M.A. Drake

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid-based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR-based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products.

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

Reduced- and low-fat cheese technology: A review

During the past 15 years, the demand for reduced- and low-fat cheeses has increased dramatically. Unfortunately, fat plays a key role in the flavor, body and texture of cheese. Many commercial reduced- and low-fat cheeses as a result exhibit poor flavor and texture. Intense research in the area of reduced- and low-fat cheese technology has produced three basic strategies to combat problems associated with fat reduction.

Rapid Detection and Identification of Lactobacillus spp. in Dairy Products by Using the Polymerase Chain Reaction

Species-specific primers for use in the polymerase chain reaction (PCR) were designed to differentially amplify DNA from the common dairy lactobacillus species Lactobacillus casei , Lactobacillus delbrueckii , Lactobacillus helveticus , and Lactobacillus acidophilus . A method for rapid extraction of bacterial DNA from dairy products was developed. The sensitivity of bacterial DNA extraction from food and subsequent amplification by PCR was 100 cells total. Lactobacillus DNA was extracted and identified from commercial yoghurts, acidophilus milk, and cheeses. The methodology allows the presumptive identification of dairy lactobacilli in less than 6 hours.

Aeromonas Species in Foods

Aeromonas species have been recognized as potential or emerging foodborne pathogens for more than 20 years. Aeromonads are estuarine bacteria and are ubiquitous in fresh water, fish and shellfish, meats, and fresh vegetables. Actual sourced foodborne outbreaks are few, but epidemiological evidence suggests that the bacterium can cause self-limiting diarrhea, with children being the most susceptible population. Most aeromonads are psychrotrophic and can grow in foods during cold storage. Aeromonads are not resistant to food processing regimes and are readily killed by heat treatment. A host of virulence factors are present, but the exact role of each in human disease has not been fully elucidated.

Sensory and mechanical aspects of cheese texture

Producing high quality dairy products requires precise control over factors determining product appearance, flavor and texture. Food texture is analyzed by descriptive sensory analysis. This method uses terms that depict the textural sensations perceived from first bite through mastication and swallowing. One component of sensory texture is mechanical properties, which are determined by empirical or fundamental methods. However, if one wants to understand the molecular basis of texture, then fundamental tests are required. Fundamental rheological properties are linked to network models, such as those for rubber elasticity or filled gels. These models predict how network interactions will alter rheological properties, providing a link from molecular interactions to sensory texture. In general, sensory and rheological terms that relate to the overall firmness and resiliency of cheese are highly correlated. However, sensory terms that describe the breakdown pattern, adhesiveness and cohesiveness of cheese, are weakly, if at all, correlated with rheological properties.

The impact of agglomeration and storage on flavor and flavor stability of whey protein concentrate 80% and whey protein isolate.

The impact of agglomeration on flavor and flavor stability of whey protein concentrates 80% (WPC80) and whey protein isolates (WPI) has not been widely addressed. This study examined the impact of agglomeration on the flavor and flavor stability of commercial WPC80 and WPI across 18 mo of storage. Duplicate agglomerated and nonagglomerated WPC80 and WPI were collected from 4 facilities and stored at 21 degrees C, 50% relative humidity. Volatile analysis using solid phase microextraction (SPME) with gas chromatography-mass spectrometry (GC-MS) and descriptive sensory analysis were conducted every 2 mo. Solubility index, bulk volume, dispersibility, moisture, and color (L, a, b) were tested every 3 or 6 mo. Consumer acceptance testing with protein beverages was conducted with fresh and stored whey proteins. Higher intensities and more rapid development of lipid oxidation flavors (cardboard, raisin/brothy, cucumber, and fatty) were noted in agglomerated powders compared to nonagglomerated powders (P < 0.05). Volatile analysis results confirmed sensory results, which indicated increased formation of aldehydes and ketones in agglomerated products compared to nonagglomerated powders (P < 0.05). Consumer acceptance scores for protein beverages were lower for beverages made with agglomerated WPC80 stored for 12 mo and agglomerated or nonagglomerated WPI stored for 18 mo compared to fresh products while trained panelists detected differences among beverages and rehydrated proteins earlier. Agglomeration with or without lecithin decreased the storage stability of whey proteins. These results indicate that the optimum shelf life at 21 degrees C for nonagglomerated whey proteins is 12 to 15 mo and 8 to 12 mo for agglomerated whey proteins.

Molecular beacon polymerase chain reaction detection of Escherichia coli O157 :H7 in milk

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.

Invited review: Annatto usage and bleaching in dairy foods

Annatto is a yellow/orange colorant that is widely used in the food industry, particularly in the dairy industry. Annatto, consisting of the carotenoids bixin and norbixin, is most commonly added to produce orange cheese, such as Cheddar, to achieve a consistent color over seasonal changes. This colorant is not all retained in the cheese, and thus a percentage remains in the whey, which is highly undesirable. As a result, whey is often bleached. Hydrogen peroxide and benzoyl peroxide are the 2 bleaching agents currently approved for bleaching whey in the United States. Recent studies have highlighted the negative effect of bleaching on whey flavor while concurrently there is a dearth of current studies on bleaching conditions and efficacy. Recent international mandates have placed additional concern on the use of benzoyl peroxide as a bleaching agent. This review discusses the advantages, disadvantages, regulatory concerns, flavor implications, and optimal usage conditions of 2 widely used bleaching agents, hydrogen peroxide and benzoyl peroxide, as well as a few alternative methods including lipoxygenase, peroxidase, and lactoperoxidase systems.

Impact of Fat Reduction on Flavor and Flavor Chemistry of Cheddar Cheeses

A current industry goal is to produce a 75 to 80% fat-reduced Cheddar cheese that is tasty and appealing to consumers. Despite previous studies on reduced-fat cheese, information is critically lacking in understanding the flavor and flavor chemistry of reduced-fat and nonfat Cheddar cheeses and how it differs from its full-fat counterpart. The objective of this study was to document and compare flavor development in cheeses with different fat contents so as to quantitatively characterize how flavor and flavor development in Cheddar cheese are altered with fat reduction. Cheddar cheeses with 50% reduced-fat cheese (RFC) and low-fat cheese containing 6% fat (LFC) along with 2 full-fat cheeses (FFC) were manufactured in duplicate. Cheeses were ripened at 8°C and samples were taken following 2 wk and 3, 6, and 9 mo for sensory and instrumental volatile analyses. A trained sensory panel (n=10 panelists) documented flavor attributes of cheeses. Volatile compounds were extracted by solid-phase microextraction or solvent-assisted flavor evaporation followed by separation and identification using gas chromatography-mass spectrometry and gas chromatography-olfactometry. Selected compounds were quantified using external standard curves. Sensory properties of cheeses were distinct initially but more differences were documented as cheeses aged. By 9 mo, LFC and RFC displayed distinct burnt/rosy flavors that were not present in FFC. Sulfur flavor was also lower in LFC compared with other cheeses. Forty aroma-active compounds were characterized in the cheeses by headspace or solvent extraction followed by gas chromatography-olfactometry. Compounds were largely not distinct between the cheeses at each time point, but concentration differences were evident. Higher concentrations of furanones (furaneol, homofuraneol, sotolon), phenylethanal, 1-octen-3-one, and free fatty acids, and lower concentrations of lactones were present in LFC compared with FFC after 9 mo of ripening. These results confirm that flavor differences documented between full-fat and reduced-fat cheeses are not due solely to differences in matrix and flavor release but also to distinct differences in ripening biochemistry, which leads to an imbalance of many flavor-contributing compounds.