Is the microbiological method good for the determination of biotin in baby food

The detection methods of free biotin in infant milk powder include microbiological method, chromatographic method, fluorescence method, ELISA method, etc. The microbiological method is characterized by high sensitivity, and the microbiological method is usually adopted in China's national standard and internationally for the determination. However, due to the differences in the working conditions of each laboratory, it is usually not possible to obtain credible results in strict accordance with the national standard test conditions, with a high failure rate and high testing costs [1-3]. Therefore, the author's laboratory to simplify and optimize the test conditions, in order to improve the quality of the test and the accuracy of the test results, reduce the cost of testing.　1 Materials and methods 1.1 Test materials The test strain is Lactobacillus plantarum (ATCC8014), provided by the American Model Culture Collection. The test medium was Lactobacillus broth medium (Qingdao Haibo) and free biotin assay medium (Bidi Pharmaceutical Instrument Company). Biotin standard (Shanghai Amp). Instruments and equipment for testing: TU-1901 dual-beam ultraviolet-visible spectrophotometer (Beijing Pulse Analytical); LRH-250 biochemical incubator (Guangdong Medical Instrument Factory); SIK-202 purification workbench (made by Sujing Group Antei); YXQ-LS-75S vertical pressure steam sterilizer (Shanghai Boxun).　1.2 Test methods 1.2.1 Production of standard curve. Sample processing and standard curve production and sample determination steps refer to GB5413.19-2010, inoculation using a disposable sterile syringe vertically suspended drops of 1 drop of bacterial solution. The concentration of the working solution of the standard curve c=0.2 ng/mL. 1.2.2 Influence of the standard curve solution preparation method. Use Lactobacillus broth medium to passivate the strain, prepare bacterial suspension with the 4th generation strain, bacterial suspension absorbance A=0.57, since the standard intermediate solution, respectively, using ultrapure water, 50% ethanol solution preparation, respectively, the standard curve production, each way to repeat the test 3 times.　1.2.3 Effect of bacterial suspension concentration. Use Lactobacillus broth medium to passivate the strain, use ultrapure water to prepare the standard curve working solution, prepare a series of concentration of bacterial suspensions with the 4th generation strain, the absorbance of bacterial suspensions (A) were 0.14, 0.22, 0.36, 0.50, 0.64, 0.79, respectively, and respectively, standard curves were made.　1.2.4 Effect of number of passages. Lactobacillus broth medium was used to passivate the strains, and ultrapure water was used to prepare the working solution for the standard curve, and the bacterial suspensions were prepared with the 1st, 2nd, 3rd, 4th, 5th, and 6th generation strains, and the absorbance of the bacterial suspensions was 0.52, 0.54, 0.56, 0.54, 0.60, and 0.55, respectively, and the standard curves were made, respectively.　2 Results and analysis In order to examine the influence of various factors on the linearity of the standard curve, the influence of the standard solution preparation method, the selection of the transmitting medium, the number of transmissions and the concentration of bacterial suspension on the linearity of the test curve were considered.　2.1 The effect of the preparation of standard curve solution The standard curve of the test using 50% ethanol solution to prepare the standard working solution is basically not linear, and cannot meet the linear requirements of the test, while the standard curve of the test using ultrapure water to prepare the standard solution has greatly improved the linearity of the standard curve, and the highest can reach 0.991 2 (Table 1, Table 2), and the results of the three times are in line with the requirements of the test. GB5413.19 requires the use of 50% ethanol solution to prepare the standard working solution. GB5413.19 requires the use of 50% ethanol solution to prepare the standard working solution, but due to the high temperature during the sterilization process and the airtightness of the test tube caused by ethanol volatilization, resulting in uneven changes in the volume of the test tube resulting in poor curve linearity, and at the same time, because of the growth of Lactobacillus plantarum can be inhibited by ethanol itself, the ethanol in the standard working solution to varying degrees to affect the test of the final microbial growth is also a cause of poor linearity or no linearity. The use of ultrapure water preparation can avoid these problems and effectively improve the linearity of the curve.　2.2 Influence of bacterial suspension concentration The curve linearity of bacterial suspension absorbance (A) in the range of 0.50 to 0.79 meets the test requirements, and R2 does not change much in this range. Although the linearity of bacterial suspension absorbance reaches 0.79 basically meets the test requirements, the growth in the test tube is too large, and the absorbance is too large, which has a certain impact on the test results, and it is not suitable for controlling the time of the test, so bacterial suspension absorbance in the range of 0.50 to 0.64 is more suitable. 0.50～0.64 is more appropriate (Figure 1). Bacterial suspension concentration is too low, the poor scalability may be the highest concentration tube growth termination time is too long, too long may lead to the death of Lactobacillus plantarum and can not completely consume the highest concentration tube biotin.　2.3 The effect of the number of generations 1 to 5 generations of the strain can meet the test requirements, more than 5 generations after the curve linearity decreased significantly, probably because with the increase in the number of generations, the strain mutation, specificity changes, which can not normally reflect the specificity of Lactobacillus plantarum on the demand for biotin, and can not be linear growth, resulting in the test is not linear or poor linearity (Figure 2).　3 Conclusion and Discussion The standard curve was prepared by using ultrapure water to prepare the standard curve working solution, the absorbance of bacterial suspension A=0.57, and the bacterial suspension prepared by the 4th generation of bacterial strain to make the standard curve, and the curve y=1.023 7x+0.180 8, R2=0.996 2. The different samples were measured and spiked tests were done, and the average spiked recoveries were 92%, and the range of spiked recoveries was from 85% to 97%, and for the same samples The coefficient of variation was 3.56% for 10 replicates of the same sample. Under the conditions of using ultrapure water to prepare the working solution for the standard curve, the absorbance A of bacterial suspension in the range of 0.50~0.64, and not more than 5 generations of strains to prepare bacterial suspensions, the accuracy of the test was higher and the reproducibility was better, and at the same time, the use of 5 concentration points to make the curve not only reduced the workload but also lowered the cost of testing compared with that of the national standard method, therefore, this test condition has a certain degree of feasibility.　In the test process, the cleaning of utensils, the selection of commercial culture medium, the use of Lactobacillus broth medium, activation, the standard working solution is now used, the syringe drops of bacteria must be suspended vertically, as well as the control of the incubation time is the key to the success or failure of the test.