Differential Apoptotic Effects of Bee Product Mixtures on Normal and Cancer Hepatic Cells.

Most effective anticancer drugs normally generate considerable cytotoxicity in normal cells; therefore, the preferential activation of apoptosis in cancer cells and the reduction of toxicity in normal cells is a great challenge in cancer research. Natural products with selective anticancer properties used as complementary medicine can help to achieve this goal. The aim of the present study was to analyze the effect of the addition of bee products [propolis (PR) or royal jelly (RJ) or propolis and royal jelly (PR+RJ), 2-10%] to thyme (TH) and chestnut honeys (CH) on the differential anticancer properties, mainly the cytotoxic and pro-apoptotic effects, in normal and cancer hepatic cells. The cytotoxic effects of samples were analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (0-250 mg/mL) and the effects on apoptosis were analyzed using cell cycle analysis, TdT-dUTP terminal nick-end labeling (TUNEL) assay, DR5 (Death Receptor 5) and BAX (BCL-2-Associated X) activation, and caspases 8, 9, and 3 activities. Both honey samples alone and honey mixtures had no or very little apoptotic effect on normal cells. Antioxidant honey mixtures enhanced the apoptotic capacity of the corresponding honey alone via both extrinsic and intrinsic pathways. Of all the samples, chestnut honey enriched with 10% royal jelly and 10% propolis (sample 14, CH+10RJ+10PR) showed the highest apoptotic effect on tumor liver cells. The enrichment of monofloral honey with bee products could be used together with conventional anticancer treatments as a dietary supplement without side effects. On the other hand, it could be included in the diet as a natural sweetener with high added value.

Formation of Listeria monocytogenes persister cells in the produce-processing environment.

Persisters are a subpopulation of growth-arrested cells that possess transient tolerance to lethal doses of antibiotics and can revert to an active state under the right conditions. Persister cells are considered as a public health concern. This study examined the formation of persisters by Listeria monocytogenes (LM) in an environment simulating a processing plant for leafy green production. Three LM strains isolated from California produce-processing plants and packinghouses with the strongest adherence abilities were used for this study. The impact of the cells' physiological status, density, and nutrient availability on the formation of persisters was also determined. Gentamicin at a dose of 100 mg/L was used for the isolation and screening of LM persisters. Results showed that the physiological status differences brought by culture preparation methods (plate-grown vs. broth-grown) did not impact persister formation (P > 0.05). Instead, higher persister ratios were found when cell density increased (P < 0.05). The formation of LM persister cells under simulated packinghouse conditions was tested by artificially inoculating stainless steel coupons with LM suspending in media with decreasing nutrient levels: brain heart infusion broth (1366 mg/L O2), produce-washing water with various organic loads (1332 mg/L O2 and 652 mg/L O2, respectively), as well as sterile Milli-Q water. LM survived in all suspensions at 4 °C with 85 % relative humidity for 7 days, with strain 483 producing the most persister cells (4.36 ± 0.294 Log CFU/coupon) on average. Although persister cell levels of LM 480 and 485 were reasonably steady in nutrient-rich media (i.e., BHI and HCOD), they declined in nutrient-poor media (i.e., LCOD and sterile Milli-Q water) over time. Persister populations decreased along with total viable cells, demonstrating the impact of available nutrients on the formation of persisters. The chlorine sensitivity of LM persister cells was evaluated and compared with regular LM cells. Results showed that, despite their increased tolerance to the antibiotic gentamicin, LM persisters were more susceptible to chlorine treatments (100 mg/L for 2 min) than regular cells.

Protective Effect of Thyme and Chestnut Honeys Enriched with Bee Products against Benzo(a)pyrene-Induced DNA Damage.

The aim of the present study was to validate the cytotoxicity, genotoxicity, and preventive potential against benzo(a)pyrene (BaP)-induced DNA damage of nine samples of thyme and chestnut honeys enriched with bee products (royal jelly and propolis, 2-10%). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (0-250 mg/mL) to select nontoxic concentrations, and DNA damage (0.1-10 µg/mL) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Treatment with honey samples or royal jelly and propolis did not affect the viability of HepG2 cells up to 100 and 50 mg/mL, respectively. Treatment with 100 µM BaP significantly increased (p ≤ 0.001) the levels of the DNA strand breaks. None of the tested concentrations (0.1-10 µg/mL) of the honey samples (thyme and chestnut), royal jelly, and propolis caused DNA damage per se. All tested samples at all the concentrations used decreased the genotoxic effect of BaP. In addition, all mixtures of thyme or chestnut honeys with royal jelly or propolis showed a greater protective effect against BaP than the samples alone, being the thyme and chestnut honey samples enriched with 10% royal jelly and 10% propolis the most effective (70.4% and 69.4%, respectively). The observed protective effect may be associated with the phenolic content and antioxidant capacity of the studied samples. In conclusion, the thyme and chestnut honey samples enriched with bee products present potential as natural chemoprotective agents against the chemical carcinogen BaP.

Enhancement of the Antioxidant Capacity of Thyme and Chestnut Honey by Addition of Bee Products.

Honey consumption and imports have increased in recent years, and it is considered by consumers to be a healthy alternative to more commonly used sweeteners. Honey contains a mixture of polyphenols and antioxidant compounds, and the botanical origin and geographical area of collection play an important role on its chemical composition. The present study investigated the physicochemical properties, total phenolic content and antioxidant capacity of Spanish thyme honey and chestnut honey, and their mixtures with royal jelly (2% and 10%) and propolis (2% and 10%). The analysis of the physicochemical parameters of both honey samples showed values within the established limits. Propolis showed the highest value of total phenolic content (17.21-266.83 mg GAE/100 g) and antioxidant capacity (DPPH, ORAC and ABTS assays; 0.63-24.10 µg eq. Tx/g, 1.61-40.82 µg eq. Tx/g and 1.89-68.54 µg eq. Tx/g, respectively), and significantly reduced ROS production in human hepatoma cells. In addition, mixtures of honey with 10% of propolis improved the results obtained with natural honey, increasing the value of total phenolic content and antioxidant capacity. A significant positive correlation was observed between total phenolic compounds and antioxidant capacity. Therefore, the antioxidant capacity could be attributed to the phenolic compounds present in the samples, at least partially. In conclusion, our results indicated that thyme and chestnut honey supplemented with propolis can be an excellent natural source of antioxidants and could be incorporated as a potential food ingredient with biological properties of technological interest, added as a preservative. Moreover, these mixtures could be used as natural sweeteners enriched in antioxidants and other bioactive compounds.

Effect of pulsed light treatment on Listeria inactivation, sensory quality and oxidation in two varieties of Spanish dry-cured ham.

The efficacy of pulsed light (PL) for the surface decontamination of ready-to-eat dry-cured ham was studied in two Spanish varieties, Serrano and Iberian. Listeriainnocua was inoculated on the surface of ham slices that were vacuum-packaged and flashed with 2.1, 4.2 and 8.4 J/cm2. Survivors were enumerated immediately after treatment. Peroxide values, sensory analysis and volatile profile were investigated during storage at 4 and 20 °C. Inactivation of Listeria was higher in Iberian (ca. 2 log cfu/cm2) than in Serrano ham (ca. 1 log cfu/cm2) with 8.4 J/cm2. PL did not increase the peroxide values above the usual levels reported in dry-cured ham, and no rancid notes were observed in the sensory analysis. PL-treated samples showed an increase in the concentration of some volatile compounds, such as methional, dimethyl disulfide and 1-octen-3-one, which imparted slight sulfur and metallic notes, although they disappeared during storage.

Characterization of damage on Listeria innocua surviving to pulsed light: Effect on growth, DNA and proteome.

The effect of pulsed light treatment on the lag phase and the maximum specific growth rate of Listeria innocua was determined in culture media at 7 °C. Fluences of 0.175, 0.350 and 0.525 J/cm2 were tested. The lag phase of the survivors increased as fluence did, showing significant differences for all the doses; an 8.7-fold increase was observed at 0.525 J/cm2. Pulsed light decreased the maximum specific growth rate by 38% at the same fluence. Both parameters were also determined by time-lapse microscopy at 25 °C in survivors to 0.525 J/cm2, with an increase of 13-fold of the lag phase and a 45% decrease of the maximum specific growth rate. The higher the fluence, the higher the variability of both parameters was. To characterize pulsed light damage on L. innocua, the formation of dimers on DNA was assessed, and a proteomic study was undertaken. In cells treated with 0.525 J/cm2, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were detected at 5:1 ratio. Pulsed light induced the expression of three proteins, among them the general stress protein Ctc. Furthermore, treated cells showed an up-regulation of proteins related to metabolism of nucleotides and fatty acids, as well as with translation processes, whereas flagellin and some glucose metabolism proteins were down-regulated. Differences in the proteome of the survivors could contribute to explain the mechanisms of adaptation of L. innocua after pulsed light treatment.

Effect of nitrate and nitrite on Listeria and selected spoilage bacteria inoculated in dry-cured ham.

The effect of nitrate and the combination of nitrate/nitrite on Listeria innocua (as surrogate of Listeria monocytogenes). And two selected spoilage microorganisms (Proteus vulgaris and Serratia liquefaciens) was studied in dry-cured ham. Hams were manufactured with different concentrations of curing agents: KNO3 (600 and 150mg/kg) alone or in combination with NaNO2 (600 and 150mg/kg). The addition of 500mg/kg of sodium ascorbate was also evaluated in a batch with 600mg/kg of nitrate and nitrite. The target microorganisms were inoculated by injection in semimembranosus, biceps femoris and in the shank, prior to curing. P. vulgaris and S. liquefaciens were controlled by temperature and aw, respectively, and no effect of nitrate/nitrite was observed. The presence of nitrite in the curing mix reduced L. innocua in semimembranosus, which population was 1.5logcfu/g lower at the end of resting (p<0.05), while at the end of the process it was more frequently detected in the no- and low-nitrite added hams. None of the treatments was able to control Listeria in deeper areas of ham. The addition of sodium ascorbate to the curing mix containing the highest amount of nitrate and nitrite did not show any effect on the microorganisms studied.

A study on the toxigenesis by Clostridium botulinum in nitrate and nitrite-reduced dry fermented sausages.

Nitrite has been traditionally used to control Clostridium botulinum in cured meat products. However, in the case of dry fermented sausages, environmental factors such as pH, aw and the competitive microbiota may exert a more relevant role than nitrite in the inhibition of the growth and toxin production by C. botulinum. In this challenge test study, two varieties of Mediterranean dry sausages (salchichón and fuet) were inoculated with spores of C. botulinum Group I (proteolytic) and C. botulinum Group II (nonproteolytic). Sausages were prepared with 150 mg/kg of NaNO3 and 150 mg/kg of NaNO2 (maximum ingoing amounts allowed by the European Union regulation), with a 25% and 50% reduction, and without nitrate/nitrite. The initial pH in both products was 5.6, and decreased to values below 5.0 in salchichón and to 5.2 in fuet. Lactic acid bacteria counts reached 8-9 log cfu/g after fermentation. The aw decreased from initial values of 0.96 to about 0.88-0.90 at the end of ripening. Botulinum neurotoxin was not detected in any of the sausages, including those manufactured without nitrate and nitrite. Despite the environmental conditions were within the range for germination and growth of C. botulinum Group I during the first 8 days of the ripening process in fuet and 10-12 days in salchichón, acidity, aw and incubation temperature combined to inhibit the production of toxin, independently of the concentration of curing agents. Although decreasing or even removing nitrate/nitrite from the formula did not compromise safety regarding C. botulinum in the conditions tested in this study, their antimicrobial role should not be underestimated in the case that other hurdles could fail or other ripening conditions were used, and also considering the effect of nitrite on other pathogens.

Estimation of the growth kinetic parameters of Bacillus cereus spores as affected by pulsed light treatment.

Quantitative microbial risk assessment requires the knowledge of the effect of food preservation technologies on the growth parameters of the survivors of the treatment. This is of special interest in the case of the new non-thermal technologies that are being investigated for minimal processing of foods. This is a study on the effect of pulsed light technology (PL) on the lag phase of Bacillus cereus spores surviving the treatment and the maximum growth rate (µmax) of the survivors after germination. The D value was estimated as 0.35 J/cm(2) and our findings showed that PL affected the kinetic parameters of the microorganism. A log linear relationship was observed between the lag phase and the intensity of the treatment. Increasing the lethality lengthened the mean lag phase and proportionally increased its variability. A polynomial regression was fitted between the µmax of the survivors and the inactivation achieved. The µmax decreased as intensity increased. From these data, and their comparison to published results on the effect of heat and e-beam irradiation on B. cereus spores, it was observed that the shelf-life of PL treated foods would be longer than those treated with heat and similar to irradiated ones. These findings offer information of interest for the implementation of PL for microbial decontamination in the food industry.

Survival of Listeria innocua in dry fermented sausages and changes in the typical microbiota and volatile profile as affected by the concentration of nitrate and nitrite.

The involvement of nitrate and nitrite in the formation of N-nitrosamines in foods is a matter of great concern. This situation has led to revise the real amount of nitrate and nitrite needed in meat products to exert proper technological and safety activities, and also to extensive research to find alternatives to their use. The present study addresses the possibility of reducing the ingoing amounts of these additives below the legal limits established by the current European regulations. Different concentrations of nitrate and nitrite were tested on Spanish salchichón-type dry fermented sausages concerning their role in the microbiota and volatile profile. Sausages were manufactured with the maximum ingoing amounts established by the EU regulations (150 ppm NaNO(3) and 150 ppm NaNO(2)), a 25% reduction and a 50% reduction; control sausages with no nitrate/nitrite addition were also prepared. The mixtures were inoculated with 5 log cfu/g of Listeria innocua as a surrogate for Listeria monocytogenes. L. innocua numbers in the final product were approximately 1.5 log cfu/g lower in the batch with the maximum nitrate/nitrite concentration when compared to 25 and 50% reduced batches, and about 2 log cfu/g in comparison to the control sausages. The final numbers of catalase-positive cocci were 1 log cfu/g higher in the 50% nitrate/nitrite reduced batch and 2 log cfu/g higher in the control sausages, compared to products manufactured with the maximum nitrate/nitrite concentration. This increase was related to a higher amount of volatile compounds derived from carbohydrate fermentation and amino acid degradation. Sausages with no addition of nitrate/nitrite showed higher amount of volatiles from lipid oxidation. Enterobacteriaceae counts reached detectable values (1-2 log cfu/g) in both nitrate/nitrite reduced sausages and in the control batch, while these organisms were not detected in the batch with the maximum ingoing amount. Nitrate and nitrite exerted a significant effect on the typical microbiota of dry fermented sausages and effectively contributed to control Listeria. These considerations should be taken into account in view of a future restriction in the use of these curing additives.