Survival and Virulence of Listeria monocytogenes during Storage on Chocolate Liquor, Corn Flakes, and Dry-Roasted Shelled Pistachios at 4 and 23°C

ABSTRACT The survival and virulence of Listeria monocytogenes was assessed during storage on three low-moisture foods (LMFs): chocolate liquor, corn flakes, and shelled, dry-roasted pistachios (water activity [aw] of 0.18, 0.27, and 0.20, respectively). The LMFs were inoculated with a four-strain cocktail of L. monocytogenes at 8 log CFU/g, dried, held until the aw stabilized, and then stored at 4°C and 25 to 81% relative humidity (RH) and at 23°C and 30 to 35% RH for at least 336 days. At 4°C, L. monocytogenes remained stable on the LMFs for at least 336 days. At 23°C, L. monocytogenes levels declined on the chocolate liquor, corn flakes, and pistachios at initial rates of 0.84, 0.88, and 0.32 log CFU/g/month, respectively. After 8 months at 23°C, L. monocytogenes levels on the chocolate liquor and corn flakes decreased to below the limit of detection (i.e., 0.48 log CFU/g). Relative populations of each strain were assessed before storage (i.e., day 0) and after 6 and 12 months of storage at 23 and 4°C, respectively. Generally, a decline in the relative level of the serotype 1/2a strain was observed during storage, coupled with the relative increase in other strains, depending on the LMF and storage temperature. The total viable populations of L. monocytogenes determined by the PMAxx quantitative PCR method after >12 months of storage at 4°C were significantly (1.8- to 3.7-log) higher than those obtained by plating on tryptic soy agar with yeast extract. Decreases in the culturable population of L. monocytogenes during storage on the LMFs were the result of both cellular inactivation and transition to a viable-but-nonculturable state. The surviving cells, specifically after long-term storage at 4°C on the chocolate liquor and pistachios, remained infectious and capable of intracellular replication in Caco-2 enterocytes. These results are relevant for predictive modeling used in microbial health risk assessments and support the addition of LMFs to food safety questionnaires conducted during listeriosis outbreaks. HIGHLIGHTS

Impact of chocolate liquor on vascular lesions in apoE-knockout mice

Cocoa polyphenols are thought to reduce the risk of cardiovascular diseases. Thus, cocoa-containing foods may have significant health benefits. Here, we studied the impact of chocolate liquor on vascular lesion development and plaque composition in a mouse model of atherosclerosis. Apolipoprotein E (apoE)-knockout mice were assigned to two groups and fed a Western diet that contained 250 g/kg of either chocolate liquor or a polyphenol-free isoenergetic control paste for 16 weeks. In addition to fat, protein, and fibers, the chocolate liquor contained 2 g/kg of polyphenols. Compared with the control group, mice fed the chocolate liquor had larger plaque areas in the descending aorta and aortic root, which were attributed to a higher mass of vascular smooth muscle cells (VSMCs) and collagen. Vascular lipid deposits and calcification areas did not differ between the two groups. The aortic tissue level of interleukin-6 (IL-6) mRNA was 5-fold higher in the mice fed chocolate liquor than in the control mice. Chocolate-fed mice exhibited an increased hepatic saturated to polyunsaturated fatty acid ratio than the controls. Although the chocolate liquor contained 14 µg/kg of vitamin D2, the chocolate liquor-fed mice did not have measurable 25-hydroxyvitamin D2 in the serum. These mice even showed a 25% reduction in the level of 25-hydroxyvitamin D3 compared with the control mice. Overall, present data may contribute to our understanding how chocolate constituents can impact vascular lesion development.

Modified Method for Extracting Light Filth from Chocolate Liquor by Using 2% Igepal CO 630 as Detergent Solution: Collaborative Study

A collaborative study was conducted among 3 laboratories comparing 2 detergent solutions, 2% Igepal CO 630 and the AOAC-approved 2% sodium lauryl sulfate, in the analysis of extraneous materials in chocolate liquor. Each laboratory tested a minimum of 8 samples per detergent solution. No significant differences were found between the numbers of rodent hairs and insect fragments recovered with the 2 detergent solutions and, in addition, igepal produced cleaner filter papers. The 2% Igepal CO 630 detergent solution has been adopted as a substitute for 2% sodium lauryl sulfate in 44.006(b) for chocolate liquor.

Comparison of Igepal and Sodium Lauryl Sulfate as Defatting Agents in Extraneous Materials Analysis of Chocolate Liquor

A detergent solution, 2% igepal CO 630, was compared with the AOAC-approved 2% sodium lauryl sulfate in the extraneous materials analysis of chocolate liquor. Thirty-two liquor samples were analyzed by one technician using both detergent solutions. No appreciable difference was noted between insect fragment and rodent hair counts obtained with the 2 detergent solutions. Igepal costs less and its use results in fewer filter papers to read. A wide range of insect fragment and rodent hair contamination should be studied with this reagent.

Collaborative Study of Methods for the Determination of Fat in Cacao Products

A collaborative study was conducted comparing the AOAC methods (12.022 Method I and 12.023 Method II) for the determination of fat in cacao products with the OICC Soxhlet method and a rapid refractometric method. Six samples of cacao products (breakfast cocoa, vegetable fat coating, cacao nibs, milk chocolate, sweet chocolate, and chocolate liquor) were analyzed by 7 collaborators. Results indicate that the OICC method gives better precision and somewhat lower results than the AOAC method. It is recommended (1) that the OICC method as presented, using a final HC1 concentration of 4N in the digestion step, be adopted as official first action to replace 12.023 Method II, which will be deleted official final action by suspension of the rules; (2) that study be continued to accumulate data needed to correlate the results of the analysis of cacao products by the OICC method and 12.022 Method I; and (3) that 12.022 be qualified by a statement “(Not applicable to cacao nibs unless finely ground)”.