Yeast biofilms on abiotic surfaces: Adhesion factors and control methods.

Biofilms are highly resistant to antimicrobials and are a common problem in many industries, including pharmaceutical, food and beverage. Yeast biofilms can be formed by various yeast species, including Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans. Yeast biofilm formation is a complex process that involves several stages, including reversible adhesion, followed by irreversible adhesion, colonization, exopolysaccharide matrix formation, maturation and dispersion. Intercellular communication in yeast biofilms (quorum-sensing mechanism), environmental factors (pH, temperature, composition of the culture medium), and physicochemical factors (hydrophobicity, Lifshitz-van der Waals and Lewis acid-base properties, and electrostatic interactions) are essential to the adhesion process. Studies on the adhesion of yeast to abiotic surfaces such as stainless steel, wood, plastic polymers, and glass are still scarce, representing a gap in the field. The biofilm control formation can be a challenging task for food industry. However, some strategies can help to reduce biofilm formation, such as good hygiene practices, including regular cleaning and disinfection of surfaces. The use of antimicrobials and alternative methods to remove the yeast biofilms may also be helpful to ensure food safety. Furthermore, physical control measures such as biosensors and advanced identification techniques are promising for yeast biofilms control. However, there is a gap in understanding why some yeast strains are more tolerant or resistant to sanitization methods. A better understanding of tolerance and resistance mechanisms can help researchers and industry professionals to develop more effective and targeted sanitization strategies to prevent bacterial contamination and ensure product quality. This review aimed to identify the most important information about yeast biofilms in the food industry, followed by the removal of these biofilms by antimicrobial agents. In addition, the review summarizes the alternative sanitizing methods and future perspectives for controlling yeast biofilm formation by biosensors.

Microbial resistance to sanitizers in the food industry: review.

Hygiene programs which comprise the cleaning and sanitization steps are part of the Good Hygiene Practices (GHP) and are considered essential to ensure food safety and quality. Inadequate hygiene practices may contribute to the occurrence of foodborne diseases, development of microbial resistance to sanitizers, and economic losses. In general, the sanitizer resistance is classified as intrinsic or acquired. The former is an inherent characteristic, naturally present in some microorganisms, whereas the latter is linked to genetic modifications that can occur at random or after continuous exposure to a nonnormal condition. The resistance mechanisms can involve changes in membrane permeability or in the efflux pump, and enzymatic activity. The efflux pump mechanism is the most elucidated in relation to the resistance caused by the use of different types of sanitizers. In addition, microbial resistance to sanitizers can also be favored in the presence of biofilms due to the protection given by the glycocalyx matrix and genetic changes. Therefore, this review aimed to show the main microbial resistance mechanisms to sanitizers, including genetic modifications, biofilm formation, and permeability barrier.

The effects of time and relative humidity on dry-aged beef: Traditional versus special bag.

The objective of this study was to evaluate the effects of relative humidity (RH) and different dry aging methods on the quality of beef. Sixteen loins, from eight carcasses, were used in this experiment. Each pair of loin was cut into eight sections with equal size, which were evenly assigned to eight treatments, by the combination of two dry aging methods (traditional and highly moisture-permeable special bag), two relative humidity (65 and 85% RH) and two aging times (21 and 42 days). At 85% RH, neither special bag nor the traditional dry aging methods were viable, since samples presented high microbiological counts, mucus and bad odor. At 65% RH, Enterobacteriaceae and lactic acid bacteria were not detected in any treatment. The highest aerobic plate count and psychrotrophic count were observed in the samples of the traditional dry-aged process whereas the special bag showed the greatest mold and yeast count. Regarding dry aging in special bag, there was a reduction in the weight loss (P < 0.05) and no change in the physical-chemical characteristics (P > 0.05) compared to traditional dry aging. The values of pH, moisture and Warner-Bratzler shear force were not affected (P > 0.05) by aging method and relative humidity. Thus, the results indicate that high RH should be avoided for both dry aging methods. Furthermore, the special bag dry aging can be considered an alternative to produce dry-aged beef, as it reduces weight losses even at conditions of lower relative humidity.

Using chitosan and radish powder to improve stability of fermented cooked sausages.

Radish powder and chitosan were evaluated in fermented cooked sausages to replace sodium nitrite. Treatments of fermented cooked sausages were prepared and evaluated during the ripening process and storage time: control (150 ppm nitrite, CONT); and two levels of chitosan (0.25%, CHI1) and (0.5%, CHI2) with radish powder (0.5%) and without the addition of nitrite. During storage, pieces sliced or not were also evaluated regarding the physicochemical, microbiological and sensory. Pure chitosan was evaluated regarding determination of MIC and MBC to Enterobacter aerogenes, Listeria innocua and Lactobacillus rhamnosus, and antioxidant capacity. The addition of chitosan increased aw and decreased weight loss of fermented sausages along processing, but pH was not affected during ripening. Except for aroma, sensory attributes were affected by the addition of chitosan. The addition of 0.5% radish powder and 0.25% chitosan showed promising results to development of nitrite-free fermented cooked sausages, mainly regarding their physicochemical and microbiological stability.

Genetic diversity, antimicrobial resistance and virulence profile of Salmonella isolated from the peanut supply chain.

Thirty-Eight Salmonella isolates recovered from different stages of the peanut supply chain in three Brazilian States (São Paulo, Minas Gerais and Bahia) were subtyped by pulsed-field gel electrophoresis (PFGE) and characterized by phenotypic and genotypic tests for antimicrobial resistance and virulence genes. The isolates were distributed into seven PFGE pulsotypes. All the isolates were resistant to sulfonamide. However, only one isolate from a production site in Minas Gerais had resistance to two types of antimicrobials (sulfonamide and ampicillin). Furthermore, the isolates had intermediary resistance to kanamycin (16/38), streptomycin (14/38) and ceftazidime (12/38). Four isolates had the antimicrobial resistance gene related to phenicols (floR) and 37 related to aminoglycosides (strA). The blashv gene related to ß-lactams was detected in isolates recovered from all the production regions. Six virulence genes (invA, sefA, sivH, mgtC, ssaQ and agfA) were observed in all isolates. The sopE gene was detected in 24 isolates, avrA in 12. The gtgB, ipfA and rck genes were not detected. The results showed that the pulsotype 1 was restricted to Minas Gerais whereas the pulsotype 7 was present in São Paulo and Bahia. In addition, most of the isolates were not multidrug resistant.

The biodiversity of Aspergillus section Flavi and aflatoxins in the Brazilian peanut production chain.

A total of 119 samples of peanut were collected throughout the peanut production chain in São Paulo State, Brazil. The peanut samples were directly plated for determination of percentages of infection and a polyphasic approach was used to identify Aspergillus section Flavi species. Further, the potential for aflatoxin production by the isolates was tested using the agar plug technique and the presence of aflatoxins in peanuts was assessed using an immunoaffinity column followed by quantification using HPLC with reverse phase column and fluorescence detection. The limit of detection and quantification were 0.05 and 0.17µg/kg for total aflatoxins, respectively. Four species of Aspergillus section Flavi were isolated: A. caelatus (11), A. flavus (515), A. parasiticus (17) and A. tamarii (13). All isolates of A. parasiticus were able to produce aflatoxin B and G whereas aflatoxin B was produced by 50% of A. flavus isolates. Aflatoxins were found in 12 samples at concentrations ranging from 0.3 to 100µg/kg. The data reported in this study add information on the occurrence and biodiversity of fungi in peanuts at several stages of the production chain. The occurrence of aflatoxins is also of major relevance for continuous monitoring and assessment of likely exposure of consumers to aflatoxins through consumption of peanuts.

Behavior of Salmonella during fermentation, drying and storage of cocoa beans.

Due to cocoa being considered a possible source of Salmonella contamination in chocolate, the behavior of Salmonella during some cocoa pre-processing stages (fermentation, drying and storage) was investigated. The fermentation process was carried out on a pilot scale (2 kg beans/box) for 7 days. Every day a fermentation box was inoculated with a Salmonella pool (ca. 4 log MPN/g). The results showed that Salmonella did not affect (P>0.05) the growth of the main microorganism groups involved in cocoa fermentation. On the other hand, the pathogen was influenced (P<0.05) by yeast, acetic acid bacteria and pH. In spite of Salmonella showing counts ≤ 1 log MPN/g in the first days, at the end of fermentation it grew in all samples, reaching counts as high as 7.49 log MPN/g. For drying and storage, cocoa beans were inoculated during the fermentation (experiment A) or during the drying (experiment B). In these stages the decline of the water activity affected the pathogen behavior. In experiment A during the drying, Salmonella count increased in most of the samples. In experiment B either a slight growth or no growth in the samples inoculated up to 48 h was observed, whereas the other samples showed reductions from the initial count. After 30 days of storage at room temperature, the water activity decreased to 0.68, and reductions of Salmonella ranged from 0.93 to 2.52 log MPN/g. Despite the reductions observed during the storage, the pathogen was detected even after 120 days. Therefore, the results showed that Salmonella growth or survival depends on when the contamination occurs.

Inactivation of Salmonella during cocoa roasting and chocolate conching.

The high heat resistance of Salmonella in foods with low water activity raises particular issues for food safety, especially chocolate, where outbreak investigations indicate that few colony-forming units are necessary to cause salmonellosis. This study evaluated the efficiency of cocoa roasting and milk chocolate conching in the inactivation of Salmonella 5-strain suspension. Thermal resistance of Salmonella was greater in nibs compared to cocoa beans upon exposure at 110 to 130°C. The D-values in nibs were 1.8, 2.2 and 1.5-fold higher than those calculated for cocoa beans at 110, 120 and 130°C. There was no significant difference (p>0.05) between the matrices only at 140°C. Since in the conching of milk chocolate the inactivation curves showed rapid death in the first 180 min followed by a lower inactivation rate, and two D-values were calculated. For the first time interval (0-180 min) the D-values were 216.87, 102.27 and 50.99 min at 50, 60 and 70°C, respectively. The other D-values were determined from the second time interval (180-1440 min), 1076.76 min at 50°C, 481.94 min at 60°C and 702.23 min at 70°C. The results demonstrated that the type of matrix, the process temperature and the initial count influenced the Salmonella resistance.

Pesquisa de enterobactérias em chocolates / Detection of enterobacteria in chocolates

O chocolate é um dos produtos responsáveis pelos maiores surtos de salmonelose em humanos, contudo pouco se sabe sobre a presença de agentes enteropatogênicos em chocolate produzido no Brasil. Neste contexto, este trabalho pesquisou a presença de enterobactérias totais, coliformes, Escherichia coli e Salmonella em 65 amostras de chocolate (22 ao leite, 22 branco, 17 meio amargo e 4 em pó) comercializadas em Campinas/SP. As amostras apresentaram valores médios de pH entre 5,8 e 7,5 e atividade de água entre 0,31 e 0,59. Não foram isolados coliformes termotolerantes, E. coli e Salmonella nas amostras analisadas. Porém, enterobactérias totais foram detectadas em 22,7% das amostras de chocolate ao leite e branco e em 11,7% das amostras de chocolate meio amargo. Os coliformes totais foram isolados, respectivamente, em 13,6% e 4,5% das amostras de chocolate ao leite e branco. Enterobactérias totais e coliformes não foram detectados nas amostras de chocolate em pó. A contaminação encontrada reforça a necessidade de implementação de rigoroso programa de boas práticas de fabricação pelas indústrias processadoras de chocolate para garantir a segurança microbiológica dos produtos manufaturados.

Enterobacteriaceae in processed cocoa products

Little is known of the presence of Salmonella in Brazilian cocoa, which justifies the present work that had the aim of checking the presence of total Enterobacteriaceae, coliforms, Escherichia coli and Salmonella in semi-processed cocoa products. A total of 150 samples of cocoa products from two different cocoa-processing manufacturers were analyzed (30 samples of nibs, 30 of liquor, 30 of cocoa cake, 30 of cocoa butter and 30 of cocoa powder). The samples of processed cocoa products had pH values between 5.31 and 7.35, and water activity ranging from 0.29 to 0.52. E. coli and Salmonella were not detected in any of the samples analyzed. Regarding the other analyzed microorganisms, 17 of the nibs contained total Enterobacteriaceae, 20 showed total coliforms, while thermotolerant coliforms were detected in one sample (0.6 log MPN/g). Seven percent of liquor and cocoa powder samples showed total coliforms. In cocoa cake, the same percentage in regard to total Enterobacteriaceae was observed. Total Enterobacteriaceae and total coliforms were detected in one sample of cocoa butter. Despite the low contamination observed, these results indicate failure in quality programs of the manufactures studied, since these bacteria are easily inactivated by thermal and sanitizer process.

Antimicrobial activity of Enterococcus faecium FAIR-E 198 against gram-positive pathogens

This study investigated the antimicrobial activity of Enterococcus faecium FAIR-E 198 against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Using the critical-dilution method, the bacteriocin produced by E. faecium FAIR-E 198 inhibited all L. monocytogenes strains evaluated (1,600 to 19,200 AU mL-1). However, none of the B. cereus and S. aureus strains investigated were inhibited. The maximum activity of this bacteriocin (800 AU mL-1) was observed in MRS broth, while the activity in milk was 100 AU mL-1. In the co-cultivation test in milk, B. cereus K1-B041 was reduced to below the detection limit (1.00 log CFU mL-1) after 48 h. E. faecium reduced the initial L. monocytogenes Scott A population by 1 log CFU mL-1 after 3 h at 35ºC. However, the pathogen regained growth, reaching 3.68 log CFU mL-1 after 48 h. E. faecium did not influence the growth of S. aureus ATCC 27154 during the 48 h of co-cultivation. Therefore, it can be concluded that the effectiveness of the antimicrobial activity of E. faecium FAIR-E 198 is strictly related to the species and strain of the target microorganism and to the culture medium.

Antimicrobial activity of Enterococcus Faecium Fair-E 198 against gram-positive pathogens.

This study investigated the antimicrobial activity of Enterococcus faecium FAIR-E 198 against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Using the critical-dilution method, the bacteriocin produced by E. faecium FAIR-E 198 inhibited all L. monocytogenes strains evaluated (1,600 to 19,200 AU mL(-1)). However, none of the B. cereus and S. aureus strains investigated were inhibited. The maximum activity of this bacteriocin (800 AU mL(-1)) was observed in MRS broth, while the activity in milk was 100 AU mL(-1). In the co-cultivation test in milk, B. cereus K1-B041 was reduced to below the detection limit (1.00 log CFU mL(-1)) after 48 h. E. faecium reduced the initial L. monocytogenes Scott A population by 1 log CFU mL(-1) after 3 h at 35°C, However, the pathogen regained growth, reaching 3.68 log CFU mL(-1) after 48 h. E. faecium did not influence the growth of S. aureus ATCC 27154 during the 48 h of co-cultivation, Therefore, it can be concluded that the effectiveness of the antimicrobial activity of E. faecium FAIR-E 198 is strictly related to the species and strain of the target microorganism and to the culture medium.

Estudo da correlaçäo linear entre a contagem padräo em placa, a contagem de psicrotróficos e a prova da redutase em leite cru resfriado / Study of good lineal correlation between standard count in plate, psychotrophics count and redutase test in raw milk

Foram analisadas 30 amostras de leite cru tipos B e C, estocados em tanques resfriadores a 4§C por 24 h após a ordenha, provenientes de propriedades rurais da bacia leiteira de Juiz de Fora - MG, com o objetivo de avaliar a correlaçäo linear existente entre a contagem padräo em placa (CPP), a contagem de psicotróficos e a prova de redutase. A contagem de psicotróficos encontrada foi, em média, 79 por cento da contagem de mesófilos, sendo que 20 por cento das amostras apresentaram contagem de psicotróficos superior à CPP, o que evidencia a tendência de substituiçäo da microbiota mesofílica pela psicrotrófica, devido ao armazenamento e ao transporte refrigerados do leite. Com relaçäo ao coeficiente de correlaçäo (r), este demonstrou haver correlaçäo linear entre a CPP e a contagem de psicotróficos (r=0,90), porém tanto a CPP quanto a contagem de psicotróficos näo apresentaram correlaçäo linear com a prova da redutase. Contudo, ao se adotar os padröes estabelecidos pela legislaçäo vigente para a comparaçäo dos resultados obtidos na CPP com os resultados da prova da redutase, notou-se 85 por cento de concordância entre eles.