Improving and Comparing Probiotic Plate Count Methods by Analytical Procedure Lifecycle Management.

Probiotics are live microorganisms that confer a health benefit to the host when administered in adequate amounts. This definition links probiotic efficacy to microbial viability. The current gold standard assay for probiotic potency is enumeration using classical microbiology plating-based procedures, yielding results in colony-forming units (CFU). One drawback to plating-based procedures is high variability due to intrinsic and extrinsic uncertainties. These uncertainties make comparison between analytical procedures challenging. In this article, we provide tools to reduce measurement uncertainty and strengthen the reliability of probiotic enumerations by using analytical procedure lifecycle management (APLM). APLM is a tool that uses a step-by-step process to define procedure performance based on the concept that the reportable value (final CFU result) must be fit for its intended use. Once the procedure performance is defined, the information gathered through APLM can be used to evaluate and compare procedures. Here, we discuss the theory behind applying APLM and give practical information about its application to CFU enumeration procedures for probiotics using a simulated example and data set. Data collected in a manufacturer's development laboratory is included to support application of the concept. Implementation of APLM can lead to reduced variability by identifying specific factors (e.g., the dilution step) with significant impact on the variability and providing insights to procedural modifications that lead to process improvement. Understanding and control of the analytical procedure is improved by using these tools. The probiotics industry can confidently apply the information and analytical results generated to make decisions about processes and formulation, including overage requirements. One benefit of this approach is that companies can reduce overage costs. More reliable procedures for viable cell count determinations will improve the quality evaluation of probiotic products, and hence manufacturing procedures, while ensuring that products deliver clinically demonstrated beneficial doses.

Improving End-User Trust in the Quality of Commercial Probiotic Products.

In a rapidly growing global probiotic market, end-users have difficulty distinguishing between high quality and poor quality products. This ambiguity threatens the trust consumers and healthcare providers have in probiotic products. To address this problem, we recommend that companies undergo third-party evaluations to certify probiotic quality and label accuracy. In order to communicate about product quality to end-users, indication of certification on product labels is helpful, although not all manufacturers choose to use this approach. Herein we discuss: third-party certification, the process of setting standards for identity, purity, and quantification of probiotics; some emerging methodologies useful for quality assessment; and some technical challenges unique to managing quality of live microbial products. This review provides insights of an Expert Panel engaged in this process and aims to update the reader on relevant current scientific methodologies. Establishing validated methodologies for all aspects of quality assessment is an essential component of this process and can be facilitated by established organizations, such as United States Pharmacopeia. Emerging methodologies including whole genome sequencing and flow cytometry are poised to play important roles in these processes.

Bacillus cereus food poisoning and its toxins.

The genus Bacillus includes members that demonstrate a wide range of diversity from physiology and ecological niche to DNA sequence and gene regulation. The species of most interest tend to be known for their pathogenicity and are closely linked genetically. Bacillus anthracis causes anthrax, and Bacillus thuringiensis is widely used for its insecticidal properties but has also been associated with foodborne disease. Bacillus cereus causes two types of food poisoning, the emetic and diarrheal syndromes, and a variety of local and systemic infections. Although in this review we provide information on the genus and a variety of species, the primary focus is on the B. cereus strains and toxins that are involved in foodborne illness. B. cereus produces a large number of potential virulence factors, but for the majority of these factors their roles in specific infections have not been established. To date, only cereulide and the tripartite hemolysin BL have been identified specifically as emetic and diarrheal toxins, respectively. Nonhemolytic enterotoxin, a homolog of hemolysin BL, also has been associated with the diarrheal syndrome. Recent findings regarding these and other putative enterotoxins are discussed.

Rates of Thermal Inactivation of Listeria monocytogenes in Beef and Fermented Beaker Sausage.

Rates of thermal inactivation of a five-strain mixture of Listeria monocytogenes were determined in ground beef roast and fermented beaker sausage. Studies were also done on ground beef contaminated with L. monocytogenes Scott A from an experimentally infected cow. D-values for the five-strain mixture at 54.4, 57.2, 60.0, and 62.8°C were 22.4, 15.7, 4.47, and 2.56 min, respectively, for ground beef roast. D-values for fermented beaker sausage at 48.9, 51.7, 54.4, and 60.0°C were 98.6, 44.4, 20.1, 11.2, and 9.13 min, respectively. D-values for the single strain of L. monocytogenes mixture in ground beef from the infected cow were about two to four times less at equivalent temperatures than those of the five-strain L. monocytogenes mixture in ground beef roast. Results from the five-strain mixture indicate that L. monocytogenes is about four times more heat resistant that Salmonella in ground beef roast.

Comparison of Procedures for Isolating Listeria monocytogenes in Soft, Surface-Ripened Cheese.

Ninety samples of soft, surface-ripened cheese from a lot previously identified to contain Listeria were assayed for Listeria monocytogenes by three procedures. These included: (a) cold enrichment, (b) the Food and Drug Administration enrichment procedure, and (c) the selective enrichment procedure of Doyle and Schoeni (Appl. Environ. Microbiol. 15:1127, 1986). L. monocytogenes was isolated from 41 of the 90 cheese samples. The organism was isolated from only 9 of the 41 L. monocytogenes -positive samples by more than one procedure. Most isolations (21) were made by the cold enrichment procedure, with 16 and 13 isolations made by the FDA and Doyle-Schoeni procedures, respectively. In most instances, the organism was isolated from a cheese sample by only one procedure.