Culture‐Dependent and Culture‐Independent Nucleic‐Acid‐Based Methods Used in the Microbial Safety Assessment of Milk and Dairy Products

Despite great advances in the diagnostics and better awareness for food safety and security worldwide, significant numbers of foodborne outbreaks have been traced back to the consumption of milk and dairy products contaminated with pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Campylobacter spp., and pathogenic Escherichia coli. Several culture‐dependent and culture‐independent nucleic acid‐based methods have been proposed to identify, detect, and type milk‐ and dairyborne pathogenic bacteria. In our review, we will provide an overview on why it is of utmost importance to ascertain the presence of pathogenic microorganisms in milk and milk products; thereafter, we will describe the most commonly used culture‐dependent and culture‐independent methods, as well as the most attractive ones with regard to their future exploitation, providing the reader with new insights into how and when they can be exploited to ensure the enumeration, and accurate detection at both species and strain level of the most important milk‐ and dairyborne pathogenic bacteria, even if in a viable but nonculturable state.     

Determination of Spoilage Microbiota of Pacific White Shrimp During Ambient and Cold Storage Using Next‐Generation Sequencing and Culture‐Dependent Method

This study was conducted to determine the initial and spoilage microbiota of Pacific white shrimp during ambient and cold storage using next‐generation sequencing (NGS) and a culture‐dependent method. The quality changes were also evaluated by sensory analysis and total volatile basic nitrogen (TVB‐N) values. After 1 d of storage, the psychrotrophic bacteria were only 5.97 log CFU/g, accounting for 1.1% of the mesophilic bacteria counts (7.94 log CFU/g). The psychrotrophic bacteria counts exceeded the counts of mesophilic bacteria for shrimp stored at 4 °C after 6 d of storage, indicating that psychrotrophic bacteria became predominant. The NGS was used to identify the bacterial species in samples stored at 25 and 4 °C. The results showed that the dominant microorganisms were Vibrio at 25 °C, and Acinetobacter, Psychrobacter, and Shewanella at 4 °C. By the culture‐dependent method based on 16S rRNA gene and VITEK®2 CompactA system, it showed that the dominant microorganisms were Proteus spp. at 25 °C, and Shewanella putrefaciens, Acinetobacter johnsonii, and Aeromonas sobria at 4 °C. In conclusion, differences in results of microbiota analyzed by culture dependent and independent approaches were observed. The combination of both methodologies may provide more comprehensive information about the dominant spoilage microbiota in Pacific white shrimp during ambient and cold storage.     

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture‐Dependent and DGGE Approaches

Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture‐dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture‐dependent analysis, the populations of yeasts and LAB were at the level of 10⁵ to 10⁷ and 10⁶ to 10⁷ cfu/g, respectively. Similarly, the results of RT‐qPCR showed that the values of total yeasts and LAB populations were in the range of 10⁶ to 10⁷ and 10⁷ to 10⁸ copies/g, respectively. Using culture‐dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR‐DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species.     

Degradation of N‐(1‐Deoxy‐d‐xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐xylulos‐1‐yl)proline using thermal treatment

The formation and degradation of N‐(1‐Deoxy‐d ‐xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d ‐xylulos‐1‐yl)proline, derived from the secondary amine Maillard reaction in xylose‐amino acid model solutions, were detailed in this study. The identification and quantitative analysis of N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline were carried out using high‐performance anion‐exchange chromatography and high‐performance liquid chromatography. The formation of intermediate and advanced products derived from N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline was also tested using an UV‐Vis spectrophotometer to gain a better comparing of the degradation process of the two important Maillard reaction products using thermal treatment. Results showed that the degradation of N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine was more significant than N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline. Moreover, xylose was tested in the degradation products of both N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline, which indicated that the degradation of N‐substituted 1‐amino‐1‐deoxyketoses was a reversible reaction to form reducing sugar.     

Culture‐Independent Rapid Detection Methods for Bacterial Pathogens and Toxins in Food Matrices

Rapid detection of bacterial pathogens and toxins in foods is necessary to provide real‐time results to mitigate foodborne illness outbreaks. Cultural enrichment methods, although the most widely used, are time‐consuming and therefore inadequate for rapid pathogen detection from food samples. The development of novel “rapid” detection methods has decreased detection time dramatically. This review presents an overview of detection methods for various foodborne pathogens, including Listeria monocytogenes, Salmonella enterica, and shiga toxin‐producing Escherichia coli, and bacterial toxins in food matrices, with emphasis on those methods which do not require cultural enrichment. Discussed methods include nucleic acid‐, immunological‐, and biosensor‐based techniques. A summary of each type of detection method is given, including referenced methods from the literature. Since these discussed methods do not require cultural enrichment, there is a higher probability of interference from the food matrices. Therefore, the review also discusses the potential interference of food components on detection methods and addresses preprocessing strategies to overcome matrix associated inhibition and to concentrate low quantities of pathogens and toxins in food. Development of rapid and sensitive detection technologies advances and ensures public health safety and security.     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.