This study established a fluorescence-based quantitative real-time polymerase chain reaction method for the detection of caprine-derived ingredients in milk powder. The specificity, sensitivity, and stability of the caprine-specific primers were evaluated. By linear fitting of the difference in cycle threshold (ΔCt) as a function of the mixing proportion between goat and horse milk powder, a calibration curve for the relative quantitation of caprine-derived ingredients in milk powder was established as follows: y = 0.720 9x + 5.651 9 (R2 = 0.987 5), and the minimum detection limit of this method was 0.000 1 ng/μL. The recoveries of the proposed method were 96.22%-112.00%, and the inter- and intra-group coefficient of variation was ≤ 0.79% and ≤ 1.77%, respectively.

An uncertainty evaluation method for the microbiological determination of biotin in infant formula was developed in order to control the quality of test results and improve the detection accuracy, a method for uncertainty evaluation of milk powder by method was established. According to the requirements of the Evaluation and Expression of Uncertainty in Measurement (JJF 1059.1—2012) and the Guide to the Evaluation and Expression of Uncertainty in Measurement (GB/T 27418—2017), the uncertainty components were estimated for the determination of biotin in infant formula by the method specified in the National Food Safety Standard Determination of Biotin in Foods (GB 5009.259—2016). It was found that the greatest uncertainty resulted from standard curve fitting and repeatability test. The uncertainty for the biotin content of three infant formula samples was as follows: sample A (19.87 ± 2.84) μg/100 g, B (30.35 ± 4.41) μg/100 g, and C (53.15 ± 7.15) μg/100 g (in the 95% confidence interval, coverage factor (k) = 2). As the content of biotin in samples increased, the expanded uncertainty also increased.

A droplet digital polymerase chain reaction (ddPCR) method for the quantitative detection Acetobacter aceti in fermented milk was established. Specific primers and probes were designed according to the internally transcribed spacer (ITS) gene sequence of Acetobacter aceti, and annealing temperature was optimized. The specificity of the method was verified by applying it on various strains, the limit of detection (LOD) was determined for artificially inoculated Acetobacter aceti, and the absolute quantification was systematically investigated by comparing the results of ddPCR and the counting results. The experimental results showed that the optimal annealing temperature was 54.6 ℃, the method had strong specificity and high sensitivity, and the LOD was 7.2 × 101 CFU/mL. The quantitative deviation rate was 23.73%. This method can meet the demand for quantitative detection of Acetobacter aceti in fermented milk.

A quality control sample for the qualitative analysis of Listeria monocytogenes was developed by vacuum freezing drying technology, and the homogeneity and stability of the quality control sample were systematically analyzed. By optimizing the type of lyophilized matrix, the optimal conditions for the development of quality control samples were obtained. The homogeneity and stability of the developed quality control sample were verified by counting the number of cells as well as using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The quality control sample was white in color, spherical in shape and uniform in size. The results of cell counting for uniformity validation showed F = 0.567, which was less than the critical value, indicating good uniformity. In the transportation stability test, the quality control sample remained stable at 37 and 25 ℃. In the storage stability test, the resurrection rate of the quality control sample was 101.5% after 28 days of storage at ?20 ℃, and 99.6% after 28 days of storage at 4 ℃, indicating that the sample has good uniformity and stability, and can be used as a positive quality control sample for the detection and quality control of Listeria monocytogenes.

In order to evaluate the application value of ready-to-use Salmonella quality control samples in matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) identification of Salmonella in milk-containing formula foods for special medical purposes, this experiment evaluated the uniformity and stability of the quality control samples. After being stored at 20 ℃ for 0, 14 and 28 days, these samples were identified by mass spectrometry and the VITEK automatic microbial analysis system. The results showed that the quality control samples had good stability and uniformity, and their mass spectra were similar to each other. The VITEK system confirmed their identity as Salmonella. In conclusion, the ready-to-use Salmonella quality control strains can be used for the detection of Salmonella in milk-containing formula foods for special medical purposes.