Water activity relations of spoilage fungi associated with smoke-dried catfish (Clarias gariepinus) sold in some open markets in Nigeria.

Spoilage fungi were isolated from the skin and the muscle of smoke-dried catfish samples, which were products of traditional and improved processing methods. The samples significantly different mean aw of 0.85 and 0.81 respectively [F (1, 3) = 0.014, P = 0.018], when they were checked immediately after purchase from selected open markets. The isolated spoilage fungi were identified by their phenotypic appearance and morphological features under microscope, with reference to standard identification guidelines. The isolates comprised of Aspergillus fumigatus, A. niger, A. flavus and Penicillium species. Effects of water activity (aw) on growth and sporulation of these species at ambient temperature (25 ± 5 °C) were studied on standard media (aw = 0.995) or media in which aw was modified using NaCl as follows: 0.98, 0.94, 0.86 and 0.80. All the isolates could grow in the range of the aw studied and there were statistically significant variabilities in the rates of growth among species [F (7, 50) = 63.34, P = 0.001] and in relation to media aw [F (28, 50) = 4.055, P = 0.001]. There was no limitant aw found in the studied conditions (0.995-0.80 aw x fluctuating ambient temperature, 25 ± 5 °C), as all the isolates were fast growing. The aw of the fish samples from the improved processing line was lower than those from the traditional processing lines. However, the aw of all the tested the samples was above the Codex Standard for smoked fish, smoke-flavoured fish and smoke-dried fish, which is 0.75 aw or less (10% moisture or less), as necessary to control bacteria pathogens and spoilage fungi. The results indicated that the open market fish samples may pose serious health risks if they are consumed after a short-term storage. Research Highlights Tolerance of smoke-dried fish spoilage fungi to water stress Effect of aw on growth characteristics of smoke-dried fish spoilage fungi Water activity relations of some spoilage fungi from dried fish.

Antifungal Activity of Volatile Organic Compounds Produced by Bacillus methylotrophicus and Bacillus thuringiensis against Five Common Spoilage Fungi on Loquats.

Loquat fruit is one of the most perishable fruits in China, and has a very limited shelf life because of mechanical injury and microbial decay. Due to an increasing concern about human health and environmental security, antagonistic microorganisms have been a potential alternative for fungicides to control postharvest diseases. In this work, the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus methylotrophicus BCN2 and Bacillus thuringiensis BCN10 against five postharvest pathogens isolated from loquat fruit, Fusarium oxysporum, Botryosphaeria sp., Trichoderma atroviride, Colletotrichum gloeosporioides, and Penicillium expansum were evaluated by in vitro and in vivo experiments. As a result, the VOCs released by BCN2 and BCN10 were able to suppress the mycelial growth of all targeted pathogens according to inhibition ratio in the double petri-dish assay as well as disease incidence and disease diameter on loquat fruits. The main volatile compounds were identified by solid-phase microextraction (SPME)-gas chromatography. These VOCs produced by the two strains played complementary roles in controlling these five molds and enabled loquat fruits to keep fresh for ten days, significantly. This research will provide a theoretic foundation and technical support for exploring the functional components of VOCs applicable in loquat fruit preservation.

Identification of endophytic bacteria in medicinal plants and their antifungal activities against food spoilage fungi.

Endophytes are fungi or bacteria living in the intracellular parts of the plants. In this study, 12 medicinal plants were examined for the presence of endophytic bacteria. Antifungal activities of the isolates were determined against Aspergillus flavus PTCC 5006, Penicillium citrinum PTCC 5304, Aspergillus fumigatus PTCC 5006, Fusarium oxysporum MTCC 284, and Rhizopus stolonifer. In order to determine the metabolites characteristics of these isolates, catalase and proteolytic enzyme treatments were assessed. Moreover, approximate molecular weights of the antifungal substance were measured by fractionation method and the volatile compounds were determined by using GC-mass spectroscopy. Finally, 16s rRNA gene sequencing confirmed the strain of the bacteria. Twenty-one endophytic bacteria, out of a total of 82 isolates, showed antifungal activities against all five spoilage fungi. The results of the PCR assay revealed two species: Bacillus pumilus and B. safensis. Proteolytic enzyme activities and the fractionation of the supernatants proved that more than one compound was responsible for the antifungal activities. This compound could be proteins, peptides, and other low-molecular compounds, such as Butanal, 3-methyl-, Propene, 2-butene, 2-heptanone, 6-methyl-5-methylene-, and 6-oxabicyclo[3.1.0] hexane, which all were identified in the headspace of the GC-mass spectroscopy.

In vitro antifungal, probiotic, and antioxidant functional properties of a novel Lactobacillus paraplantarum isolated from fermented dates in Saudi Arabia.

BACKGROUND: Fermented foods produced using dates are used in Gulf countries as beneficial and healthful foods. The beneficial microbial flora in fermented dates contributes to maintaining the nutritional properties of dates by preventing the growth of spoilage fungi. Here, we examined the antifungal, probiotic, and antioxidant properties of the novel Lactobacillus strain D-3 isolated from fermented dates. RESULTS: Analyzing the morphological, physiological, and biochemical characteristics of this strain demonstrated that it was similar to Lactobacillus species, and molecular-level amplification of the 16S rRNA gene showed that it belonged to Lactobacillus paraplantarum. Under shake flask cultivation using date juice, the strain produced significant amounts of ethanol and lactic, succinic, and acetic acids. Purification of benzoic acid extracted from the extracellular fermentation medium was confirmed by nuclear magnetic resonance, and infrared and mass spectral data revealed minimum inhibitory concentration values of 10, 20, 10, 5, and 10 mg mL-1 for Aspergillus fumigates, Curvularia lunata, Fusarium oxysporum, Gibberella moniliformis, and Penicillium chrysogenum, respectively. The strain showed several advantages, including the ability to survive under conditions similar to the gastrointestinal tract (low pH, bile salts, and antimicrobial susceptibility) and high levels of extracellular enzyme activities. The strain's growth patterns under various concentrations of H2 O2 and its scavenging properties towards hydroxyl radical (64.85%) and DPPH (84.97%) were also interesting properties. CONCLUSION: The antifungal, probiotic, and antioxidant properties of L. paraplantarum D3 may provide health benefits to consumers. © 2017 Society of Chemical Industry.

Synergistic antifungal effects of the preservative ammonium propionate and medium chain fatty acids against dormant and germinating conidia, germ tubes and hyphae of Aspergillus chevalieri, a feed spoilage fungus.

In feed, propionic acid is the weak organic acid of choice to prevent growth of spoilage fungi. For safe and easy industrial handling this antifungal agent is applied in the presence of neutralizing ammonium, which however has the disadvantage to negatively affect the efficacy of fungus-inhibiting properties of the formulation. In the present study we investigated the impact of medium chain fatty acids (MCFA) on the antifungal efficacy of an ammonium propionate formulation on dormant- and germinating conidia as well as germ tubes and hyphae of Aspergillus chevalieri, a xerophilic fungus predominant on moulded feed. Dormant conidia were not affected by 32 mM of ammonium propionate after a 28 h-treatment in demi water. Similar results were obtained with solely 0.52 mM MCFA. However, the combination of both components nearly eradicated formation of colonies from these conidia and was accompanied by distortion of the cellular structure as was visible with light- and transmission electron microscopy. Germination of conidia, characterised by swelling and germ tube formation, was significantly decreased in the presence of 16 mM ammonium propionate and 0.26 mM MCFA, while the latter component itself did not significantly decrease germination. We conclude that a combination of ammonium propionate and MCFA had a synergistic antifungal effect on dormant and germinating conidia. When the combination of ammonium propionate and MCFA was tested on hyphae for 30 min, we observed that cell death was significantly increased in comparison to components alone. Treatment of the hyphae with 16 mM of ammonium propionate caused aberrant mitochondria, as evidenced by irregularly shaped and enlarged mitochondria that contained electron-dense inclusions as observed by transmission electron microscopy. When the combination of ammonium propionate and MCFA was applied against the hyphae, more severe cell damage was observed, with signs of autophagy. Summarised, our results demonstrate synergistic antifungal effects of ammonium propionate and medium chain fatty acids on fungal survival structures, during their germination and after a short (sudden) treatment of growing cells. This is of potential importance for several areas of feed and food storage and shelf-life.

In situ control of food spoilage fungus using Lactobacillus acidophilus NCDC 291.

A challenge for food industry today is to produce minimally processed food, without use of chemical preservatives and little compromise on nutritional status. Lactobacillus acidophilus NCDC 291 can be directly added to food where it enhances shelf life by competing with other microflora (both bacterial and fungal) for food and also by production of antimicrobial metabolites as bacteriocins. Comprehensive studies have demonstrated the in vitro activity of bacteriocins. However their role in preventing fresh food spoilage needs more elucidation. The present study was conducted to evaluate the efficacy of the whole cells of this organism as biopreservative agent against fungi. Four most commonly occurring spoilage fungi were isolated and were identified as Fusarium, Alternaria, Penicillium and Aspergillus. Growth of all of them was inhibited in in vitro studies, (approximately 33-43% decrease in mycelial dry weight basis between test and control). In situ biopreservation of Indian cheese and raw poultry meat was attempted and the colony count of Alternaria was significantly (p < 0.05, Bonferroni Holm) reduced in presence of L. acidophilus. Dip and Keep approach of preservation for Mangifera and Momordica were carried out in which microbial spoilage was not observed up to 6 days.

Diversity of Filamentous Fungi Associated with Dairy Processing Environments and Spoiled Products in Brazil.

Few studies have investigated the diversity of spoilage fungi from the dairy production chain in Brazil, despite their importance as spoilage microorganisms. In the present study, 109 filamentous fungi were isolated from various spoiled dairy products and dairy production environments. The isolates were identified through sequencing of the internal transcribed spacer (ITS) region. In spoiled products, Penicillium and Cladosporium were the most frequent genera of filamentous fungi and were also present in the dairy environment, indicating that they may represent a primary source of contamination. For dairy production environments, the most frequent genera were Cladosporium, Penicillium, Aspergillus, and Nigrospora. Four species (Hypoxylon griseobrunneum, Rhinocladiella similis, Coniochaeta rosae, and Paecilomyces maximus) were identified for the first time in dairy products or in dairy production environment. Phytopathogenic genera were also detected, such as Montagnula, Clonostachys, and Riopa. One species isolated from the dairy production environment is classified as the pathogenic fungi, R. similis. Regarding the phylogeny, 14 different families were observed and most of the fungi belong to the Ascomycota phylum. The understanding of fungal biodiversity in dairy products and environment can support the development of conservation strategies to control food spoilage. This includes the suitable use of preservatives in dairy products, as well as the application of specific cleaning and sanitizing protocols designed for a specific group of target microorganisms.

Occurrence of Aflatoxins and Ochratoxin A during Merkén Pepper Powder Production in Chile.

Berry fruits of Capsicum annuum L. cv. "Cacho de Cabra" are used for the manufacture of a traditional pepper powder known as Merkén. In the present study, aflatoxins (AFs) and ochratoxin A (OTA) contamination in berry fruits of C. annuum was determined at harvest, drying, and smoking stages of Merkén production, in cumin and coriander seeds used as Merkén ingredients, and in the final packaged Merkén produced by local farmers. Additionally, Merkén samples from local markets in the region of La Araucanía (Chile) were also evaluated. Chromatographic analysis was based on a qualitative method. AFs and OTA were not detected on pepper pods and seeds. There was no detection of AFs and OTA on cultured Aspergillus and Penicillium strains isolated from pepper pods, cumin and coriander seeds and Merkén. The lack of AFs/OTA-producers among the isolated fungal species can explain and support the absence of contamination in pepper pods. In contrast, the AFB1 was detected in 75% of Merkén obtained from farmers and 46% of Merkén samples purchased from local markets; while OTA was detected in 100% of Merkén samples obtained from farmers and local markets. In the Merkén production chain, the harvest and post-harvest are key stages for fungal growth while the commercialization stage is highly susceptible to AFs and OTA contamination.

Evolving challenges and strategies for fungal control in the food supply chain.

Fungi that spoil foods or infect crops can have major socioeconomic impacts, posing threats to food security. The strategies needed to manage these fungi are evolving, given the growing incidence of fungicide resistance, tightening regulations of chemicals use and market trends imposing new food-preservation challenges. For example, alternative methods for crop protection such as RNA-based fungicides, biocontrol, or stimulation of natural plant defences may lessen concerns like environmental toxicity of chemical fungicides. There is renewed focus on natural product preservatives and fungicides, which can bypass regulations for 'clean label' food products. These require investment to find effective, safe activities within complex mixtures such as plant extracts. Alternatively, physical measures may be one key for fungal control, such as polymer materials which passively resist attachment and colonization by fungi. Reducing or replacing traditional chlorine treatments (e.g. of post-harvest produce) is desirable to limit formation of disinfection by-products. In addition, the current growth in lower sugar food products can alter metabolic routing of carbon utilization in spoilage yeasts, with implications for efficacy of food preservatives acting via metabolism. The use of preservative or fungicide combinations, while involving more than one chemical, can reduce total chemicals usage where these act synergistically. Such approaches might also help target different subpopulations within heteroresistant fungal populations. These approaches are discussed in the context of current challenges for food preservation, focussing on pre-harvest fungal control, fresh produce and stored food preservation. Several strategies show growing potential for mitigating or reversing the risks posed by fungi in the food supply chain.

Comparative Growth Inhibition of Bread Spoilage Fungi by Different Preservative Concentrations Using a Rapid Turbidimetric Assay System.

Bread and intermediate moisture bakery products are mainly spoiled by yeasts and filamentous fungi. The inoculum load and preservation system used determines their shelf life. To extend the shelf life of such commodities, the use of chemical preservatives is the most common way to try and control the initiation of mold spoilage of bread. This study has utilized a rapid turbidimetric assay system (Bioscreen C) to examine the temporal efficacy of calcium propionate (CP) and potassium sorbate (PS) for controlling the growth of important bread spoilage fungi. The objectives were to compare the temporal growth of strains of three important spoilage fungi Hyphopichia burtonii (HB17), Paecilomyces variotii (PV11), and Penicillium roqueforti (PR06) isolated from visibly molded bread to (a) different concentrations of CP and PS (0-128 mM), (b) temperatures (25°C, 30°C), (c) water activity (aw; 0.95, 0.97), and (d) pH (5.0, 5.5). All three abiotic factors, pH, aw, and temperature, and preservative concentrations influenced the relative growth of the species examined. In general, PS was more effective than CP in inhibiting the growth of the strains of these three species. In addition, the Time to Detection (TTD) for the efficacy of the preservatives under the interacting abiotic factors was compared. The strain of Paecilomyces variotii (PV10) was the most tolerant to the preservatives, with the shortest TTD values for both preservatives. P. roqueforti was the most sensitive with the longest TTD values under all conditions examined. These results are discussed in the context of the evolution of resistance to food-grade preservatives by such spoilage fungi in bakery products.

Mycobiota in Chilean chilli Capsicum annuum L. used for production of Merkén.

This work aims to provide the first study on the mycobiota present in Chilean pepper Capsicum annuum L. cv. "Cacho de Cabra" throughout the early production stages. Two hundred and forty berry fruits were sampled: 1) at the ripe fruits harvest day; 2) during drying; and 3) smoking processes. A total of 192 strains, encompassing 11 genera and 44 species, were identified through analysis of ß-tubulin (benA) gene and internal transcribed spacer of ribosomal DNA (ITS) region. All collection points showed samples with high fungal contamination, but the mycobiota composition varied as a result of different environmental conditions. Alternaria spp. and Fusarium spp. were predominantly isolated from fresh fruits of C. annuum. Penicillium spp. was the most frequent genus in all analysed points. Penicillium brevicompactum and P. crustosum were the most abundant species. Among Aspergillus, A. niger and A. flavus were dominant after the drying phase. In our study, none of the analysed strains of Penicillium (113) and Aspergillus (35) produced Ochratoxin A at detectable levels. The broad characterization of the fungal community of C. annuum carried out in this study, could be a guideline for future mycotoxin analyses performed directly on the pod. Understanding the role and dynamics of mycobiota and its relationship with the toxins present in this substrate, will be useful to establish and improve control measures considering the specificities of each point in the C. annuum production chain.

Identification and Toxigenic Potential of Fungi Isolated from Capsicum Peppers.

Capsicum peppers are among the most popular horticultural crops produced and consumed worldwide. This study aimed to assess the occurrence of spoilage fungi responsible for post-harvest losses in the most common varieties of Capsicum peppers collected from retail markets in Nigeria and Ghana. Forty fungal isolates belonging to 7 families, 8 genera, and 17 species were identified on the basis of morphology, culture characteristics, and DNA sequencing of the internal transcribed spacer (ITS) region. Aspergillus spp. (42.5%), Fusarium spp. (22.5%), and Colletotrichum spp. (15%) were found to be the predominant fungal pathogens. Furthermore, potential ability of the isolated mycotoxigenic fungi to produce some major mycotoxins was analyzed using high-performance liquid chromatography (HPLC). Among the 22 isolates analyzed, 11 strains belonging to the genera of Aspergillus, Fusarium, and Penicillium were found to be able to produce mycotoxins, such as aflatoxin B1, gliotoxin, deoxynivalenol, and citrinin. A better understanding of the role of fungal contaminants in pepper fruits, especially the prevalence of mycotoxigenic fungi and their associated mycotoxigenic potential, will assist in the development of management strategies to control mycotoxin contamination and to reduce toxicological risks related to pepper consumption by humans and animals.

Overview of Fungi and Mycotoxin Contamination in Capsicum Pepper and in Its Derivatives.

Capsicum products are widely commercialised and consumed worldwide. These substrates present unusual nutritional characteristics for microbial growth. Despite this, the presence of spoilage fungi and the co-occurrence of mycotoxins in the pepper production chain have been commonly detected. The main aim of this work was to review the critical control points, with a focus on mycotoxin contamination, during the production, storage and distribution of Capsicum products from a safety perspective; outlining the important role of ecophysiological factors in stimulating or inhibiting mycotoxin biosynthesis in these food commodities. Moreover, the human health risks caused by the ingestion of peppers contaminated with mycotoxins were also reviewed. Overall, Capsicum and its derivative-products are highly susceptible to contamination by mycotoxins. Pepper crop production and further transportation, processing and storage are crucial for production of safe food.

The fungal threat to global food security.

Fungi threaten the security of food supply to human populations on several fronts. They destroy up to 30 % of crop products through disease and spoilage processes, while mycotoxin-producing fungi and opportunistic pathogens endanger food safety. Control of these fungi is vital for improving food security, but current measures are inadequate and further challenges due to human-population growth and climate change are escalating. Investment and innovation in research on strategies to control fungal growth, harnessed through international, inter-disciplinary collaboration across socio-economic boundaries, provides one key to rising to this challenge.

Comparative Analysis of the Composition and Active Property Evaluation of Certain Essential Oils to Assess their Potential Applications in Active Food Packaging.

The antifungal, antibacterial, and antioxidant activity of four commercial essential oils (EOs) (thyme, clove, rosemary, and tea tree) from Romanian production were studied in order to assess them as bioactive compounds for active food packaging applications. The chemical composition of the oils was determined with the Folin-Ciocâlteu method and gas chromatography coupled with mass spectrometry and flame ionization detectors, and it was found that they respect the AFNOR/ISO standard limits. The EOs were tested against three food spoilage fungi-Fusarium graminearum, Penicillium corylophilum, and Aspergillus brasiliensis-and three potential pathogenic food bacteria-Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes-using the disc diffusion method. It was found that the EOs of thyme, clove, and tea tree can be used as antimicrobial agents against the tested fungi and bacteria, thyme having the highest inhibitory effect. Concerning antioxidant activity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) methods, it has been established that the clove oil exhibits the highest activity because of its high phenolic content. Promising results were obtained by their incorporation into chitosan emulsions and films, which show potential for food packaging. Therefore, these essential oils could be suitable alternatives to chemical additives, satisfying the consumer demand for naturally preserved food products ensuring its safety.

Evaluation of antioxidant and antifungal properties of the traditional plants against foodborne fungal pathogens.

OBJECTIVE: To determine the antioxidant and antifungal activities of the essential oils from five aromatic herbs, including Thymus vulgaris, Chamaemelum nobile, Ziziphora clinopodioides, Zingiber officinale and Cuminum cyminum, against different Aspergillus and Penicillium species. METHODS: The oils were subjected to screening for their possible antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The susceptibility test for the oils was carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) using microdilution method. RESULTS: The values of the essential oils in DPPH assay were as follows: T. vulgaris (450.11±5.23 µg/mL), Ch. nobile (602.73±4.8 µg/mL), Ziz. clinopodioides (1238.82±9.3 µg/mL), Cu. cyminum (1255.52±8.92 µg/mL) and Zin. officinale (5595.06±8.24 µg/mL). Our findings also indicated a strong activity against tested fungi for the oil of T. vulgaris (1250 µg/mL), followed by Cu. cyminum (1416 µg/mL), Zin. officinale (1833 µg/mL), Ziz. clinopodioides (2166 µg/mL) and Ch. nobile (3750 µg/mL). This study confirmed the excellent antifungal and antioxidant properties of the essential oils, especially T. vulgaris, against foodborne pathogenic fungi. CONCLUSION: Owing to their strong protective features, these oils could be used in ethno-medicine as preventers of lipid peroxidation and cellular damage, and in food industries as preservers of foodstuffs against spoilage fungi. Also, they could be the candidates to develop new antibiotics and disinfectants to control infective agents.