Antifungal Mechanism of Ruta graveolens Essential Oil: A Colombian Traditional Alternative against Anthracnose Caused by Colletotrichum gloeosporioides.

Here, we report for the first time on the mechanisms of action of the essential oil of Ruta graveolens (REO) against the plant pathogen Colletotrichum gloeosporioides. In particular, the presence of REO drastically affected the morphology of hyphae by inducing changes in the cytoplasmic membrane, such as depolarization and changes in the fatty acid profile where straight-chain fatty acids (SCFAs) increased by up to 92.1%. In addition, REO induced changes in fungal metabolism and triggered apoptosis-like responses to cell death, such as DNA fragmentation and the accumulation of reactive oxygen species (ROS). The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, ß-galactosidase, ß-glucosidase, and N-acetyl-ß-glucosaminidase, was significantly reduced in the presence of REO. In addition, C. gloeosporioides activated naphthol-As-BI phosphohydrolase as a mechanism of response to REO stress. The data obtained here have shown that the essential oil of Ruta graveolens has a strong antifungal effect on C. gloeosporioides. Therefore, it has the potential to be used as a surface disinfectant and as a viable replacement for fungicides commonly used to treat anthracnose in the postharvest testing phase.

Green Management of Postharvest Anthracnose Caused by Colletotrichum gloeosporioides.

Fruits and vegetables are constantly affected by postharvest diseases, of which anthracnose is one of the most severe and is caused by diverse Colletotrichum species, mainly C. gloeosporioides. In the last few decades, chemical fungicides have been the primary approach to anthracnose control. However, recent trends and regulations have sought to limit the use of these substances. Greener management includes a group of sustainable alternatives that use natural substances and microorganisms to control postharvest fungi. This comprehensive review of contemporary research presents various sustainable alternatives to C. gloeosporioides postharvest control in vitro and in situ, ranging from the use of biopolymers, essential oils, and antagonistic microorganisms to cultivar resistance. Strategies such as encapsulation, biofilms, coatings, compounds secreted, antibiotics, and lytic enzyme production by microorganisms are revised. Finally, the potential effects of climate change on C. gloeosporioides and anthracnose disease are explored. Greener management can provide a possible replacement for the conventional approach of using chemical fungicides for anthracnose postharvest control. It presents diverse methodologies that are not mutually exclusive and can be in tune with the needs and interests of new consumers and the environment. Overall, developing or using these alternatives has strong potential for improving sustainability and addressing the challenges generated by climate change.

Yeasts associated with the worker caste of the leaf-cutting ant Atta cephalotes under experimental conditions in Colombia.

Yeasts isolated from the worker caste of the Colombian leaf-cutting ant, Atta cephalotes (Hymenoptera: Myrmicinae) were cultured and identified by molecular methods. Abundant, persistent, and omnipresent species were classified as "prevalent". Experimental data were compared with information gathered from published reports on the yeast species composition in other leaf-cutting ant species. Diversity analysis was conducted using diversity values (q0, q1, and q2) to compare the richness and abundance of yeasts present in different leaf-cutting ant species. Clustering analysis was carried out to assess the similarity of yeast community according to ant species. The yeast species composition was highly variable among the ant species. A. laevigata and A. capiguara showed the highest degree of similarity and differed from the group composed by A. cephalotes, A. sexdens, A. sexdens rubropilosa, and A. texana. The isolation of dominant yeasts in different ant castes within the different compartments of a colony strongly suggests that the identified microorganisms are not transient but are native to the soil surrounding ant colonies and the substrates used by the ants to grow their fungal cultivars. It is apparent that the ant-fungus mutualism does not operate in an environment devoid of other microbes, but rather that the association must be seen within the context of a background of other microorganisms, particularly the dominant yeasts.

Colombian Essential Oil of Ruta graveolens against Nosocomial Antifungal Resistant Candida Strains.

Drug resistance in antifungal therapy, a problem unknown until a few years ago, is increasingly assuming importance especially in immunosuppressed patients and patients receiving chemotherapy and radiotherapy. In the past years, the use of essential oils as an approach to improve the effectiveness of antifungal agents and to reduce antifungal resistance levels has been proposed. Our research aimed to evaluate the antifungal activity of Colombian rue, Ruta graveolens, essential oil (REO) against clinical strains of Candida albicans, Candida parapsilopsis, Candida glabrata, and Candida tropicalis. Data obtained showed that C. tropicalis and C. albicans were the most sensitive strains showing minimum inhibitory concentrations (MIC) of 4.1 and 8.2 µg/mL of REO. Time-kill kinetics assay demonstrated that REO showed a fungicidal effect against C. tropicalis and a fungistatic effect against C. albicans. In addition, an amount of 40% of the biofilm formed by C. albicans was eradicated using 8.2 µg/mL of REO after 1 h of exposure. The synergistic effect of REO together with some antifungal compounds was also investigated. Fractional inhibitory concentration index (FICI) showed synergic effects of REO combined with amphotericin B. REO Lead a disruption in the cellular membrane integrity, consequently resulting in increased intracellular leakage of the macromolecules, thus confirming that the plasma membrane is a target of the mode of action of REO against C. albicans and C. tropicalis.

Exploring the Capability of Yeasts Isolated from Colombian Fermented Cocoa Beans to Form and Degrade Biogenic Amines in a Lab-Scale Model System for Cocoa Fermentation.

Yeast starters for cocoa fermentation are usually tested according to their enzymatic activities in terms of mucilage degradation and flavor improvement, disregarding their influence on the production or elimination of toxic compounds as biogenic amines (BAs), important for human health. In this work, we tested 145 strains belonging to 12 different yeast species and isolated from the Colombian fermented cocoa beans (CB) for their capability of producing BAs in vitro. Sixty-five strains were able to decarboxylate at least one of the amino acids tested. Pichia kudriavzevii ECA33 (Pk) and Saccharomyces cerevisiae 4 (Sc) were selected to evaluate their potential to modulate BAs, organic acids, and volatile organic compounds (VOCs) accumulation during a simulated cocoa fermentation. The growth of Sc or Pk in the presence of CB caused a significant reduction (p < 0.05) of 2-phenylethylamine (84% and 37%) and cadaverine (58% and 51%), and a significant increase of tryptamine and putrescine with a strong influence of temperature in BA formation and degradation. In addition, our findings pointed out that Pk induced a major production of fatty acid- and amino acid-derived VOCs, while Sc induced more VOCs derived from fatty acids metabolism. Our results suggest the importance of considering BA production in the choice of yeast starters for cocoa fermentation.

Functional Biodiversity of Yeasts Isolated from Colombian Fermented and Dry Cocoa Beans.

Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.

Effect of Fermentation, Drying and Roasting on Biogenic Amines and Other Biocompounds in Colombian Criollo Cocoa Beans and Shells.

The composition of microbiota and the content and pattern of bioactive compounds (biogenic amines, polyphenols, anthocyanins and flavanols), as well as pH, color, antioxidant and reducing properties were investigated in fermented Criollo cocoa beans and shells. The analyses were conducted after fermentation and drying (T1) and after two thermal roasting processes (T2, 120 °C for 22 min; T3, 135 °C for 15 min). The fermentation and drying practices affected the microbiota of beans and shells, explaining the great variability of biogenic amines (BAs) content. Enterobacteriaceae were counted in a few samples with average values of 103 colony forming units per gram (CFU g-1), mainly in the shell, while Lactobacillus spp. was observed in almost all the samples, with the highest count in the shell with average values of 104 CFU g-1. After T1, the total BAs content was found to be in a range of 4.9÷127.1 mg kg-1DFW; what was remarkable was the presence of cadaverine and histamine, which have not been reported previously in fermented cocoa beans. The total BAs content increased 60% after thermal treatment T2, and of 21% after processing at T3, with a strong correlation (p < 0.05) for histamine (ß = 0.75) and weakly correlated for spermidine (ß = 0.58), spermine (ß = 0.50), cadaverine (ß = 0.47) and serotonine (ß = 0.40). The roasting treatment of T3 caused serotonin degradation (average decrease of 93%) with respect to unroasted samples. However, BAs were detected in a non-alarming concentration (e.g., histamine: n.d ÷ 59.8 mg kg-1DFW; tyramine: n.d. ÷ 26.5 mg kg-1DFW). Change in BAs level was evaluated by principal component analysis. PC1 and PC2 explained 84.9% and 4.5% of data variance, respectively. Antioxidant and reducing properties, polyphenol content and BAs negatively influenced PC1 with both polyphenols and BA increasing during roasting, whereas PC1 was positively influenced by anthocyanins, catechin and epicatechin.

Bioactive micro-constituents of ackee arilli (Blighia sapida K.D. Koenig).

Ackee (Blighia sapida K. D. Koenig) is an exotic fruit widely consumed in the Caribbean countries. While there is extensive research on the presence of hypoglycin A, other bioactive compounds have not been studied. We identified and quantified the changes in bioactive molecules (total phenol, ascorbic acid, hypoglycin A, squalene, D: A-Friedooleanan-7-ol, (7.alpha.), and oleic acid), antioxidant potential, and volatile compounds during two stages of ripe. A clear reduction in hypoglycin A, ascorbic acid, and total polyphenols during the maturation process were observed. On the contrary, oleic acid, squalene, and D: A-Friedooleanan-7-ol, (7.alpha.) contents increased about 12, 12, and 13 times, respectively with advancing maturity. These bioactive molecules were positively correlated with radical scavenging (DDPH and ABTS). Solid phase microextraction (SPME) and gas chromatography coupled mass spectrometry (GC/MS) analysis revealed more than 50 compounds with 3-penten-2-one and hexanal as the major compounds in the fully ripe stage. The results suggested that ripe ackee arilli could serve as an appreciable source of natural bioactive micro-constituents.

Colletotrichum Gloesporioides Inhibition In Situ by Chitosan-Ruta graveolens Essential Oil Coatings: Effect on Microbiological, Physicochemical, and Organoleptic Properties of Guava (Psidium guajava L.) during Room Temperature Storage.

Guava is a fruit appreciated worldwide for its high content of bioactive compounds. However, it is considered a highly perishable fruit, generally attacked by pathogenic species such as the fungi Colletotrichum gloeosporioides, which causes anthracnosis. To diminish the losses caused by pathogenic fungi, coatings of chitosan (CS) with Ruta graveolens essential oil (RGEO) in different concentrations (0.5, 1.0, 1.5% v/v) were applied in situ and their effects on the physical properties and microbiological quality of the guavas were studied. The CS+RGEO coated fruits exhibited better physicochemical behavior and lower microbiological decay as compared to the uncoated guavas, demonstrating the effectiveness of the coatings, especially those with 1.5% of RGEO content. All the fruits coated had greater acceptance and quality than the controls, being more those with essential oil incorporation. In situ investigation of C. gloesporioides infection of guavas demonstrated that the CS+RGEO coated guavas showed a high percentage of inhibition in the development of anthracnose lesions. In the present investigation, an alternative method has been proposed to extend the stability of the guavas fruit up to 12 days with application in the food industry.

Chitosan films incorporated with Thymus capitatus essential oil: mechanical properties and antimicrobial activity against degradative bacterial species isolated from tuna (Thunnus sp.) and swordfish (Xiphias gladius).

Chitosan-based coatings and films have been widely studied, demonstrating to be an efficient and eco-friendly approach to extend the shelf life of food products. The effect of incorporating Thymus capitatus essential oil (TCEO) at different concentrations (0.5, 1.0, and 1.5% w/w) on physical, mechanical and antimicrobial properties of chitosan films was studied. The antimicrobial activity of the films was evaluated by agar diffusion method, against 23 spoiling microorganisms isolated from tuna and swordfish (ten Shewanella baltica, one S. morhuae, one S. putrefaciens, two Pseudomonas fluorescens, two P. fragi, five Serratia spp., one Aeromonas molluscorum, and one Acinetobacter radioresistens) and Shewanella putrefaciens ATCC 49138. The films exerted antimicrobial activity against all the tested strain, although not proportional to increasing TCEO concentration. In particular, S. baltica was the most sensitive species and the inhibition was stable after 72 h. In general, TCEO incorporation in chitosan films, significantly (p < 0.05) decreased the water permeability (from 0.577 ± 0.060 gmm/kPahm2 at 61% R.U. for chitosan to 0.487 ± 0.037 gmm/kPahm2 for the film with 1.5% TCEO), the elongation at brake (from 27.322 ± 2.35% for chitosan to 14.695 ± 3.99% for the film with 1.5% TCEO) and increased the tensile strength (from 1.697 ± 0.16% for chitosan to 19.480 ± 2.86% for the film with 1.5% TCEO). Moisture content and water contact angle of the films also showed a similar trend with TCEO introduction, because of crosslinking reaction among the polymer chains and TCEO components. Scanning electron microscopy confirmed structure-properties relationships. These results suggest chitosan films incorporated with TCEO as an alternative treatment to inhibit the growth of degradative bacteria with potential application in the fish industry. The importance of testing more than one strain of the same bacteria species to evaluate the effectiveness of chitosan-essential oils coatings was also demonstrated.

Exploring the Bacterial Microbiota of Colombian Fermented Maize Dough "Masa Agria" (Maiz Añejo).

Masa Agria is a naturally fermented maize dough produced in Colombia, very common in the traditional gastronomy. In this study we used culture-dependent and RNA-based pyrosequencing to investigate the bacterial community structure of Masa Agria samples produced in the south west of Colombia. The mean value of cell density was 7.6 log CFU/g of presumptive lactic acid bacteria, 5.4 log cfu/g for presumptive acetic bacteria and 5.6 og CFU/g for yeasts. The abundance of these microorganisms is also responsible for the low pH (3.1-3.7) registered. Although the 16S rRNA pyrosequencing revealed that the analyzed samples were different in bacteria richness and diversity, the genera Lactobacillus, Weissella, and Acetobacter were predominant. In particular, the most common species were Lactobacillus plantarum and Acetobacter fabarum, followed by L. fermentum, L. vaccinostercus, and Pediococcus argentinicus. Several microorganisms of environmental origin, such as Dechloromonas and most of all Sphingobium spp., revealed in each sample, were detected, and also bacteria related to maize, such as Phytoplasma. In conclusion, our results elucidated for the first time the structures of the bacterial communities of Masa Agria samples obtained from different producers, identifying the specific dominant species and revealing a complete picture of the bacterial consortium in this specific niche. The selective pressure of tropical environments may favor microbial biodiversity characterized by a useful technological potential.

Yeasts from Colombian Kumis as source of peptides with Angiotensin I converting enzyme (ACE) inhibitory activity in milk.

This study investigated the possibility of using yeast strains in fermented milks to obtain products with high Angiotensin I-converting enzyme (ACE) inhibitory activity and low bitter taste. Ninety-three yeast strains isolated from Colombian Kumis in different geographic regions were molecularly identified, and their milk fermentation performances were determined. Molecular identification evidenced that Galactomyces geotrichum, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis, were the dominant species. Eighteen out of 93 strains produced fermented milk with ACE-inhibitory (ACEI) activity values ranging from 8.69 to 88.19%. Digestion of fermented milk samples by pepsin and pancreatin demonstrated an increase in ACEI activity, with C. lusitaniae KL4A as the best producer of ACEI peptides. Moreover, sensory analysis of the products containing the major ACE-inhibitory activity pointed out that P. kudriavzevii KL84A and Kluyveromyces marxianus KL26A could be selected as potential adjunct starter cultures in Kumis, since they made a considerable contribution to the ACE inhibitory activity and produced fermented milk without bitter taste. In this study we observed that Colombian Kumis can be an excellent vehicle for the isolation of yeasts with a potential to enhance bioactive peptides produced during milk fermentation.

Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population.

Kumis is a traditional fermented cow milk produced and consumed in South West Colombia. The main objective of this research was to studied the enterococcal population, present in 13 kumis samples traditionally manufactured, for their role as beneficial organisms or opportunistic pathogens. The molecular identification of 72 isolates evidenced that Enterococcus faecalis and E. faecium were the dominant species. The genes gelE, esp, asa1, cyl and hyl, all associated with virulence factors in enterococci, were detected in 30 isolates, while 42 were free of virulence determinants. Skim milk media were fermented by all the different isolates and further tested for proteolysis (free NH(3) groups), Angiotensin-I Converting Enzyme (ACE) inhibitory activity and biogenic amines production. Nine E. faecalis and two E. faecium strains produced fermented milk with ACE-inhibitory activity values ranging from 39.7% to 84.35% .The digestion of fermented milk samples by pepsin and pancreatin evidenced an increase in ACE inhibitory activity, with E. faecalis KE09 as the best producer (IC50 = 14.25 µg ml(-1)). Moreover, the strains showed a very low tyrosine decarboxylase activity and did not produce histamine during 48 h fermentation in milk. This study underlines the that Colombian kumis is a good source of not virulent enterococci able to produce fermented milks with ACE-inhibitory activity.

Detection and identification of wild yeasts in Champús, a fermented Colombian maize beverage.

The aim of this study was to identify and characterise the predominant yeasts in Champús, a traditional Colombian cereal-based beverage with a low alcoholic content. Samples of Champús from 20 production sites in the Cauca Valley region were analysed. A total of 235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var. kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hanseniaspora spp. Model Champús systems were inoculated with single strains of some isolated sporogenus species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champús strains produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-Saccharomyces yeasts from Champús can exert a relevant influence on the sensory characteristics of the fermented beverage. The Champús strains could thus represent an important source for new yeast biotypes with potential industrial applications.