Effects of growth conditions on the resistance of some pathogenic and spoilage species to high pressure homogenization

R. LANCIOTTI, F. GARDINI, M. SINIGAGLIA AND M.E. GUERZONI. 1996. The effects of chemicophysical growth conditions such as pH, temperature and water activity ( a _w) on lethal high homogenization pressure effects on Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Yarrowia lipolytica were investigated. The results, though based on standard media, emphasize the importance of food system composition and its thermal history on the high pressure tolerance of the microbial population.     

Evaluation of Fourier-transform infrared spectroscopy for data capture in predictive microbiology

Chemical changes in the medium, induced by the fermentative species Lactobacillus plantarum and Lactobacillus brevis and by the enzymatic action of a proteolytic, spoilage species, Yarrowia lipolytica, were analysed using Fourier-transform i.r. spectroscopy (FTIR). Changes in the absorbance data over time could be modelled using one of the more current predictive, mathematical models of microbial growth, such as the Gompertz equation. Moreover, a linear correlation between FTIR data (expressed as absorbance of some selected peaks) and viability data (expressed as log₁₀ c.f.u./g or ml) was observed during the fermentation process, both for L. plantarum and L. brevis.     

Antimicrobial Activity of Aroma Compounds against Saccharomyces cerevisiae and Improvement of Microbiological Stability of Soft Drinks as Assessed by Logistic Regression

The combined effects of a mild heat treatment (55°C) and the presence of three aroma compounds [citron essential oil, citral, and (E)-2-hexenal] on the spoilage of noncarbonated beverages inoculated with different amounts of a Saccharomyces cerevisiae strain were evaluated. The results, expressed as growth/no growth, were elaborated using a logistic regression in order to assess the probability of beverage spoilage as a function of thermal treatment length, concentration of flavoring agents, and yeast inoculum. The logit models obtained for the three substances were extremely precise. The thermal treatment alone, even if prolonged for 20 min, was not able to prevent yeast growth. However, the presence of increasing concentrations of aroma compounds improved the stability of the products. The inhibiting effect of the compounds was enhanced by a prolonged thermal treatment. In fact, it influenced the vapor pressure of the molecules, which can easily interact within microbial membranes when they are in gaseous form. (E)-2-Hexenal showed a threshold level, related to initial inoculum and thermal treatment length, over which yeast growth was rapidly inhibited. Concentrations over 100 ppm of citral and thermal treatment longer than 16 min allowed a 90% probability of stability for bottles inoculated with 10⁵ CFU/bottle. Citron gave the most interesting responses: beverages with 500 ppm of essential oil needed only 3 min of treatment to prevent yeast growth. In this framework, the logistic regression proved to be an important tool to study alternative hurdle strategies for the stabilization of noncarbonated beverages.     

Two 2[5H]-Furanones as Possible Signaling Molecules in Lactobacillus helveticus

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.     

Effect of trans-2-hexenal on the growth of Aspergillus flavus in relation to its concentration, temperature and water activity

AIMS: The antifungal activity of trans-2-hexenal on Aspergillus flavus in a model system in relation to its concentration, incubation temperature and aw was assessed. METHODS AND RESULTS: A model describing the antifungal activity of the aldehyde in relation to these variables was obtained. CONCLUSION: According to this model, the inhibition of A. flavus was weakly dependent on the incubation temperature (at least within the range of values considered) and strongly affected by the trans-2-hexenal concentration and aw, which showed a remarkable synergistic effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-2-hexenal proved to be a molecule with remarkable antimicrobial properties, even when added in closed systems at low concentration.     

Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.     

Study of the effects of temperature, pH, NaCl, and aw on the proteolytic and lipolytic activities of cheese-related lactic acid bacteria by quadratic response surface methodology

The individual and interactive effects of temperature, pH, NaCl, and a_w on the proteolytic and lipolytic activities of Lactobacillus delbrueckii subsp. bulgaricus B397, Lactococcus lactis subsp. lactis T12, and Lb. plantarum 2739 were studied by quadratic response surface methodology. The effects on enzyme activities depended on the interactions among the independent variables, type of activity, substrate and, especially, species. The proteinase activity of strains B397 and T12 was affected differently by pH as individual or interactive terms depending on the type of substrate _sl- or β-casein. The increase of NaCl concentration (2.5–7.5%) under cheese-like conditions had a negative effect on the proteinase activity of strain T12. The effect of NaCl was related to the corresponding decrease in a_w. Aminopeptidases N and A, iminopeptidase and endopeptidase of Lc. lactis subsp. lactis T12 were strongly inhibited by pH 5–6 and NaCl concentration higher than 3.75%. The negative effects of these independent variables was in several cases enhanced by their interaction and/or by the interaction with the lowest temperatures. In contrast, the same peptidases of Lb. plantarum 2739 retained a high activity under the very hostile environmental conditions. Iminopeptidase and especially endopeptidase activities of strain 2739 were stimulated slightly by NaCl at concentrations up to 5%. Lipase/esterase activity of Lb. delbrueckii subsp. bulgaricus B397 was markedly inhibited under cheese-like conditions.     

Characterisation of yeast microbiota,chemical and sensory properties of organic and biodynamic Sangiovese red wines

Wine quality is closely linked to the fermentation step, which is driven by the microbial ecology of grape and the use of selected microbial strains as well. The microbial species developing during fermentation determines the type and concentration of many substances, which contribute to the sensory properties of wine and its safety. In this view, the present work aims to characterise the yeast microbiota, chemical and sensory properties of Sangiovese red wines obtained from both biodynamic and organic agriculture. The natural yeast populations of grape musts and their evolution during spontaneous were monitored and investigated. In addition, the volatile composition, physico-chemical and safety features (ethyl-carbamate) and sensory properties of wines were evaluated. The results showed that the yeast population was mostly related to the grape management, i.e. organic or biodynamic, while the wine composition was mainly affected by the winemaking process, and then by the grape management.     

Vanadate-induced cell growth regulation and the role of reactive oxygen species

While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (*OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O2*-), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O2*- by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O2*- in turn generated H2O2, which reacted with vanadium(IV) to generate *OH radical through a Fenton-type reaction (V(IV) + H2O2 --> V(V) +*OH + OH-). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1 expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1 levels. Superoxide dismutase, a scavenger of O2*-, or sodium formate, an inhibitor of *OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2 is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex.