Development of a Method for the Analysis of Multiclass Antibiotic Residues in Milk Using QuEChERS and Liquid Chromatography-Tandem Mass Spectrometry.

A precise and simplified method of sample preparation for the simultaneous quantification of the antibiotics ß-lactam, macrolide, tetracycline, sulfonamide, and quinolone in bovine milk was developed. The central composite design of response surface methodology was used to design and optimize the method for the determination of six different antibiotic residues in milk. The recovery of each antibiotic was studied using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. Octadecylsilane (C18), primary secondary amine (PSA), and sodium acetate (Na acetate) were the main factors affecting the recovery of each antibiotic. After optimization, the maximum predicted recovery rate was 84.18% for erythromycin under the optimized conditions of 101.20 mg C18, 52.00 mg PSA, and 1.01 g Na acetate. The recovery rates of the five other antibiotic residues ranged from 86.09% to 115.99%. The results suggested that modified QuEChERS could effectively be implemented in the analysis of antibiotic residues in milk.

A new comprehensive index for discriminating adulteration in bovine raw milk.

This paper proposes a new comprehensive index, called Q, which can effectively discriminate artificial adulterated milk from unadulterated milk. Both normal and adulterated samples of bovine raw milk were analysed by Fourier transform infrared spectroscopic instrument to measure the traditional indices of quality, including fat (FAT), protein (PRO), lactose (LAC), total solids (TS), non-fat solid (NFS), freezing point (FP) and somatic cell counts (SCC). From these traditional indices, this paper elaborates a method to build the index Q. First, correlated analysis and principle component analysis were used to select parameter pairs TS-FAT and FP-LAC as predominant variables. Second, linear-regression analysis and residual analysis are applied to determine the index Q and its discriminating ranges. The verification and two-blind trial results suggested that index Q could accurately detect milk adulteration with maltodextrin and water (as low as 1.0% of adulteration proportions), and with other nine kinds of synthetic adulterants (as low as 0.5% of adulteration proportions).

[Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing].

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture. These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship. Kefir grains can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and immunomodulatory properties. The microorganisms in Kefir grains are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. However, the microorganisms that isolate by these methods can not revert to Kefir grains which indicate that there are some other bacteria that are not isolate from it. In this study, PCR-based Denaturing gradient gel electrophoresis(DGGE) and sequence analysis of 16S ribosomal RNA gene (16S rDNA) clone libraries was used for the rapid and accurate identification of microorganisms from Kefir grains. The PCR primers were designed from conserved nucleotide sequences on region V3 of 16S rDNA with GC rich clamp at the 5'-end. PCR was performed using the primers and genomic DNAs of Kefir grains bacteria. The generated region V3 of 16S rDNA fragments were separated by denaturing gel, and the dominant 16S rDNA bands were cloned, sequenced and subjected to an online similarity search. Research has shown that regions V3 of 16S rDNAs have eight evident bands on the DGGE gel. The sequence analysis of these eight bands has indicated that they belong to different four genera, among them three sequences are similar to Sphingobacterium sp. whose similarities with database sequences are over 98%, three sequences are similar to Lactobacillus sp. whose similarities with database sequences are over 96%, the other two sequence are similar to Enterobacter sp., and Acinetobacter sp. whose similarities with database sequences are over 99% respectively. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, because when universal bacterial PCR primers are used, only the dominant microbiota of an ecosystem will be visualized on a DGGE gel, producing complex banding patterns. However, it could visualize the bacterial qualitative compositions and reveal the major species of the Kefir grains. Among them Sphingobacterium can be found in Kefir grains as the predominant flora which is reported for the first time. PCR-based DGGE and sequence analysis of 16S rDNA proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in microecosystem and probiotic products.