Control of Bacillus weihenstephanensis in Pasteurized Liquid Whole Eggs Formulated with Nisin

Bacillus weihenstephanensis can grow at refrigeration temperature and cause food poisoning. It has been isolated from liquid whole egg products. The moderate heat used for pasteurization of liquid egg products is ineffective for killing spore-forming bacteria including Bacillus. Available predictive models and a pretrial study in broth suggested the potential for growth of Bacillus spp. under the tested conditions. Hence, hurdles such as storage of product below 4°C or use of preservatives would be needed to ensure the food safety of pasteurized egg products. This study evaluated the growth inhibition of B. weihenstephanensis in pasteurized liquid whole egg product formulated with 6.25 ppm nisin during storage at refrigerated and abuse refrigerated temperatures for a total 13 weeks, in three replicate trials. At day 0, the product had a pH of 7.52±0.29, while background microflora such as aerobic plate counts, presumptive B. cereus, and yeast and molds were <10 CFU/g. Product inoculated with target 2.5 log10 CFU/g of B. weihenstephanensis, stored at 4°C for 4 weeks and subsequently at 7 or 10°C for 9 weeks exhibited no growth in all three replicate trials. Average counts reduced (p<0.05) by at least one-log10 in six weeks in all samples stored at either 7 or 10°C. Similarly, growth of total plate counts, presumptive Bacillus spp., yeast and mold counts was not observed in uninoculated controls stored at 4°C for 4 weeks and subsequently at 7 or 10°C for 9 weeks. Visual and odor evaluation performed at each sampling time point showed no abnormalities. This study assessed the efficacy of maximum allowed level of nisin for use in pasteurized liquid whole eggs and validated the inhibition of B. weihenstephanensis in the product for an extended shelf life of up to 13 weeks.

Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time

ABSTRACT Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis. Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH. IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

Phylogenetic characteristics of novel Bacillus weihenstephanensis and Pseudomonas sp. to desert locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae)

Thirty bacterial isolates were isolated from the gut contents of diseased/dead locust. Their pathogenicity was tested against 4th instar nymphs of desert locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae). Two isolates, designated DL2 and DL6, out of thirty showed the highest insecticidal activities against locust nymphs in preliminary bracketing. They were bioassayed via leaf dip and per os techniques and toxicity was determined using SAS program. The insecticidal activity of DL6 was more than DL2, whereas LC50’s values of 35 × 106 and 13 × 106 cfu’s/ml were determined for DL2 and DL6, respectively, after 48 h of leaf-dip treatment. However, LD50’s value of 53 × 106 and 26 × 106 cfu’s/ml was determined for DL2 and DL6, respectively, after 24 h of per os treatment. The relative potencies of DL6 to DL2 were (2.6 and 2.03) folds in leaf-dip and per os treatments, respectively. Biochemical characterization was conducted, using GEN III MicroPlate™ Biolog identification system and confirmed with molecular identification via 16S rDNA gene sequencing. Nucleotide sequencing of each was submitted to a gene bank and an accession number was generated for each isolate. Obtained bacterial strains DL2 and DL6 were identified as Bacillus weihenstephanensis (KY630645) and Pseudomonas sp. (KY630649), with a similarity of 100 and 75% to B. weihenstephanensis strain PHCDB9 (NR_024697) and Pseudomonas sp. strain DSM11821 (KF417541), respectively. The tested strains proved their potential to be bio-pesticide agents involved in controlling desert locust nymphs. Graphical abstract