Caffeine and Theobromine.

Two components of cocoa powder, caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine), were evaluated for their effect on growth of L. monocytogenes strain V7. Caffeine (0.5%) and theobromine (2.5%) were added singly or in combination to skim milk or a modified tryptose phosphate broth (MTPB), which were sterilized, inoculated to contain ca. 1 × 103 CFU L. monocytogenes /ml, and incubated at 30°C. Both compounds allowed some growth of the pathogen; however, longer lag phases occurred in samples with (6 to 9 h) rather than without (<3 h) caffeine. Generation times at 30°C ranged from 1.10 h (2.5% theobromine in broth) to 2.28 h (2.5% theobromine plus 0.5% caffeine in milk). Generation times were significantly (p<0.05) lengthened in the presence (2.17 h) rather than in the absence (1.2 h) of caffeine. Populations in samples without caffeine were more than ten times greater (8.57 log10 CFU/ml) than in samples with caffeine (7.21 log10 CFU/ml). Theobromine concentration (0 or 2.5%) and substrate (MTPB or skim milk) had only limited effects on growth of the pathogen. The change in pH of the medium was a function of the extent of growth of L. monocytogenes and the buffering capacity of the substrate. A smaller pH change (0.09 unit) occurred in the milk medium than in broth (0.48 unit) because of the minimal buffering capacity of the 0.2% tryptose solution. Samples with caffeine had a small decrease in pH (0.07 unit) because growth of L. monocytogenes was inhibited and thus acid production was minimal.

Inhibition of Listeria monocytogenes by Cocoa in a Broth Medium and Neutralization of this Effect by Casein.

Dutch-processed cocoa (0.75 to 10.0%, w/v), when added to a broth medium, inhibited/inactivated Listeria monocytogenes strain V7. With agitated incubation at 30°C, samples with 5.0, 7.5, and 10.0% cocoa were free of detectable viable cells (<1/ml) 15 to 24 h after inoculation to contain ca. 1 × 105 L. monocytogenes strain V7/ml. Without agitation at 30°C, presence of 0.75 to 10.0% cocoa lengthened (1.02 to 1.12 h) the generation time of the pathogen when compared to samples without cocoa (0.94 h). However, the pathogen, in samples containing cocoa, eventually reached a higher (9.05 to 9.18 log10 CFU/ml) population than in samples without cocoa (8.81 log10 CFU/ml). The lag phase of L. monocytogenes was longer with (8.15 h) rather than without (4.41 h) agitation, at a lower (ca 1×103 CFU/ml) (6.92 h) rather than a higher (ca. 1 × 105 CFU/ml) (5.65 h) inoculum level, and in the presence of 0.75% (10.52 h) rather than 0% (2.04 h) cocoa. Higher maximum populations (9.18 log10 CFU/ml) developed in samples that were agitated rather than incubated quiescently (8.39 log10 CFU/ml), and in samples with (9.23 log10 CFU/ml) rather than without (8.88 log10 CFU/ml) cocoa. Casein (1.5 or 3.0%) relieved the inhibition when incubation was quiescent and inactivation of L. monocytogenes by cocoa when incubation was with agitation. Without agitation, the lag phase of the pathogen was extended in the presence of cocoa (4.01 h) compared to samples with cocoa and casein (1.77 to 2.74 h). Casein did not significantly (p>0.05) affect the maximum population attained by the pathogen, but presence of cocoa increased the maximum population (8.86 and 9.25 log10 CFU/ml for 0 and 5.0% cocoa). With agitation, presence of 5.0% cocoa completely inactivated the pathogen; however, addition of 2.5% casein to these samples allowed growth of L. monocytogenes to a population of 9.47 log10 CFU/ml (compared to 8.90 log10 CFU/ml in broth).

Behavior of Listeria monocytogenes in the Presence of Cocoa, Carrageenan, and Sugar in a Milk Medium Incubated With and Without Agitation.

Enhanced growth of Listeria monocytogenes strain V7 in chocolate milk rather than skim milk was further investigated by testing various concentrations of cocoa powder (two types of Dutch-process, designated A and B), cane sugar, and sodium carrageenan in skim milk at 13 and 30°C with and without agitated incubation. Increasing sugar concentrations (0, 6.5, and 12.0%) were marginally significant (p = 0.06) in shortening generation times (5.17, 5.07, and 5.05 h, respectively) of the pathogen. Maximum populations attained by the pathogen were greater when cocoa (0.75% type A or B) and sugar (6.5 or 12.0%) were present. Sugar concentration affected growth of L. monocytogenes in an approximately linear relationship (8.41, 8.67, 8.82 log10 CFU/ml for 0, 6.5, and 12.0% sugar, respectively) except in samples containing only carrageenan. In this instance, presence of 6.5 and 12.0% sugar resulted in equivalent maximum populations (8.54 and 8.52 log10 CFU/ml). Three factors enhanced growth of the pathogen at 30°C: addition of cocoa, addition of sugar, and agitated rather than quiescent incubation. Without cocoa, generation times of L. monocytogenes were longer (1.04 h) compared to presence of type A (0.87 h) or B (0.90 h) cocoa. L. monocytogenes in agitated samples had shorter (0.82 h) generation times than in quiescent cultures (0.95 h). Highest populations were attained in agitated samples containing sugar and type A (9.21 log10 CFU/ml) or type B (9.22 log10 CFU/ml) cocoa compared to lowest populations in quiescent samples of skim milk (8.56 log10 CFU/ml).