In order to improve the detection efficiency of molds and yeasts in low-temperature yogurt, primers and probes were designed according to the sequences of the conserved gene elongation factor-1α (EF-1α) for molds and yeasts, respectively. A real-time fluorescence quantitative polymerase chain reaction (real-time PCR) method with TaqMan probe was established for the detection of molds and yeasts, respectively. The genomic DNA of fungi and lactic acid bacteria commonly found in low-temperature yogurt, were used as amplification templates for specificity testing. The sensitivity and repeatability were also tested, and the standard curves were constructed. The results showed that the designed primers and probes had good specificity; the sensitivity of the method was 101 and 102 CFU/mL for yeasts and molds, respectively; the coefficients of variation within and between groups in the repeatability test were less than 2%, indicating good reliability and stability; the correlation coefficients of the standard curves were higher than 0.99. Therefore, the real-time PCR method enables the rapid detection of molds and yeasts in low-temperature yogurt.

β-lactoglobulin (β-LG) is a major allergen in cow’s milk. In this study, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, circular dichroism (CD) spectroscopy, molecular docking, and indirect competitive enzyme-linked immunosorbent assay were used to explore the interaction of docosahexaenoic acid (DHA) with β-LG and its influence on the allergenicity of β-LG. The results showed that the binding of DHA to β-LG significantly changed the microenvironment of the aromatic residues of β-LG and increased its polarity. DHA bound to β-LG to form a stable complex through hydrophobic interactions, changing the secondary structure of β-LG and resulting in folding of its backbone. The relative content of β-sheet increased and then decreased with increasing concentration of DHA, while the relative content of α-helix showed an opposite trend. However, the relative contents of β-turn and random coil showed irregular changes. In addition, the antigenicity of β-LG decreased after combination with DHA, and the immunoglobulin E (IgE) binding ability inhibition rate was about 29% by 3 mmol/L DHA.

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.

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 rapid detection method based on helicase-dependent isothermal DNA amplification (HDA) was established for Lactobacillus plantarum in fermented milk. Specific primers were designed according to the scrB gene sequence of Lactobacillus plantarum (Genbank Accession NO. AJ579541.1), and the optimal concentrations of UvrD helicase and T4 gp32 in the reaction system were determined through experiments. The limit of detection (LOD), specificity, consistency and stability of the proposed method were evaluated by use of L. plantarum-spiked samples, amplification of various strains, and electrophoresis of amplified products and sequence alignment analysis, respectively. The results showed that the optimized of UvrD helicase and T4 gp32 in the reaction system were found to be 0.15 and 5.0 μg, respectively. The HDA method had high specificity with no amplification of other strains tested. The detection limit was 2.8 × 101 CFU/g. The amplified product was consistent with the designed sequence length (273 bp) and the sequence homology was 100%. In conclusion: this method is rapid, simple, sensitive and suitable for the detection of Lactobacillus plantarum 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.

A rapid detection method for Salmonella in fermented milk was developed by helicase-dependent isothermal DNA amplification (HDA). In this method, the invA gene sequence of Salmonella was used as the target gene to design specific primers, and the concentrations of UvrD helicase and T4 gp32 in the reaction system were optimized to establish the optimal reaction system. The HDA method was used to directly detect Salmonella in fermented milk, and the amplification products were detected by electrophoresis to verify the specificity of this method. The results showed that the proposed method exhibited high specificity for Salmonella in fermented milk. The optimized reaction system contained 0.10 μg of UvrD helicase and 5.0 μg of T4 gp32 per 50 μL of sample. The amplification product was consistent with the designed sequence length (304 bp). The detection limit was 2.6 × 102 CFU/g. The method can meet the requirements for the rapid detection of Salmonella in fermented milk, and thanks to its high sensitivity and easy operation, it can be used as a basic method for rapid detection of Salmonella in fermented milk.

A rapid method using helicase-dependent isothermal DNA amplification (HDA) for the detection of Gluconobacter in fermented milk was established. The ITS gene sequence (gene number KF896260.1) of Gluconobacter was selected as the target gene for design of specific primers, and the concentrations of UvrD helicase and T4 gp32 in the reaction system were optimized to establish an optimal reaction system. The HDA method was used to directly detect Gluconobacter in fermented milk. The established HDA system was used to amplify and electrophoretically detect various strains in fermented milk to verify the specificity of the method and determine its detection limit. The results showed that the detection of Gluconobacter in fermented milk by the HDA method was consistent with the designed sequence length (309 bp) and the detection limit was 4.3 × 101 CFU/g. The appropriate amounts of UvrD helicase and T4 gp32 added in the reaction system were 0.1 and 5.0 μg, respectively. The method is highly sensitive and time saving.

A helicase-dependent isothermal DNA amplification (HDA) assay was developed for the rapid and accurate detection of Gluconacetobacter in yogurt.A pair of specific oligonucleotide primers according to the 16S ribosomal RNA gene (GenBank accession number:HQ677466.1) of Gluconacetobacter was designed,and the optimal concentration of UvrD helicase and T4 gp32 in the reaction system were determined.The HAD method enabled direct detection of Gluconacetobacter in yogurt.The specificity of the method was tested by amplification and electrophoresis of various strains in yoghurt using the HAD system.The amplification product by HDA was the same as the designed gene fragment (101 bp).The sensitivity of HDA was 102 CFU/g.The optimal reaction system was established by the use of 0.1 μg of UvrD helicase and 5.0 μg of T4 gp32.In conclusion,the method was sensitive and time saving.

A method for the simultaneous determination of Pb, Cr and Hg in milk was developed by microwave digestion combined with inductively coupled plasma mass spectrometry (ICP-MS). The sample preparation procedure, the ICP-MS parameters, the internal standard and the monitor isotope were optimized. The results showed that the digestion temperature was 180 ℃. A mixed solution of 10 μg/L 72Ge and 209Bi was selected as the internal standard to eliminate the matrix effect, and 200 μg/L Au was added to the solution to eliminate the memory effect of Hg. 208Pb, 52Cr and 202Hg were monitored with ICP-MS to determine Pb, Cr and Hg, respectively. Under the optimal conditions, detection limits of 5.0 (Pb), 1.0 (Cr) and 0.5 (Hg) μg/kg were obtained. The contents of Pb, Cr and Hg in 40 batches of commercial milk were low. The detection values were smaller than 0.011 mg/kg for Pb and smaller than 0.031 mg/kg for Cr. Only 1 sample was found to contain Hg, and the detection value was 0.008 mg/kg.

Objective: A loop-mediated isothermal amplification (LAMP) assay was designed for rapid and accurate detection of Bacillus cereus in yogurt. Methods: Two pairs of oligonucleotide primers exclusively amplifying the hblA gene of Bacillus cereus were designed and Bacillus cereus could be directly detected by LAMP in yogurt, and the amplification products were detected by electrophoresis. Results: Detection of Bacillus cereus in yogurt by the LAMP assay was highly specific, the sensitivity was 6.4 CFU/mL, and the detection limit was 21 CFU/mL. Conclusion: The LAMP was fast, sensitive and useful for the detection of Bacillus cereus in yogurt.