Viable nonculturable state in Salmonella enterica sorovar Enteritidis deficient in (p)ppGpp synthesis

Salmonella enterica é um patógeno de origem alimentar capaz de entrar no estado viável não cultivável em resposta a condições ambientais adversas. S. enterica é capaz de produzir (p)ppGpp, um nucleotídeo responsável pelo controle da expressão gênica no nível da transcrição e da tradução, em condições de estresse nutricional. Em razão da expressão gênica determinada pelo acúmulo de (p)ppGpp, S. enterica suporta condições ambientais variadas. Este trabalho teve como objetivos: quantificar por PCR em tempo real a expressão dos genes mreB, relacionado ao citoesqueleto e, rpoS, que codifica genes de resposta ao estresse, de estirpes mutantes simples e duplo da Salmonella Enteritidis PT4 578 deficientes na síntese de (p)ppGpp, em razão de mutação no gene relA, que codifica uma GTP pirofosfoquinase, e,ou spoT, que codifica guanosina-3',5'-difosfato 3'-pirofosfohidrolase submetidas aos estresses nutricional e choque frio; induzir essas estirpes ao estado VNC em solução fosfato de Butterfield (BPS) acrescida ou não de cloreto de sódio a 4 ºC e avaliar a morfologia dessas estirpes em fase logarítmica de crescimento e no estado VNC. A expressão do gene mreB foi reduzida para ambas as estirpes mutantes (simples e duplo) após 25 dias em condição de estresse. No entanto, o mutante duplo apresentou um aumento na expressão do gene rpoS após 25 dias nesta mesma condição de estresse. As estirpes de Salmonella Enteritidis PT4 578 selvagem e mutantes entraram no estado VNC após incubação em BPS acrescido ou não de cloreto de sódio a 4 °C em diferentes tempos. Estirpes de Salmonella Enteritidis PT4 578 mutantes simples e duplo em fase logarítmica de crescimento apresentaram células filamentosas. Em vez disso, células VNC de mutantes simples e duplo mostraram redução no diâmetro, volume e comprimento e também transição da forma bacilar para a forma cocoide. Concluiu-se que a expressão dos genes mreB e rpoS das estirpes de Salmonella Enteritidis PT4 578 mutantes simples e duplo difere da estirpe selvagem. Houve também mudança da morfologia entre as células da fase log e células VNC das estirpes mutantes de Salmonella Enteritidis PT4 578. No entanto, a molécula de (p)ppGpp não é importante para indução e permanência no estado VNC desta bactéria.Salmonella enterica is a foodborne pathogen able to enter in viable but nonculturable state (VNC) in response to adverse environmental conditions. S. enterica is capable to produce (p)ppGpp, a nucleotide responsible for controlling gene expression at the level of transcription and translation in nutritional stress conditions. Due to gene expression determined by the accumulation of (p)ppGpp, S. enterica supports various environmental conditions. The aims of this work were to quantify by real time PCR the expression of the genes mreB, related to the cytoskeleton, and rpoS, which codifies stress response genes, in single and double mutants of Salmonella Enteritidis PT4 578 deficient in the synthesis of (p)ppGpp, due to the mutation on gene relA, which codifies a GTP pyrophosphokinase, and, or spoT, which codifies guanosine-3',5'-diphosphate 3'-pyrophosphohydrolase, submitted to nutritional stress and cold shock; to induce these strains to VNC state in Butterfield phosphate solution (BPS), in the absence or presence of sodium chloride at 4 ºC and to evaluate the morphology of these strains in logarithmic growth phase and in VNC state. The expression of gene mreB was reduced in both mutant strains (single and double) after 25 days in stress condition. However, the double mutant presented an increase in rpoS gene expression after 25 days in the same stress condition. Wild and mutant strains of Salmonella Enteritidis PT4 578 entered in VNC state after incubation in BPS in the absence or presence of sodium chloride at 4 °C, in different periods of time. Single and double mutant strains of Salmonella Enteritidis PT4 578 in logarithmic growth phase presented filamentous cells. On the other hand, VNC cells of single and double mutants showed a reduction in diameter, volume and length and also transition from bacillary to coccoid form. In conclusion, the expression of mreB and rpoS genes in single and double mutant strains of Salmonella Enteritidis PT4 578 was different when compared to the wild strain. There was also alteration in the morphology of cells in log phase when compared to the VNC cells of mutant strains of Salmonella Enteritidis PT4 578. However, the (p)ppGpp molecule is not essential for induction and permanence of this bacterium in VNC state.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Oil encapsulation techniques using alginate as encapsulating agent: applications and drawbacks

International audienceOils are used in agriculture, nutrition, food and cosmetics; however, these substances are oxidisable and may readily lose their properties. To reduce their degradation or to mask certain undesirable aspects, one strategy consists in encapsulating the oil in inert structures (capsules). The capsules are classified according to the morphology, the number of cores and size, can be produced by several techniques: jet-cutting, vibrating jet, spray-drying, dispersion and milli-microfluidic. Among the polymers used as a membrane in the capsules, alginates are used in oil encapsulation because of their high gelling capacity, biocompatibility and low toxicity. In the presence of calcium ions, the alginate macromolecules crosslink to form a three-dimensional network called hydrogel. The oil encapsulation using alginate as encapsulating material can be carried out using technologies based on the external, internal or inverse gelation mechanisms. These capsules can found applications in areas as cosmetics, textile, foods and veterinary, for example

Potential of the endogenous and artificially inserted CRISPR-Cas system for controlling virulence and antimicrobial resistance of food pathogens

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas) are prokaryotic defenses against viruses and foreign nucleic acids found in many bacterial and archaeal genomes. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. The ability of the CRISPR-Cas systems to perform sequence-specific DNA cleavage evidenced its potential for gene deletion, insertion, or regulation. This allows the food microbiota to be easily genetically modulated, including virulence or resistance gene editing from pathogens which could lead to more safe and high-quality products. This review provides insights into the CRISPR-Cas systems, followed by the understanding of the synergistic or antagonistic relationship of resistance and virulence determinants in foodborne pathogens in connection to their intrinsic CRISPR system. By employing specific examples and recently reported studies this review also widens the discussion of the CRISPR-Cas use for controlling food pathogens by editing genes associated with virulence modulation and reversal of antimicrobial resistance

Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain

Background: Campylobacter jejuni is a leading cause of bacterial enteritis worldwide. This microaerophilic bacterium can survive in aerobic environments, suggesting it has protective mechanisms against oxidative stress. The clinical C. jejuni Bf strain is characterized by an increased resistance to oxygen. This study aimed to characterize the behavior of the clinical C. jejuni Bf strain under an aerobic atmosphere and in response to ROS promoter agents. Methods: Growth was studied in both aerobic and microaerobic conditions using classic cultivable methods. Electronic microscopy and mreB gene expression were used to evaluate the morphology of this strain under aerobic conditions. The survival under oxidative stress was tested in the presence of different concentrations of hydrogen peroxide (H2O2) and paraquat (PQ). Results: The results showed that C. jejuni Bf strain can grow aerobically, unlike other strains of C. jejuni tested. Cells of C. jejuni Bf exposed to oxidative stress presented changes in morphology and the gene mreB, responsible for maintaining the bacillary cell morphology, was down‑expressed. In aerobically acclimated conditions, C. jejuni Bf exhibited a higher survival rate of 52% in the presence of H2O2 (1mM) compared to the reference strain NCTC 11168. Concentrations above 1mM PQ were lethal for the reference strain but not for C. jejuni Bf. Conclusions: Taken together, these data highlight the resistance to oxidative stress conditions of C. jejuni Bf, indicating that this microorganism seems more adapted to survival in hostile environmental conditions

Induction of the viable but nonculturable state of Salmonella enterica serovar Enteritidis deficient in (p)ppGpp synthesis.

Salmonella enterica enters a viable but nonculturable (VBNC) state in adverse environmental conditions. Under nutritional stress, RelA and SpoT proteins synthesize (p)ppGpp, a molecule that controls the expression of genes important for the survival of a cell under those conditions. This study aimed to verify the involvement of (p)ppGpp in the entry of S. enterica serovar Enteritidis PT4 578 cells into the VBNC state by evaluating ?relA and ?relA?spoT mutants. The wild-type and mutant strains deficient in (p)ppGpp synthesis were subjected to osmotic, nutritional and cold stress, after which the cells entered a VBNC state at different time periods, concurrent with reductions in cell diameter, volume and length, and conversion from a bacillary to a coccoid form. No difference in the culturability or cell viability of the wild-type and single and double mutants was observed. Expression of the rpoS gene was increased in the double mutant, while both mutant strains presented a reduction in mreB gene expression after 25 days under conditions of nutritional and cold stress. Surprisingly, (p)ppGpp was not necessary for induction of the VBNC state in Salmonella PT4 578 cells, but may be associated with regulation of genes that control septum formation during cell division, maintaining the bacillary cell morphology (mreB) and stress response (rpoS). Our findings contribute to the understanding of the mechanisms of resistance and survival of Salmonella under adverse conditions

Uso de ferramentas matemáticas em processos de secagem de leite e soro

Em 2014, a produção global de leite foi 892 milhões de toneladas, tendo um aumento de 3,3% comparado a 2013. Devido à perecibilidade do leite, boa parte da produção é destinada à fabricação de produtos lácteos desidratados. Em 2014, aproximadamente 50 milhões de toneladas de leite foram destinados à produção de leite em pó integral (The World Dairy Situation, 2015). Na fabricação deste tipo de produto algumas técnicaspodem ser aplicadas como “roller drying” (secagem em tambor), mas majoritariamente o “spray-drying” (secagem por atomização). Na secagem em tambor, o produto é seco por contato direto com a parede aquecida do rolo de aço inoxidável. Eles são indicados para secagem de alimentos com elevada viscosidade. Tem eficiência energética entre 300 a 1000 KWh.ton-1 de água removida, fácil operação e manutenção (WALSTRA, WOUTERS, GEURTS, 2006). Por outro lado, alguns produtos com elevados teores de açúcar podem ficar aderidos ao rolo ou apresentar sabor cozido devido ao contato direto com a superfície aquecida (JUMING, FENG, SHEN, 2003). Apesar das vantagens de ambas as técnicas, a secagem por atomização é a mais utilizada pelas indústrias de laticínios para fabricação de produtos em pó. O consumo energético deste sistema é aproximadamente um décimo do consumo da liofilização 1000 a 2000 kWh·ton-1 de água removida) e, contrariamente a técnica de secagem em tambor, os produtos não alcançam altas temperaturas durante a evaporação da água. Entretanto, essa ainda é uma das operações unitárias de maior custo energético da indústria de laticínios (SCHUCK et al., 2013; GHARSALLAOUI et al., 2007) e, por essa razão, a otimização dos parâmetros de secagem é a melhor estratégia para diminuir os gastos na produção

Oil encapsulation techniques using alginate as encapsulating agent: applications and drawbacks

International audienceOils are used in agriculture, nutrition, food and cosmetics; however, these substances are oxidisable and may readily lose their properties. To reduce their degradation or to mask certain undesirable aspects, one strategy consists in encapsulating the oil in inert structures (capsules). The capsules are classified according to the morphology, the number of cores and size, can be produced by several techniques: jet-cutting, vibrating jet, spray-drying, dispersion and milli-microfluidic. Among the polymers used as a membrane in the capsules, alginates are used in oil encapsulation because of their high gelling capacity, biocompatibility and low toxicity. In the presence of calcium ions, the alginate macromolecules crosslink to form a three-dimensional network called hydrogel. The oil encapsulation using alginate as encapsulating material can be carried out using technologies based on the external, internal or inverse gelation mechanisms. These capsules can found applications in areas as cosmetics, textile, foods and veterinary, for example

Powder properties and influencing factors

International audience3.1 Water activity is related to water vapor pressure in a material, and it is a useful parameter relating to glass transition, water content and water plasticization. The glass transitions of dairy solids, particularly that of lactose, determine the dehydration characteristics and successful spray-drying conditions of dairy liquids. Water and glass transition relationships assist in the selection of spray-drying parameters as well as required storage conditions. A critical water activity and glass transition-related strength of dairy solids are useful in the formulation of dairy food with desired physicochemical stability in processing and storage.3.2 Caking is a common root of many problems encountered when handling powders and can lead to unwanted shutdowns, client claims and product recycling/rejection due to the clogging of conveying lines, feeding devices and storage silos. Caking can be defined as a formation of material bonds at the points of contact between particles that leads to undesired and uncontrolled agglomeration of powders. It is a very complex process bringing into play a multitude of interactions and mechanisms resulting from variations in environmental conditions, in particular those of temperature and relative humidity. The objective of this chapter is to describe the fundamental mechanisms of powder caking and especially those involved in the caking of dairy powders.3.3 This chapter presents the structural changes at protein/aggregate scale and the changes of functionalities (water-holding capacity, viscosity, foaming, emulsifying and gelling properties) resulting from the pre-texturizing of whey proteins by heating in dry state. The first part of this chapter describes the main chemical/structural modifications occurring during protein dry-heating (limited protein unfolding, Maillard reaction, dehydration reactions) and the specificities of the aggregation reactions that are controlled by molecular mobility (or a w ) and the presence of heat-sensitive compounds (type and amount of sugars). Then, the second part of the chapter describes the functionalities of the pre-texturized whey protein by dry-heating. The change of functionalities can be huge, as shown for the modification of viscosity of protein solutions after whey protein dry-heating at alkaline pH (protein particles are able to trap up to 30g of water per g of protein). The strength of the whey protein gels is increased two-fold by the dry-heating treatment, favoring the formation of soluble aggregates. The change in the foaming properties after whey protein dry-heating is much more limited.3.4 The perceived quality of dairy powders obtained by spray-drying depends on their functional properties, i.e., their ability to flow easily and rehydrate quickly without lump formation. These requirements can be achieved provided that the physicochemical properties of the powders are mastered by an adequate choice of formulation and spray-drying conditions. On one hand, this chapter presents the impact of the spray-drying conditions on the physicochemical characteristics of the resulting dairy powders and highlights the tremendous role of the evaporation rate in the particle structure and surface composition. On the other hand, the influence of the main powder physical properties (particle size, shape, microstructure and surface mechanical properties) on flowing properties and rehydration capacity is discussed through examples of dairy powders produced by spray drying. In order to complete this discussion of the link between spray-drying conditions, physicochemical properties and functionalities, the main analytical methods used to assess spray-dried dairy powder characteristics are briefly reviewed throughout the chapter.3.5 Initial milk contamination may occur due to intramammary infections, microorganisms on the external udder surface, poorly sanitized handler hands and milking equipment. During the refrigeration, storage and transport stages, lapses in hygienic-sanitary conditions can lead to milk contamination from pathogenic and spoilage microorganisms.Once raw milk reaches the processing plant for dehydration, it undergoes processes including cooling, heat treatments, evaporative procedures and membrane filtration in order to inhibit microbial growth and destroy microorganisms. Following thermal treatments and membrane filtration, caution should be taken to ensure the hygienic-sanitary conditions of equipment and utensils to avoid product recontamination. All the stages of dried dairy products (from milking to packaging) should be monitored to guarantee the quality and microbiological safety of these foods

Powder properties and influencing factors

International audience3.1 Water activity is related to water vapor pressure in a material, and it is a useful parameter relating to glass transition, water content and water plasticization. The glass transitions of dairy solids, particularly that of lactose, determine the dehydration characteristics and successful spray-drying conditions of dairy liquids. Water and glass transition relationships assist in the selection of spray-drying parameters as well as required storage conditions. A critical water activity and glass transition-related strength of dairy solids are useful in the formulation of dairy food with desired physicochemical stability in processing and storage.3.2 Caking is a common root of many problems encountered when handling powders and can lead to unwanted shutdowns, client claims and product recycling/rejection due to the clogging of conveying lines, feeding devices and storage silos. Caking can be defined as a formation of material bonds at the points of contact between particles that leads to undesired and uncontrolled agglomeration of powders. It is a very complex process bringing into play a multitude of interactions and mechanisms resulting from variations in environmental conditions, in particular those of temperature and relative humidity. The objective of this chapter is to describe the fundamental mechanisms of powder caking and especially those involved in the caking of dairy powders.3.3 This chapter presents the structural changes at protein/aggregate scale and the changes of functionalities (water-holding capacity, viscosity, foaming, emulsifying and gelling properties) resulting from the pre-texturizing of whey proteins by heating in dry state. The first part of this chapter describes the main chemical/structural modifications occurring during protein dry-heating (limited protein unfolding, Maillard reaction, dehydration reactions) and the specificities of the aggregation reactions that are controlled by molecular mobility (or a w ) and the presence of heat-sensitive compounds (type and amount of sugars). Then, the second part of the chapter describes the functionalities of the pre-texturized whey protein by dry-heating. The change of functionalities can be huge, as shown for the modification of viscosity of protein solutions after whey protein dry-heating at alkaline pH (protein particles are able to trap up to 30g of water per g of protein). The strength of the whey protein gels is increased two-fold by the dry-heating treatment, favoring the formation of soluble aggregates. The change in the foaming properties after whey protein dry-heating is much more limited.3.4 The perceived quality of dairy powders obtained by spray-drying depends on their functional properties, i.e., their ability to flow easily and rehydrate quickly without lump formation. These requirements can be achieved provided that the physicochemical properties of the powders are mastered by an adequate choice of formulation and spray-drying conditions. On one hand, this chapter presents the impact of the spray-drying conditions on the physicochemical characteristics of the resulting dairy powders and highlights the tremendous role of the evaporation rate in the particle structure and surface composition. On the other hand, the influence of the main powder physical properties (particle size, shape, microstructure and surface mechanical properties) on flowing properties and rehydration capacity is discussed through examples of dairy powders produced by spray drying. In order to complete this discussion of the link between spray-drying conditions, physicochemical properties and functionalities, the main analytical methods used to assess spray-dried dairy powder characteristics are briefly reviewed throughout the chapter.3.5 Initial milk contamination may occur due to intramammary infections, microorganisms on the external udder surface, poorly sanitized handler hands and milking equipment. During the refrigeration, storage and transport stages, lapses in hygienic-sanitary conditions can lead to milk contamination from pathogenic and spoilage microorganisms.Once raw milk reaches the processing plant for dehydration, it undergoes processes including cooling, heat treatments, evaporative procedures and membrane filtration in order to inhibit microbial growth and destroy microorganisms. Following thermal treatments and membrane filtration, caution should be taken to ensure the hygienic-sanitary conditions of equipment and utensils to avoid product recontamination. All the stages of dried dairy products (from milking to packaging) should be monitored to guarantee the quality and microbiological safety of these foods