Changes in GC-MS metabolite profile, antioxidant capacity and anthocyanins content during fermentation of fine-flavor cacao beans from Ecuador.

The fermentation of fine-flavor cacao beans is a key process contributing to the enhancement of organoleptic attributes and monetary benefits for cacao farmers. This work aimed to describe the dynamics of the gas chromatography-mass spectrometry (GC-MS) metabolite profile as well as the antioxidant capacity and anthocyanin contents during fermentation of fine-flavor cacao beans. Samples of Nacional x Trinitario cacao beans were obtained after 0, 24, 48, 72, 96, and 120 hours of spontaneous fermentation. Total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and total anthocyanin content were measured by ultraviolet-visible (UV-Vis) spectrophotometry. Volatiles were adsorbed by headspace solid phase microextraction (HS-SPME) while other metabolites were assessed by an extraction-derivatization method followed by gas chromatography-mass spectrometry (GC-MS) detection and identification. Thirty-two aroma-active compounds were identified in the samples, including 17 fruity, and 9 floral-like volatiles as well as metabolites with caramel, chocolate, ethereal, nutty, sweet, and woody notes. Principal components analysis and Heatmap-cluster analysis of volatile metabolites grouped samples according to the fermentation time. Additionally, the total anthocyanin content declined during fermentation, and FRAP-TPC values showed a partial correlation. These results highlight the importance of fermentation for the improvement of the fine-flavor characteristics of cacao beans.

Microbial Diversity and Contribution to the Formation of Volatile Compounds during Fine-Flavor Cacao Bean Fermentation.

Cacao demand is continuously increasing, and variations in cacao prices have been associated with the aroma of fermented cacao beans. However, the role of microorganisms in the formation of volatile-aroma compounds during fermentation remains unclear. Microbial diversity in Nacional × Trinitario cacao was characterized during spontaneous fermentation by using culture-based methods and next-generation sequencing (NGS) of DNA amplicons. Cacao beans that were spontaneously fermented for 0, 24, 48, 72 and 96 h were UV-sterilized prior to the inoculation of the microbial isolates obtained by the culture-based methods. The volatile formation in inoculated cacao beans was evaluated by GC-MS. The species isolated during fermentation included yeast, such as Saccharomyces cerevisiae and Candida metapsilosis; lactic acid bacteria (LAB), such as Limosilactobacillus fermentum and Liquorilactobacillus nagelii; acetic acid bacteria (AAB), such as Acetobacter pasteurianus, Acetobacter ghanensis and Acetobacter syzygii, as well as other species, such as Bacillus subtilis and Bacillus amyloliquefaciens. Additionally, NGS revealed an abundance of environmental microorganisms, including Escherichia spp., Pantoea spp., Staphylococcus spp., Botrytis spp., Tetrapisispora spp. and Pichia spp., among others. During the lab-scale fermentation, the inoculation of S. cerevisiae mostly yielded alcohols, while LAB and AAB produced volatiles associated with floral, almond and fruity notes throughout the fermentation, but AAB also produced acetic acid with a sour aroma. Similarly, the inoculation of C. metapsilosis and Bacillus spp. in 96 h fermented cacao beans yielded esters with floral aromas. This is the first report describing the role of microorganisms in volatile formation during fine-flavor cacao fermentation.

Plants and Natural Products with Activity against Various Types of Coronaviruses: A Review with Focus on SARS-CoV-2.

COVID-19 is a pandemic disease caused by the SARS-CoV-2 virus, which is potentially fatal for vulnerable individuals. Disease management represents a challenge for many countries, given the shortage of medicines and hospital resources. The objective of this work was to review the medicinal plants, foods and natural products showing scientific evidence for host protection against various types of coronaviruses, with a focus on SARS-CoV-2. Natural products that mitigate the symptoms caused by various coronaviruses are also presented. Particular attention was placed on natural products that stabilize the Renin-Angiotensin-Aldosterone System (RAAS), which has been associated with the entry of the SARS-CoV-2 into human cells.

GC-MS metabolite profiling for specific detection of dwarf somaclonal variation in banana plants.

PREMISE OF THE STUDY: The production of banana (Musa spp.; Musaceae) plants is affected by various types of somaclonal variations (SV), including dwarfism. However, methods for specific detection of SV are still scarce. To overcome this, a metabolite-based method for detection of dwarf variants was evaluated. METHODS: The gas chromatography-mass spectrometry (GC-MS) metabolite profile of dwarf banana variants was investigated and compared to that of normal-healthy (N) and cucumber mosaic virus (CMV)-infected plants using principal components analysis and partial least squares discriminant analysis (PLS-DA). RESULTS: Significant differences among the sample groups were observed in 82 metabolites. Rhamnose was exclusively present in dwarf plants but allothreonine and trehalose were present in all but SV samples. Cellobiose was only detected in N plants, while 45 other metabolites, including methyl-glucopyranoside, allopyranose, lactose, phenylalanine, and l-lysine were detected in all but CMV-infected samples. PLS-DA models were able to detect SV, CMV, and N plants with 100% accuracy and specificity. DISCUSSION: The GC-MS metabolite profile can be used for the rapid, specific detection of SV at early plant production stages. This is the first metabolite-based characterization and detection of somaclonal variation in plants.

Chemical, antimicrobial, and molecular characterization of mortiño (Vaccinium floribundum Kunth) fruits and leaves.

Fruits and leaves of Vaccinium spp. are known for their high content of bioactive compounds, but the chemical and biological characteristics of mortiño (Vaccinium floribundum Kunth) have not been fully described. In this study, the levels of polyphenols, antioxidant capacity, anthocyanins, antimicrobial activity, and genetic variability were determined in mortiño plants. The Folin-Ciocalteu's, ABTS scavenging, pH differential, and well diffusion methods were used to evaluate the levels of polyphenols, antioxidant capacity, anthocyanins, and antimicrobial activity, respectively. The genetic variability was evaluated by sequencing of the matK and rbcl DNA regions. Polyphenol content was up to 229.81 mg gallic acid equivalents/100 g, the average antioxidant capacity was 11.01 mmol Trolox equivalents/100 g, and anthocyanin content was up to 1,095.39 mg/100 g. Mortiño extracts significantly inhibited the growth of Gram-negative bacteria including Burkholderia gladioli, Burkholderia cepacia, Salmonella Typhimurium, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus, Escherichia coli, and Pseudomona aeruginosa, as well as Gram-positive bacteria such as Probionibacterium propionicum, Staphylococcus aureus, and Enterococcus faecalis showing greater inhibition halos than those produced by the antibiotic ampicillin. A polymorphic nucleotide was found in position 739 of the matK region. This study shows the potential of mortiño for the food and pharmaceutical industries.

Survival of Salmonella enterica Typhimurium in water amended with manure.

Outbreaks of Salmonella enterica have been associated with water sources. Survival of S. enterica in various environments has been studied but survival in water has rarely been attempted. In two separate experiments, we examined the survival of S. enterica Typhimurium in clean spring water at various eutrophication levels and temperatures. In the first experiment, lasting for 135 days, survival of S. enterica (10(10) CFU/ml) in water with 0, 50, 100, 500, and 1,000 mg/liter of added carbon at 7, 17, and 27°C was monitored weekly. In the second experiment, lasting for 3 weeks, survival of S. enterica in water at 0, 100, and 200 mg/ liter of added carbon and 27°C was studied daily. Each experiment had four replicates. Dissolved organic carbon was measured daily in each experiment. At the beginning, midpoint, and end of the survival study, microbial communities in both experiments were assessed by denaturing gradient gel electrophoresis (DGGE). Even at minimal carbon concentrations, S. enterica survived for at least 63 d. Survival of Salmonella was highly dependent on eutrophication levels (as measured by dissolved organic carbon) and temperature, increasing at high eutrophication levels, but decreasing at high temperatures. Survival was also strongly affected by microbial competition or predation.

Occurrence and population density of Campylobacter jejuni in irrigation ponds on produce farms in the Suwannee River Watershed.

Campylobacter spp., especially Campylobacter jejuni, are common causal agents of gastroenteritis globally. Poultry, contaminated water, and fresh produce are considered to be the main sources for infection by this pathogen. In this study, occurrence and population density of C. jejuni from vegetable irrigation ponds in the Suwannee River watershed were investigated and the relationship to environmental factors was analyzed. Two water samples were collected from each of 10 ponds every month from January 2011 to February 2012. Campylobacter jejuni was detected by quantitative real-time PCR. Nine of the 10 ponds were positive for C. jejuni some of the time with an overall prevalence of 19.3%. The highest counts were obtained in spring 2011. Oxidation-reduction potential and total nitrogen concentration were positively correlated (P < 0.05) with mean population and occurrence of C. jejuni, while temperature and dissolved oxygen percent saturation (DO%) were negatively correlated with mean population (P < 0.05). Presence of this pathogen was related to bacterial community composition. No correlations were found between C. jejuni and fecal indicators. Increasing DO% of irrigation water and limiting nitrogen pollution in the ponds are suggested to reduce the contamination risk of C. jejuni in a major fruit and vegetable growing area.

Factors affecting the occurrence of Escherichia coli O157 contamination in irrigation ponds on produce farms in the Suwannee River Watershed.

Outbreaks of enteritis caused by Escherichia coli O157 associated with fresh produce have resulted in questions about the safety of irrigation water; however, associated risks have not been systematically evaluated. In this study, the occurrence and distribution of the human pathogen E. coli O157 from vegetable irrigation ponds within the Suwannee River Watershed in Georgia were investigated, and the relationship to environmental factors was analyzed. Surface and subsurface water samples were collected monthly from 10 vegetable irrigation ponds from March 2011 to February 2012. Escherichia coli O157 was isolated from enriched filtrates on CHROMagar and sorbitol MacConkey agar media and confirmed by an agglutination test. Presence of virulence genes stx1, stx2 , and eae was tested by polymerase chain reaction. In addition, 27 environmental variables of the sampled ponds were measured. Denaturing gradient gel electrophoresis was conducted for the analysis of bacterial communities in the water samples. Biserial correlation coefficients were calculated to evaluate the log10 colony-forming unit per millilitre correlations between the environmental factors and the occurrence of E. coli O157. Stepwise and canonical discriminant analyses were used to determine the factors that were associated with the presence and absence of E. coli O157 in water samples. All 10 ponds were positive for E. coli O157 some of the time, mainly in summer and fall of 2011. The temporal distribution of this bacterium differed among the 10 ponds. Temperature, rainfall, populations of fecal coliform, and culturable bacteria were positively correlated with the occurrence of E. coli O157 (P < 0.05), while the total nitrogen concentration, oxidation-reduction potential, and dissolved oxygen concentration were negatively correlated with the occurrence of this pathogen (P < 0.05). Temperature and rainfall were the most important factors contributing to the discrimination between samples with and without E. coli O157, followed by bacterial diversity and culturable bacteria population density. Bacterial numbers and diversity, including fecal coliforms and E. coli O157, increased after rainfall (and possibly runoff from pond margins) in periods with relatively high temperatures, suggesting that prevention of runoff may be important to minimize the risk of enteric pathogens in irrigation ponds.

Organically managed soils reduce internal colonization of tomato plants by Salmonella enterica serovar Typhimurium.

A two-phase experiment was conducted twice to investigate the effects of soil management on movement of Salmonella enterica Typhimurium in tomato plants. In the first phase, individual leaflets of 84 tomato plants grown in conventional or organic soils were dip inoculated two to four times before fruiting with either of two Salmonella Typhimurium strains (10(9) CFU/ml; 0.025% [vol/vol] Silwet L-77). Inoculated and adjacent leaflets were tested for Salmonella spp. densities for 30 days after each inoculation. Endophytic bacterial communities were characterized by polymerase chain reaction denaturing gradient gel electrophoresis before and after inoculation. Fruit and seed were examined for Salmonella spp. incidence. In phase 2, extracted seed were planted in conventional soil, and contamination of leaves and fruit of the second generation was checked. More Salmonella spp. survived in inoculated leaves on plants grown in conventional than in organic soil. The soil management effect on Salmonella spp. survival was confirmed for tomato plants grown in two additional pairs of soils. Endophytic bacterial diversities of tomato plants grown in conventional soils were significantly lower than those in organic soils. All contaminated fruit (1%) were from tomato plants grown in conventional soil. Approximately 5% of the seed from infested fruit were internally contaminated. No Salmonella sp. was detected in plants grown from contaminated seed.

Ingress of Salmonella enterica Typhimurium into tomato leaves through hydathodes.

Internal contamination of Salmonella in plants is attracting increasing attention for food safety reasons. In this study, three different tomato cultivars "Florida Lanai", "Crown Jewel", "Ailsa Craig" and the transgenic line Sp5 of "Ailsa Craig" were inoculated with 1 µl GFP-labeled Salmonella Typhimurium through guttation droplets at concentrations of 10(9) or 10(7) CFU/ml. Survival of Salmonella on/in tomato leaves was detected by both direct plating and enrichment methods. Salmonella cells survived best on/in the inoculated leaves of cultivar "Ailsa Craig" and decreased fastest on/in "Florida Lanai" leaves. Increased guttation in the abscisic acid over-expressing Sp5 plants may have facilitated the entrance of Salmonella into leaves and the colonization on the surface of tomato leaves. Internalization of Salmonella Typhimurium in tomato leaves through guttation drop inoculation was confirmed by confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can enter tomato leaves through hydathodes and move into the vascular system, which may result in the internal translocation of the bacteria inside plants.

Adhesion and splash dispersal of Salmonella enterica Typhimurium on tomato leaflets: effects of rdar morphotype and trichome density.

Salmonella enterica strains with rdar (red dry and rough) and saw (smooth and white) morphotypes have previously been associated with tomato outbreaks but the dispersal mechanisms of these morphotypes are still poorly understood. In this study, Salmonella adhesion was distinguished from attachment by comparing different contact periods. Initial adhesion of rdar and saw morphotypes of Salmonella was compared in relation to tomato plants with different leaf trichome densities. Trichome densities were increased or reduced by treatment with jasmonic or salicylic acid, respectively. The overall effect of Salmonella morphotype and trichome density on splash dispersal was assessed in a rain simulator and correlated to cell hydrophobicity and initial adhesion. The presence of the rdar morphotype increased initial adhesion at high trichome densities but not at low trichome densities. Attachment of the rdar strain occurred after 30s contact time regardless of trichome density. Splash dispersal was slightly further for the saw morphotype than the rdar morphotype of S. enterica at all trichome densities. Salmonella cells of both morphotypes survived significantly better on the surface of high trichome density leaflets.

Growth of Clostridium perfringens during cooling of refried beans.

Outbreaks of Clostridium perfringens have been associated with dishes containing refried beans from food service establishments. However, growth of C. perfringens in refried beans has not been investigated, and predictive models have not been validated in this food matrix. We investigated the growth of C. perfringens during the cooling of refried beans. Refried beans (pinto and black, with and without salt added) were inoculated with 3 log CFU/g C. perfringens spores and incubated isothermally at 12, 23, 30, 35, 40, 45, and 50°C. The levels of C. perfringens were monitored 3, 5, 8, and 10 h after inoculation, and then fitted to the Baranyi primary model and the Rosso secondary model prior to solving the Baranyi differential equation. The final model was validated by dynamic cooling experiments carried out in stockpots, thus mimicking the worst possible food service conditions. All refried beans samples supported the growth of C. perfringens, and all models fit the data with pseudo-R(2) values of 0.95 or greater and mean square errors of 0.3 or lower. The estimated maximum specific growth rates were generally higher in pinto beans, with or without salt added (2.64 and 1.95 h(-1), respectively), when compared with black beans, with or without salt added (1.78 and 1.61 h(-1), respectively). After 10 h of incubation, maximum populations of C. perfringens were significantly higher in samples with no salt added (7.9 log CFU/g for both pinto and black beans) than in samples with salt added (7.3 and 7.2 log CFU/g for pinto and black beans, respectively). The dynamic model predicted the growth of C. perfringens during cooling, with an average root mean squared error of 0.44. The use of large stockpots to cool refried beans led to an observed 1.2-log increase (1.5-log increase predicted by model) in levels of C. perfringens during cooling. The use of shallower pans for cooling is recommended, because they cool faster, therefore limiting the growth of C. perfringens.

Salmonella can reach tomato fruits on plants exposed to aerosols formed by rain.

Outbreaks of Salmonella enterica have been associated with tomatoes and traced back to production areas but the spread of Salmonella in agricultural fields is still poorly understood. Post-rain Salmonella transfer from a point source to the air and then to tomato plants was evaluated. GFP-labeled kanamycin-resistant S. enterica serovar Typhimurium (10(8)CFU/mL) with and without expression of the rdar morphotype (rough colonies; cells with fimbriae and cellulose) was used as the point source in the center of a rain simulator. Rain intensities of 60 and 110 mm/h were applied for 5, 10, 20, and 30 min. Petri dishes with lactose broth and tomato plants with fruit (50-80 cm high) were placed in the simulator after the rain had ceased. Salmonella recovery from air was maximum (300 CFU/plate) after a rain episode of 60 mm/h for 10 min at distances of at least 85.5 cm above the source and when the rdar morphotype strain was used. Small scale experiments showed that the smooth-colony strain without fimbriae precipitated from the air in significantly higher numbers than the rdar strain. Transfer of aerial Salmonella with the rdar morphotype to tomato fruits on plants followed a beta distribution (2.5950, 4.7393) within the generalized range from 0 to 30 min of rain. Results show for the first time that Salmonella may transfer from rain to the air and contaminate tomato fruits at levels that could possibly be infectious to humans.

Dispersal of Salmonella Typhimurium by rain splash onto tomato plants.

Outbreaks of Salmonella enterica have increasingly been associated with tomatoes and traced back to production areas, but the spread of Salmonella from a point source onto plants has not been described. Splash dispersal by rain could be one means of dissemination. Green fluorescent protein-labeled, kanamycin-resistant Salmonella enterica sv. Typhimurium dispensed on the surface of plastic mulch, organic mulch, or soil at 108 CFU/cm² was used as the point source in the center of a rain simulator. Tomato plants in soil with and without plastic or organic mulch were placed around the point source, and rain intensities of 60 and 110 mm/h were applied for 5, 10, 20, and 30 min. Dispersal of Salmonella followed a negative exponential model with a half distance of 3 cm at 110 mm/h. Dispersed Salmonella survived for 3 days on tomato leaflets, with a total decline of 5 log and an initial decimal reduction time of 10 h. Recovery of dispersed Salmonella from plants at the maximum observed distance ranged from 3 CFU/g of leaflet after a rain episode of 110 mm/h for 10 min on soil to 117 CFU/g of leaflet on plastic mulch. Dispersal of Salmonella on plants with and without mulch was significantly enhanced by increasing rain duration from 0 to 10 min, but dispersal was reduced when rainfall duration increased from 10 to 30 min. Salmonella may be dispersed by rain to contaminate tomato plants in the field, especially during rain events of 10 min and when plastic mulch is used.

GC-MS metabolomic differentiation of selected citrus varieties with different sensitivity to citrus huanglongbing.

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The rapid identification of tolerant varieties is considered a critical step towards controlling HLB. GC-MS metabolite profiles were used to differentiate HLB-tolerant citrus varieties 'Poncirus trifoliata' (TR) and 'Carrizo citrange' (CAR) from HLB-sensitive varieties 'Madam Vinous sweet orange' (MV) and 'Duncan' grapefruit (DG). PCR analyses revealed that MV was the most sensitive variety followed by DG and the tolerant varieties CAR and TR. Metabolomic multivariate analysis allowed classification of the cultivars in apparent agreement with PCR results. Higher levels of the amino acids l-proline, l-serine, and l-aspartic acid, as well as the organic acids butanedioic and tetradecanoic acid, and accumulation of galactose in healthy plants were characteristic of the most sensitive variety MV when compared to all other varieties. Only galactose was significantly higher in DG when compared to the tolerant varieties TR and CAR. The tolerant varieties showed higher levels of l-glycine and mannose when compared to sensitive varieties MV and DG. Profiling of the sensitive varieties MV and DG over a 20-week period after inoculation of those with the HLB-containing material revealed strong responses of metabolites to HLB infection that differed from the response of the tolerant varieties. Significant changes of l-threonine level in the leaves from old mature flushes and l-serine, l-threonine, scyllo-inositol, hexadecanoic acid, and mannose in the leaves from young developing flushes were observed in MV. Significant changes in myo-inositol in old flushes and l-proline, indole, and xylose in new flushes were observed in DG.

Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants.

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 10(9) CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 10(4) CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 10(6) CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.