Decimal reduction energies of UV-C-irradiated spoilage yeasts in coconut liquid endosperm.

The ultraviolet-C (UV-C) decimal reduction energy (DUV-C) values of 17 spoilage yeasts and their composited inoculum were determined in coconut liquid endosperm (pH 5.26, 5.8 °Brix, 0.04% malic acid, 0.17% w/v insoluble solids). Growth kinetic parameters of all the test yeast strains were first established to standardize the growth stage of the cells prior to inactivation studies. Approximately 4.0 to 5.0 log CFU/mL cells in the mid-stationary growth phase (30.3 to 39.9 h, 25 °C) were suspended in 4 mL turbulent flowing juice and subjected to UV-C irradiation at a surface irradiance range of 3.42 to 4.99 mW/cm2. Survivor populations after exposure to predetermined UV-C energy were enumerated, and were used to derive the DUV-C values using the linear regression and Baranyi and Roberts (1994) model fitting. Results show that the yeast strains exhibited either log-linear or biphasic inactivation behavior with inactivation lag. The most UV-C resistant spoilage yeast was found to be Cryptococcus albidus (LJY1) with DUV-C values of 122.72 and 214.89 mJ/cm2 determined from linear regression and model-fitting, respectively. The least UV-C resistant was Torulaspora delbrueckii (LYJ5) with a DUV-C of 17.34 (linear regression) and 17.35 mJ/cm2 (model-fitting). The DUV-C values determined from the model fitting were generally greater than those calculated from linear regression, although only those determined for C. albidus were significantly different. To the investigators' knowledge, this is the first report of the UV-C inactivation kinetic parameters of Kluyveromyces marxianus, Trichosporon cutaneum, Pichia anomala, and Meyerozyma guilliermondii and C. albidus in coconut liquid endosperm. The results of this study can be used in the establishment and validation of UV-C process schedules for coconut liquid endosperm and other similar commodities.

Individual and combined efficacies of mild heat and ultraviolet-c radiation against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in coconut liquid endosperm.

This study determined the inactivation kinetic parameters of selected pathogens in heat, ultraviolet-C and combined heat-UV-C treated coconut liquid endosperm. Separate cocktails of Escherichia coli O157:H7, Salmonella enterica serovars, and Listeria monocytogenes strains were inoculated into coconut liquid endosperm (pH 5.15, TSS 4.4oBx, TA 0.062% malic acid, extinction coefficient (ε) at 254 nm of 0.0154 cm-1) for inactivation studies. Result showed that all organisms generally exhibited a log-linear heat inactivation behavior (R2 0.81-0.99). The E. coli O157:H7 cocktail (D55 = 19.75 min, D57 = 10.79 min, D60 = 3.38 min, and D63 = 0.46 min) was found to be significantly more resistant (P > 0.05) than the tested cocktail of L. monocytogenes (D55 = 11.68 min, D57 = 4.53 min, D60 = 1.82 min and D63 = 0.26 min) and S. enterica cocktail (D55 = 3.08 min, D57 = 2.60 min, D60 = 0.89 min and D63 = 0.25 min). Despite the differences in DT values, computed z values for L. monocytogenes cocktail (5.12 ±â€¯0.43 °C) and E. coli O157:H7 cocktail (4.95 ±â€¯0.12 °C) were not significantly different (P > 0.05), but were both significantly (P < 0.05) lower than that of S. enterica cocktail (7.10 ±â€¯0.15 °C). All test organisms also exhibited a generally log-linear UV-C inactivation behavior (R2 0.90-0.99) with E. coli O157:H7 cocktail (DUV-C = 25.26 mJ/cm2) demonstrating greatest resistance to UV-C than S. enterica (DUV-C = 24.65 mJ/cm2) and L. monocytogenes (DUV-C = 17.30 mJ/cm2) cocktails. The D55 values of each organism cocktail were used to calculate for the 3-log reduction heating process schedules, during which UV-C treatments were simultaneously applied. Lethal rates (F values) calculations in the combined processes revealed that within the 3-log reduction heating processes, co-exposure of UV-C resulted in 5.62 to 6.20 log reductions in the test organism populations. Heating caused 69.3, 97.2, and 67.4% of the reduction in E. coli O157:H7, S. enterica and L. monocytogenes cocktails, respectively. These results can be used as baseline data in the establishment of mild heat treatment in combination with UV-C process schedules for coconut liquid endosperm and other similar products.

Exploring the Impacts of Postharvest Processing on the Microbiota and Metabolite Profiles during Green Coffee Bean Production.

The postharvest treatment and processing of fresh coffee cherries can impact the quality of the unroasted green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through two different wet and dry methods to monitor differences in the microbial community structure and in substrate and metabolite profiles. The changes were followed throughout the postharvest processing chain, from harvest to drying, by implementing up-to-date techniques, encompassing multiple-step metagenomic DNA extraction, high-throughput sequencing, and multiphasic metabolite target analysis. During wet processing, a cohort of lactic acid bacteria (i.e., Leuconostoc, Lactococcus, and Lactobacillus) was the most commonly identified microbial group, along with enterobacteria and yeasts (Pichia and Starmerella). Several of the metabolites associated with lactic acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in the mucilage were also found in the endosperm. During dry processing, acetic acid bacteria (i.e., Acetobacter and Gluconobacter) were most abundant, along with Pichia and non-Pichia (Candida, Starmerella, and Saccharomycopsis) yeasts. Accumulation of associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the drying outer layers of the coffee cherries. Consequently, both wet and dry processing methods significantly influenced the microbial community structures and hence the composition of the final green coffee beans. This systematic approach to dissecting the coffee ecosystem contributes to a deeper understanding of coffee processing and might constitute a state-of-the-art framework for the further analysis and subsequent control of this complex biotechnological process. IMPORTANCE: Coffee production is a long process, starting with the harvest of coffee cherries and the on-farm drying of their beans. In a later stage, the dried green coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm, postharvest processing method applied can impact the quality of the green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through wet and dry processing in four distinct variations. The microorganisms present and the chemical profiles of the coffee beans were analyzed throughout the postharvest processing chain. The up-to-date techniques implemented facilitated the investigation of differences related to the method applied. For instance, different microbial groups were associated with wet and dry processing methods. Additionally, metabolites associated with the respective microorganisms accumulated on the final green coffee beans.

PvPGIP2 Accumulation in Specific Floral Tissues But Not in the Endosperm Limits Fusarium graminearum Infection in Wheat.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most destructive fungal diseases of wheat worldwide. The pathogen infects the spike at flowering time and causes severe yield losses, deterioration of grain quality, and accumulation of mycotoxins. The understanding of the precise means of pathogen entry and colonization of floral tissue is crucial to providing effective protection against FHB. Polygalacturonase (PG) inhibiting proteins (PGIPs) are cell-wall proteins that inhibit the activity of PGs, a class of pectin-depolymerizing enzymes secreted by microbial pathogens, including Fusarium spp. The constitutive expression of a bean PGIP (PvPGIP2) limits FHB symptoms and reduces mycotoxin accumulation in wheat grain. To better understand which spike tissues play major roles in limiting F. graminearum infection, we explored the use of PvPGIP2 to defend specific spike tissues. We show here that the simultaneous expression of PvPGIP2 in lemma, palea, rachis, and anthers reduced FHB symptoms caused by F. graminearum compared with symptoms in infected nontransgenic plants. However, the expression of PvPGIP2 only in the endosperm did not affect FHB symptom development, indicating that once the pathogen has reached the endosperm, inhibition of the pathogen's PG activity is not effective in preventing its further spread.

Previous physicochemical stress exposures influence subsequent resistance of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes to ultraviolet-C in coconut liquid endosperm beverage.

This study investigated the influences of prior exposures to common physicochemical stresses encountered by microorganisms in food and food processing ecologies such as acidity, desiccation, and their combinations, on their subsequent susceptibility towards UV-C treatment in coconut liquid endosperm beverage. Cocktails of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes were separately subjected to gradually acidifying environment (final pH 4.46), exposed to abrupt desiccation by suspension in saturated NaCl solution (aw=0.85) for 4, 8, and 24h, and sequential acidic and desiccated stresses before suspending in the coconut beverage for UV-C challenge. The exposure times (D) and UV-C energy dose values (DUV-C) necessary to reduce 90% of the population of the different test organisms varied with previous exposures to different sublethal stresses, indicating possible influence of implicit microbial factors towards resistance to UV-C. All tested individual and combined stresses resulted in increased resistance, albeit some were not statistically significant. Non-stressed cells had D values of 3.2-3.5s, and corresponding DUV-C values of 8.4-9.1 mJ/cm(2). Cells exposed to previous acid stress had D values of 4.1-4.8s and corresponding DUV-C values of 10.7-12.5 mJ/cm(2). Prior exposure to desiccation resulted in D values of 5.6-7.9s and DUV-C values of 14.7-20.6 mJ/cm(2), while exposure to combined acid and desiccation stresses resulted in D values of 6.1-8.1s and DUV-C values of 15.9-21.0 mJ/cm(2). The D and DUV-C values of S. enterica after previous exposure to sequential acid (24 h) and desiccation (24 h) stresses were found significantly greatest, making the organism and physiological state an appropriate reference organism for the establishment of UV-C pasteurization process for the beverage.

Identification of the Infection Route of a Fusarium Seed Pathogen into Nondormant Bromus tectorum Seeds.

The genus Fusarium has a wide host range and causes many different forms of plant disease. These include seed rot and seedling blight diseases of cultivated plants. The diseases caused by Fusarium on wild plants are less well-known. In this study, we examined disease development caused by Fusarium sp. n on nondormant seeds of the important rangeland weed Bromus tectorum as part of broader studies of the phenomenon of stand failure or "die-off" in this annual grass. We previously isolated an undescribed species in the F. tricinctum species complex from die-off soils and showed that it is pathogenic on seeds. It can cause high mortality of nondormant B. tectorum seeds, especially under conditions of water stress, but rarely attacks dormant seeds. In this study, we used scanning electron microscopy (SEM) to investigate the mode of attack used by this pathogen. Nondormant B. tectorum seeds (i.e., florets containing caryopses) were inoculated with isolate Skull C1 macroconidia. Seeds were then exposed to water stress conditions (-1.5 MPa) for 7 days and then transferred to free water. Time lapse SEM photographs of healthy versus infected seeds revealed that hyphae under water stress conditions grew toward and culminated their attack at the abscission layer of the floret attachment scar. A prominent infection cushion, apparent macroscopically as a white tuft of mycelium at the radicle end of the seed, developed within 48 h after inoculation. Seeds that lacked an infection cushion completed germination upon transfer to free water, whereas seeds with an infection cushion were almost always killed. In addition, hyphae on seeds that did not initiate germination lacked directional growth and did not develop the infection cushion. This strongly suggests that the fungal attack is triggered by seed exudates released through the floret attachment scar at the initiation of germination. Images of cross sections of infected seeds showed that the fungal hyphae first penetrated the caryposis wall, then entered the embryo, and later ramified throughout the endosperm, completely destroying the seed.

Evaluation of sodium benzoate and licorice (Glycyrrhiza glabra) root extract as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm.

UNLABELLED: This study evaluated the use of sodium benzoate (SB) and licorice root extract (LRE) as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm. Consumer acceptance scoring showed that maximum permissible supplementation (MPS) levels for SB and LRE were at 300 and 250 ppm, respectively. The MPS values were considered in the generation of a 2-factor rotatable central composite design for the tested SB and LRE concentration combinations. Liquid endosperm with various SB and LRE supplementation combinations was inoculated with E. coli O157:H7 and heated to 55°C. The susceptibility of the cells towards heating was expressed in terms of the decimal reduction time (D55 ). Response surface analysis showed that only the individual linear effect of benzoate significantly influenced D55 value, where increasing supplementation level resulted in increasing susceptibility. The results reported could serve as baseline information in further investigating other additives that could be used as heat-sensitizing agents against pathogens in heat-labile food systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Fruit juice products have been linked to outbreaks of microbial infection, where unpasteurized products were proven vectors of diseases. Processors often opt not to apply heat process to juice products as the preservation technique often compromises the sensorial quality. This work evaluated two common additives for their heat-sensitizing effects against E. coli O157:H7 in coconut liquid endosperm, the results of which may serve as baseline information to small- and medium-scale processors, and researchers in the establishment of mild heat process schedule for the test commodity and other similar products.

An assessment of the biotechnological use of hemoglobin modulation in cereals.

Non-symbiotic hemoglobin (nsHb) genes are ubiquitous in plants, but their biological functions have mostly been studied in model plant species rather than in crops. nsHb influences cell signaling and metabolism by modulating the levels of nitric oxide (NO). Class 1 nsHb is upregulated under hypoxia and is involved in various biotic and abiotic stress responses. Ectopic overexpression of nsHb in Arabidopsis thaliana accelerates development, whilst targeted overexpression in seeds can increase seed yield. Such observations suggest that manipulating nsHb could be a valid biotechnological target. We studied the effects of overexpression of class 1 nsHb in the monocotyledonous crop plant barley (Hordeum vulgare cv. Golden Promise). nsHb was shown to be involved in NO metabolism in barley, as ectopic overexpression reduced the amount of NO released during hypoxia. Further, as in Arabidopsis, nsHb overexpression compromised basal resistance toward pathogens in barley. However, unlike Arabidopsis, nsHb ectopic overexpression delayed growth and development in barley, and seed specific overexpression reduced seed yield. Thus, nsHb overexpression in barley does not seem to be an efficient strategy for increasing yield in cereal crops. These findings highlight the necessity for using actual crop plants rather than laboratory model plants when assessing the effects of biotechnological approaches to crop improvement.

Localization, morphology and transcriptional profile of Aspergillus flavus during seed colonization.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize kernels pre-harvest, creating major human health concerns and causing substantial agricultural losses. Improved control strategies are needed, yet progress is hampered by the limited understanding of the mechanisms of infection. A series of studies were designed to investigate the localization, morphology and transcriptional profile of A. flavus during internal seed colonization. Results from these studies indicate that A. flavus is capable of infecting all tissues of the immature kernel by 96 h after infection. Mycelia were observed in and around the point of inoculation in the endosperm and were found growing down to the germ. At the endosperm-germ interface, hyphae appeared to differentiate and form a biofilm-like structure that surrounded the germ. The exact nature of this structure remains unclear, but is discussed. A custom-designed A. flavus Affymetrix GeneChip® microarray was used to monitor genome-wide transcription during pathogenicity. A total of 5061 genes were designated as being differentially expressed. Genes encoding secreted enzymes, transcription factors and secondary metabolite gene clusters were up-regulated and considered to be potential effector molecules responsible for disease in the kernel. Information gained from this study will aid in the development of strategies aimed at preventing or slowing down A. flavus colonization of the maize kernel.

Mass spectrometric analysis reveals remnants of host-pathogen molecular interactions at the starch granule surface in wheat endosperm.

The starch granules of wheat seed are solar energy-driven deposits of fixed carbon and, as such, present themselves as targets of pathogen attack. The seed's array of antimicrobial proteins, peptides, and small molecules comprises a molecular defense against penetrating pathogens. In turn, pathogens exhibit an arsenal of enzymes to facilitate the degradation of the host's endosperm. In this context, the starch granule surface is a relatively unexplored domain in which unique molecular barriers may be deployed to defend against and inhibit the late stages of infection. Therefore, it was compelling to explore the starch granule surface in mature wheat seed, which revealed evidence of host-pathogen molecular interactions that may have occurred during grain development. In this study, starch granules from the soft wheat Triticum aestivum cv. AC Andrew and hard wheat T. turgidum durum were isolated and water washed 20 times, and their surface proteins were digested in situ with trypsin. The peptides liberated into the supernatant and the peptides remaining at the starch granule surface were separately examined. In this way, we demonstrated that the identified proteins have a strong affinity for the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Although most of these peptides corresponded to uncharacterized hypothetical proteins of fungal pathogens, several peptide fragments were identical to cytosolic and membrane proteins of specific microbial pathogens. During development and maturation, wheat seed appeared to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface.