Spontaneously growing fungi on the surface and processing areas of matured sheep ham and volatile compounds produced.

Raw ham is a dried and matured product traditionally made from pork leg, but other animals, such as sheep, can be used. The natural presence of bacteria and fungi in this product influences its characteristics throughout the process. This study analysed the fungal populations present during raw sheep hams' processing. Two types of products were developed: without and with the addition of seasonings. Mycological analyses were carried out from the ingredients, seasonings, facilities air, as well as on the surfaces of the hams and the air in the chamber throughout the maturation period (0, 45, 90, and 180 days) using 18 % dichloran glycerol agar and the data were submitted to Principal Component Analysis. Volatile compounds were evaluated at the end of the sheep ham manufacturing process through a gas chromatograph coupled to a mass spectrometer. At 45 days of aging, a more remarkable similarity was observed between the fungi present on the non-seasoned hams and those in the ripening chamber's air, while the seasoned hams showed a more evident relation with those fungi present in the spices. With time, the fungi in the air of the ripening chamber started to be influenced by Aspergillus ser. Aspergillus and Aspergillus ser. Rubri already installed in the seasoned hams at 45 days, and then it probably dispersed the non-seasoned ones due to the airborne spores, becoming the most prevalent in both treatments at 90 days. At the end of ripening, the mycobiota of both raw hams was composed mainly by xerophilic species of Aspergillus section Aspergillus. The total fungal count was 5.78 log CFU/cm2 for the non-seasoned and 7.19 log CFU/cm2 for the seasoned ones. A potentially ochratoxigenic Aspergillus ser. Circumdati was detected at the end of aging in raw, unseasoned hams. In conclusion, seasoning directly influences the species developing on the surface of seasoned hams throughout the ripening process, and indirectly affects the mycobiota of the non-seasoned hams when sharing the same ripening chamber. The presence of fungi in the matured sheep ham seems to contribute to the formation of volatile compounds, which are related to the sensory quality of these products.

Potential of electrolyzed water to inactivate bread and cheese spoilage fungi.

Controlling food spoilage fungi remains a challenge for food industries, and regulations on the usage of chemical disinfectants are becoming restrictive. Then, this study aimed to evaluate electrolyzed water (EW) as a sustainable alternative for food spoilage fungi inactivation. The experiment was carried out according to the protocol for testing the antifungal effects of chemical sanitizers by the European Committee for Standardization (CEN), using acidic electrolyzed water (AEW-AAC: 85 ppm; pH: 2.65; ORP: 1120 mV) and a basic electrolyzed water (BEW- pH: 11.12; ORP: -209 mV) to inactivate spoilage fungi strains from bread (Hyphopichia burtonii and Penicillium roqueforti) and cheese (P. roqueforti and Penicillium commune), besides the standard fungi for this type of essay (Candida albicans and Aspergillus brasiliensis). AEW presented a higher antifungal effect, inactivating an average of 89 % of the exposed population when compared to its respective BEW, which inactivates about 81.5 %. In general, the standard strains A. brasiliensis (ATCC 16404) and Candida albicans (ATCC 24433) were more sensitive to both AEW and BEW than the food-spoilage strains. Among those, P. roqueforti strains were the most sensitive, followed by P. commune strains, while H. burtonii strains were the most tolerant. EW can be a sustainable alternative for product surface and facility cleaning with further antifungal action when a sanitization step is not mandatory or needed. Future studies searching for conditions to improve the antifungal action of EW could make their industrial usage more viable.

Efficacy of weak acid preservatives on spoilage fungi of bakery products.

Organic acids and their salts are usually the first choice in the bread industry to restrict fungal spoilage, but their efficacy is pH-dependent and spoilage by fungi remains as a common threat. Therefore, this study aimed to evaluate the susceptibility of spoilage fungi of bakery products to acetic, sorbic, and propionic acids at different pH. Penicillium roqueforti, Penicilium paneum, Aspergillus pseudoglaucus, Aspergillus montevidensis and Hyphopichia burtonii strains isolated from spoiled products had their minimum inhibitory concentration (MIC) defined by macrodilution. The concentrations tested were: (i) sorbic acid up to 32 mM; (ii) propionic acid up to 1024 mM and (iii) acetic acid up to 800 mM with pH adjusted in 4.5, 5.0, 5.0 and 6.0 after setting the agent concentration. The lowest MICs for all agents were obtained at pH 4.5, usually doubling with every 0.5 pH increase. P. roqueforti strains isolated from spoiled products were the most resistant to all tested preservatives; while strains of the related species P. paneum, showed similar tolerance to acetic and propionic acids but was double more susceptible to sorbic acid. Strains of A. pseudoglaucus and A. montevidensis were indistinctly susceptible to the preservatives and were the most susceptible species to propionic and acetic acids. H. burtonii strains demonstrated the most variable behaviour in comparison to the other strains being the most susceptible to sorbic acid, were like Aspergillus strains regarding propionic acid, but tolerate well acetic acid. Propionic acid concentrations usually allowed in baked goods are lower than the concentrations required to inhibit the most tolerant isolates tested in this study. The same is true for sorbic acid at higher pH levels. Spoilage species of bakery ware presents a distinct susceptibility profile to the preservatives commonly used in this sector, but the high tolerance observed is a cause of concern.

Relationship between the Fungal Incidence, Water Activity, Humidity, and Aflatoxin Content in Maize Samples from the Highlands and Coast of Ecuador.

This study evaluated the fungal incidence through direct plating in Agar Dichloran Glycerol, and the presence of aflatoxins in maize samples from the Highlands and Coast of Ecuador by HPLC, investigating the influence of the temperature, altitude, water activity, and humidity of the collection regions on the maize samples' contamination using Principal Components Analysis (PCA). The overall kernel infection by fungi was usually lower in samples from the Highlands, and no aflatoxins or Aspergillus series Flavi were detected in the samples from this region. In the coastal samples, Aspergillus sp. were isolated from all samples, while the potentially aflatoxigenic A. Flavi contaminated about 80% of them. Aflatoxins were present in 50% of these samples, in ranges from 0.42 to 107.69 µg/kg. PCA was able to segregate the samples according to their collection region, and showed that the maximum and minimum temperatures are closely and positively related to the presence of A. Flavi. A highly positive relationship was also observed between the water activity of the sample and aflatoxin contamination. On the other hand, the altitude had a very strong-but negative-relationship with the variables studied. This study is relevant because data regarding fungi and aflatoxin occurrence, as well the main factor influencing the contamination of Ecuadoran maize, are scarce; it clearly shows that aflatoxins are a hazard present in maize from the Ecuadorian Coast but not the Highlands.

Fungal growth, patulin accumulation and volatile profile in 'Fuji Mishima' apples under controlled atmosphere and dynamic controlled atmosphere.

The objective of this study was to evaluate fungal and patulin contamination, together with its correlation with the volatile compounds (VCs), in 'Fuji Mishima' apples (up to 25% decayed) under controlled atmosphere (CA) and dynamic controlled atmosphere with respiratory quotient (DCA-RQ) of 1.3 combined with different partial pressures of carbon dioxide (0.8, 1.2, 1.6 and 2.0 pCO2). Fruits were stored under the above conditions for 8 months at 0.5 °C plus 7 days shelf life at 20 °C. Toxigenic fungi and patulin accumulation were found in apples from all treatments. Penicillium expansum was the most prevalent species. For all storage conditions, patulin concentrations were above the maximum level allowed in Brazil (50  µg  kg-1) with an exception of DCA-RQ1.3 + 0.8 kPa CO2. This condition, with lower pCO2, showed the lowest patulin accumulation, below the legal limit. The CA provided the highest patulin concentration (166 µg  kg-1). It was observed that fungal growth could also contribute to changes in the volatile composition. Styrene and 3-methyl-1-butanol are considered P. expansum markers in some apple cultivars and were detected in the samples. However, it was not possible to identify volatile organic compounds (VOCs) that are biomarkers from P. expansum, because there were other fungi species present in all samples. In this study, styrene, n-decanoic acid, toluene, phenol and alpha-farnesene were the compounds that showed the most positive correlation with patulin accumulation. On the other hand, a negative correlation of patulin with acids has been shown, indicating that in treatments with a higher patulin concentration there were less acidic compounds.

Influence of type, concentration, exposure time, temperature, and presence of organic load on the antifungal efficacy of industrial sanitizers against Aspergillus brasiliensis (ATCC 16404).

Parameters such as type and concentration of the active compound, exposure time, application temperature, and organic load presence influence the antimicrobial action of sanitizers, although there is little data in the literature. Thus, this study aimed to evaluate the antifungal efficacy of different chemical sanitizers under different conditions according to the European Committee for Standardization (CEN). Aspergillus brasiliensis (ATCC 16404) was exposed to four compounds (benzalkonium chloride, iodine, peracetic acid, and sodium hypochlorite) at two different concentrations (minimum and maximum described on the product label), different exposure times (5, 10, and 15 min), temperatures (10, 20, 30, and 40 °C), and the presence or absence of an organic load. All parameters, including the type of sanitizer, influenced the antifungal efficacy of the tested compounds. Peracetic acid and benzalkonium chloride were the best antifungal sanitizers. The efficacy of peracetic acid increased as temperatures rose, although the opposite effect was observed for benzalkonium chloride. Sodium hypochlorite was ineffective under all tested conditions. In general, 5 min of sanitizer exposure is not enough and >10 min are necessary for effective fungal inactivation. The presence of organic load reduced sanitizer efficacy in most of the tested situations, and when comparing the efficacy of each compound in the presence and absence of an organic load, a difference of up to 1.5 log CFU was observed. The lowest concentration recommended on the sanitizer label is ineffective for 99.9% fungal inactivation, even at the highest exposure time (15 min) or under the best conditions of temperature and organic load absence. Knowledge of the influence exerted by these parameters contributes to successful hygiene since the person responsible for the sanitization process in the food facility can select and apply a certain compound in the most favorable conditions for maximum antifungal efficacy.

Antifungal efficacy of sanitizers and electrolyzed waters against toxigenic Aspergillus.

The presence of mycotoxin-producing fungi in food production environments is a cause of concern since they can contaminate food products, synthesizing toxic compounds in later steps. To avoid this, an effective hygiene and sanitation process of the environment and equipment should be adopted, using sanitizing agents with adequate antifungal efficacy. This work evaluated the effectiveness of different chemical sanitizers: benzalkonium chloride (0.3%, 1.2%, 2%), biguanide (2%, 3.5%, 5%), iodine (0.2%, 0.6%, 1%), peracetic acid (0.3%, 0.6%, 1%), sodium hypochlorite (0.5%, 0.75%, 1%), besides a new non-polluting technology, the electrolyzed water, both the acid in the chlorine concentrations of 60, 85 and 121 ppm and the respective basic electrolyzed water formed against strains of toxigenic Aspergillus flavus, Aspergillus nomius, Aspergillus parasiticus, Aspergillus carbonarius, Aspergillus niger, Aspergillus ochraceus and Aspergillus westerdijkiae through the methodology recommended by the European Committee for Standardization. Benzalkonium chloride and iodine were the most effective sanitizers to eliminate Aspergillus from the Flavi and Nigri section. Peracetic acid showed the best elimination of the growth of Aspergillus from Circumdati section. Sodium hypochlorite, biguanide, and electrolyzed water agents were the least effective, reducing less than 3 log from initial control, not being the most suitable agents for the control of toxigenic fungi in food industries.

Comparison of electrolized water and multiple chemical sanitizer action against heat-resistant molds (HRM).

This study aimed to evaluate the sensitivity of heat-resistant molds isolated from spoiled thermally processed foods to antimicrobial compounds used for food industry sanitation. An ortho-phenylphenol-based smoke generator sanitizer, liquid chemical sanitizers (benzalkonium chloride, biguanide, iodine, peracetic acid, and sodium hypochlorite), and acidic and alkaline electrolyzed water were used against Aspergillus australensis (MB 2579; NFF 02), Aspergillus aureoluteus (NFC1), Paecilomyces fulvus (PFF 01), Paecilomyces niveus (PNT 01; PNDC 01; PNB1 01), and Paecilomyces variotii (PV 01; PV 01; PVCH 03). The fungal strains were exposed separately to liquid sanitizers and electrolyzed water in stainless steel discs for 15 min following the European Committee for Standardization (CEN) recommendations. Moreover, the fungal strains were exposed to the smoke generator sanitizer for 7 h following French protocol NF-T-72281. The best results of fungal inactivation were achieved when the highest concentration specified in the label of these sanitizers was tested. On the opposite, the lowest concentration specified in the label should be avoided since it was ineffective in most cases (94%). The ortho-phenyphenol-based smoke generator sanitizer and peracetic acid (1%) showed the best results of spore inactivation, while iodine and benzalkonium chloride achieved satisfactory results against the strains evaluated. Sodium hypochlorite and biguanide were ineffective against most of the fungi studied at all concentrations tested. Acidic and basic electrolyzed water was also ineffective to achieve the 3-log CFU reduction required in the concentrations tested. In general, Paecilomyces spp. was more sensitive than Aspergillus spp. against all sanitizers evaluated, whereas A. aureoluteus NFC1 was resistant to all agents and concentrations tested. The heat-resistant fungal strains showed varied sensitivity against the different agents. Notably, the two most effective commercial sanitizers against the heat-resistant strains were ineffective against the filamentous fungi recommended for sanitizer testing (A. brasiliensis ATCC 16404), which demonstrates the relevance of testing fungal isolates that cause spoilage to choose the most effective compound and obtain the best results of fungal control.

Spoilage fungi in a bread factory in Brazil: Diversity and incidence through the bread-making process.

This study aimed to verify the main fungal species involved in the deterioration of different types of bread and to identify the possible sources of contamination of these products. Samples of raw materials (n = 127), environmental air (n = 50) and moldy bread (n = 90) were analyzed. Aspergillus candidus, Wallemia sebi, and Penicillium roqueforti were the predominant species in the raw materials and were isolated in samples of wheat flour, in two-thirds of the samples of rye and 62.5% of the wheat flour. Penicillium roqueforti was isolated from all types of moldy bread analyzed and Hyphopichia burtoni was also present in samples of moldy wheat and rye bread. These two species were also recovered during air sampling from baking industry facilities (cooling and slice and package areas), which may be crucial for product contamination after baking. Hygienic measures to reduce airborne contamination during the cooling and packaging of food should be taken to prevent the early deterioration of bread.

Ochratoxin A production by Aspergillus westerdijkiae in Italian-type salami.

The occurrence of ochratoxin A (OTA) in matured meat products can be attributed to mycotoxin coming from raw materials and/or molds that develop on the product surface during ripening. This work aimed to evaluate OTA production by Aspergillus westerdijkiae inoculated on the surface of sausages and its diffusion into the product throughout ripening, study the effect of relative humidity (RH) on the production of this mycotoxin, and investigate the presence of OTA in dry fermented sausages that naturally present intense contamination by A. westerdijkiae. Italian-type sausages were surface inoculated with A. westerdijkiae and two distinct experiments were performed. In the first, the sausages were matured in a chamber with decreasing RH (from 95 to 75%) for 35 days. In the other, the sausages were incubated under different RH (79, 85 or 95%) for 21 days. Samples were taken at the beginning of the experiments and every 7 days, subdividing into casing, outer border, and core for analyses. Sausage samples naturally spoiled by ochratoxigenic fungi were collected during sanitary inspection. Even in the presence of A. westerdijkiae mycelia, no OTA was detected for up to 7 days of sausage maturation. On the other hand, this study demonstrated that the growth of A. westerdijkiae on salami surface produces high amounts of OTA on the casing and allows its diffusion through the casing with contamination to the outer border of sausages. In the same way, it shows that under similar water activity values of substrate, RH influences the amount of OTA produced. Conversely, OTA was restricted to the casing in the naturally contaminated sausages.

Antifungal activity of commercial sanitizers against strains of Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus: Bakery spoilage fungi.

Information on the sensitivity of spoilage fungi of bakery products to sanitizing agents is scarce in the literature. Thus, the aim of this study was to evaluate the antifungal activity of different classes of commercial sanitizers, which have permitted use in the food industry, on the main fungi involved in spoiling bakery products. The tests were carried out according to the protocol for testing the antifungal effect of chemical sanitizers of the European Committee for Standardization (CEN), with adaptations. Different strains of six isolated fungal species responsible for spoiling bakery products (Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus) were tested against five sanitizers at three concentrations: benzalkonium chloride (0.3%, 2.5%, 5%), biguanide (2%, 3.5%, 5%), peracetic acid (0.15%, 1.5%, 3%), quaternary ammonium (0.3%, 2.5%, 5%), and sodium hypochlorite (0.01%, 0.1%, 0.2%). Peracetic acid was the most effective sanitizes considering the genera, species, and concentrations evaluated, generally being capable of reductions between 2 and 4 logs of initial control tested. Biguanide should not be the compound of choice when the main goal of the bakery industry is fungal control.

Sensitivity of food spoilage fungi to a smoke generator sanitizer.

Smoke generator sanitizers are easy to handle and can access to hard-to-reach places. They are a promising alternative for controlling food and air borne fungi, which are known to cause losses in the bakery, meat, and dairy industries. Therefore, the present study aimed to evaluate the efficiency of a smoke generator sanitizer based on orthophenylphenol against ten fungal species relevant to food spoilage. The tests were carried out according to the norms by the French protocol NF-T-72281, with adaptations specific for disinfectants diffused in the air. The tests were performed in an enclosed room of approximately 32 m3. Aspergillus brasiliensis (ATCC 16404), Candida albicans (ATCC 10231), Aspergillus flavus (ATCC 9643), Aspergillus chevalieri (IMI 211382), Cladosporium cladosporioides (IMI 158517), Lichtheima corymbifera (CCT 4485), Mucor hiemalis (CCT 4561), Penicillium commune (CCT 7683), Penicillium polonicum (NGT 33/12), and Penicillium roqueforti (IMI 217568) were exposed to the smoke generator sanitizer for 7 h. The product was efficient against C. albicans and C. cladosporioides, although it was unable to reduce 4 log of the other tested species. The variable sensitivity of the fungal species to the sanitizer emphasizes the importance of confronting a target microorganism (causing problems in a specific food industry) with the sanitizer aiming to control it and obtain satisfactory results in hygiene programs.

Incidence of spoilage fungi in the air of bakeries with different hygienic status.

This study aimed to determine the level of adequacy of bakeries to the requirements of good manufacturing practices (GMP) and the fungal contamination of the environmental air of these places, verifying the existence of a correlation between both. The checklist present in Ordinance No 78/2009 of good practices based on the current legislation was used to evaluate GMP adequacy and the contamination of environmental air was determined through the air sampling method. After the application of the checklist, it was observed that 75% (n = 15) of the bakeries evaluated were classified into group 2 (good) and 25% (n = 5) as group 3 (regular). On the other hand, only five (25%) and three (15%) bakeries presented values below of 300 CFU/m3 for the fungal contamination of air in food cooling and exposure areas, respectively. Fungi isolated from these areas were mainly species commonly reported as potential spoilers of bakery products. This study revealed that in spite of the evaluated bakeries presenting conditions considered "good" by the GMP, they presented, in most cases, high counts of fungi in the cooling and exposition areas for ready products. Only a weak correlation was found between the items of GMP and the load of airborne fungi present in the bakeries. More attention should be directed to hygienic aspects related to the microbiological quality of the air of bakeries, since these micro-organisms could contaminate the surface of freshly baked products, compromising the final quality of these foods, as well as prompt its early deterioration.