving the powdery sample; uniformly mixing the liquid sample before use;
s4, preparing a liquid to be detected, wherein the liquid to be detected comprises a standard series tube and a sample series tube;
s5, sterilizing, namely covering test tubes in the S4 with test tube caps, and placing the test tubes in a sterilization kettle for sterilization;
s6, inoculating, and under the aseptic operation condition, adding an inoculating liquid into all test tubes in the S5, wherein blank inoculation and blank sample are not performed in a standard curve tube except for blank inoculation and blank sample;
s7, culturing, namely putting all the test tubes obtained in the step S6 into an incubator, and culturing for 18-20 h at constant temperature;
s8, analyzing and testing, mixing the cultured test tubes uniformly by using a vortex mixer, and testing at 550nm by using a cuvette with the thickness of 1 cm.
2. The method for detecting the content of free biotin in the infant formula food according to claim 1, comprising the following steps:
s1, preparing a standard solution, namely accurately weighing 101.41mg of a biotin standard substance, metering the volume to 1000ml by using an ethanol solution, and preparing a standard stock solution with the concentration of 101.41 mug/ml; sucking 1mL of standard stock solution, and fixing the volume to 100mL by using an ethanol solution to prepare a standard intermediate solution with the concentration of 1.0141 mu g/mL; sucking 1ml of standard intermediate solution, using an ethanol solution to fix the volume to 100ml, preparing standard working solution with the concentration of 10.141ng/ml, and preparing the working solution before use; a certain amount of standard working solution is sucked and diluted into 0.20282ng/ml high-concentration standard solution and 0.10141ng/ml low-concentration standard solution by using ethanol solution, and the standard working solution is prepared before use;
s2, preparing an inoculation liquid, transferring a strain lactobacillus plantarum into a lactobacillus agar culture medium, culturing for 20-24 h in a constant-temperature incubator at 37 +/-1 ℃, taking out, and storing in a refrigerator at 2-4 ℃ to serve as a storage strain; inoculating the stock strain into lactobacillus broth, and culturing at 36 + -1 deg.C for 16-20h to obtain activated strain; centrifuging the activated strain at 2000r/min for 2-3min, and removing supernatant; adding 1mL of sterilized NaCl solution, uniformly mixing, centrifuging for 2-3min, removing supernatant, repeating the operation for 2-3 times, and then adding sodium chloride solution for dilution to obtain inoculation liquid;
s3, processing the sample, grinding and sieving the powdery sample; uniformly mixing the liquid sample before use; accurately weighing a proper amount of sample, pouring the sample into a 250ml conical flask, adding 100ml of sulfuric acid solution, hydrolyzing at 80-100 ℃ for 1-2 h, cooling, adjusting the pH to 4.5 +/-0.2 by using an alkaline carbonate solution, transferring into a 250ml volumetric flask, fixing the volume by using water, and uniformly mixing; filtering with filter paper to obtain filtrate, discarding the first several mL, sucking 5mL of filtrate, adding 20mL of water, adjusting pH to 6.8 + -0.2 with alkali carbonate solution, transferring into 100mL volumetric flask, and fixing volume to obtain sample extractive solution;
s4, preparing a liquid to be detected, wherein the liquid to be detected comprises a standard series tube and a sample series tube;
standard series of tubes: taking test tubes C1-C10, respectively adding 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL of low-concentration standard solution and 3.0mL, 4.0mL and 5.0mL of high-concentration standard solution, supplementing water to 5.00mL, adding 5.0mL of culture solution for biotin determination, uniformly mixing, and making 3 parallel samples per gradient; wherein C1 is a non-inoculated blank and C2 is an inoculated blank;
sample series tube: taking test tubes D1-D5, adding 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of sample extracting solution respectively, supplementing water to 5.0mL, adding 5.0mL of culture solution for biotin determination, uniformly mixing, and preparing 3 parallel samples per gradient, wherein D5 is a sample blank tube;
s5, sterilizing, namely covering test tubes in the S4 with test tube caps, and placing the test tubes in a sterilization kettle for sterilization;
s6, inoculating, and under the aseptic operation condition, adding 50 mu L of inoculating liquid into all test tubes in the S5 by using a liquid transfer gun, wherein the non-inoculated blank tube C1 and the sample blank tube D5 are excluded;
s7, culturing, namely putting all the test tubes obtained in the step S6 into an incubator, and culturing for 18-20 h at the constant temperature of 36 +/-1 ℃;
and S8, analyzing and testing, namely uniformly mixing the cultured test tubes by using a vortex mixer, adjusting the light transmittance to 100% by using an inoculation blank tube C2 in a cuvette with the thickness of 1cm at 550nm, and then sequentially measuring the light absorption values of a standard series tube and a sample series tube.
3. The method for detecting the content of free biotin in an infant formula according to claim 1 or 2, wherein the volume fraction of the ethanol solution is 20% in step S1.
4. The method for detecting the content of free biotin in the infant formula according to claim 1 or 2, wherein in step S2, the absorbance of the inoculation solution is 0.5 to 0.65.
5. The method for detecting the content of free biotin in an infant formula according to claim 2, wherein in step S3, the aperture of the sieve plate during sieving is 0.3mm to 0.5 mm.
6. The method of claim 2, wherein in step S4, the raw material of the biotin-determination culture solution includes 10.0g of casamino acids, 30.0g of glucose, 16.0g of urea, 1.6g of aspartic acid, 0.8g of serine, 1.6g of glycine, 1.0g of threonine, 1.0g of alanine, 1.4g of arginine, 0.4g of tyrosine, 0.7g of valine, 0.3g of methionine, 1.0g of phenylalanine, 0.7g of isoleucine, 1.6g of leucine, 1.0g of dipotassium hydrogen phosphate, 1.0g of monopotassium phosphate, 0.4g of sodium chloride, 20.0mg of magnesium sulfate, 20.0mg of ferrous sulfate, 20.0mg of manganese sulfate, and a predetermined amount of water.
7. The method for detecting the content of free biotin in an infant formula according to claim 2, wherein in step S5, the sterilization temperature is 121 ℃ and the sterilization time is 5 min.
8. The method of claim 2, wherein the step S5 comprises the following steps:
(1) and (3) immediately taking out the culture medium from the pressure cooker after the sterilization is finished, putting the culture medium into an ice water bath for timely cooling, wherein the obtained culture medium is light yellow, and if the color of the culture medium is dark after the sterilization, the culture medium cannot be used and needs to be prepared again.
9. The method of claim 2, wherein the step S8 comprises the following steps:
(1) visual inspection is carried out on each test tube, the culture solution in the inoculation blank tube C2 is clear, and if turbidity occurs, the result is invalid;
(2) and (4) taking an inoculation blank tube C2 as a control, measuring the absorbance of the test tube with the highest concentration standard curve, re-measuring after two hours, and taking out all test tubes to measure the absorbance A if the difference of the light transmittance obtained by the two times is less than 2%.
10. A method for detecting the amount of free biotin in an infant formula according to any of claims 1 to 9, wherein the method is used for detecting the amount of free biotin in an infant formula.
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