Creativity in Scientific Research: Multidisciplinarity Fosters Depth of Ideas Among Scientists in Electronic "Brainwriting" Groups.

OBJECTIVE: The aim of this study was to examine the potential benefits of multidisciplinarity among agri-food researchers working in small groups to generate ideas to stimulate innovation in the context of a laboratory project. BACKGROUND: Research on the role of multidisciplinarity in scientific research teams remains limited, particularly regarding the generation of ideas to innovate in a real laboratory project, and on a task with a real challenge for innovation. METHOD: Researchers and agri-food research staff were assigned to small groups of either multidisciplinary or unidisciplinary composition to produce ideas on a cross-cutting theme for an innovative laboratory project using an electronic "brainwriting" application. RESULTS: A greater depth in idea generation (number of ideas per category) was observed in the multidisciplinary condition than in the unidisciplinary condition. CONCLUSION: The main benefits of this study were to experimentally examine the effects of multidisciplinarity in small scientific research groups on the production of ideas in a field study conducted on the premises of an agri-food laboratory. APPLICATION: This study provides advice on how to promote innovative projects by stimulating ideation processes, which includes constructing small multidisciplinary groups and using an electronic "brainwriting" technique.

Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese.

In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening.

Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. cremoris during milk fermentation.

BACKGROUND: Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance. This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. cremoris MG1363 at the transcriptional and metabolic levels in relation to acidification kinetics and growth conditions, especially at an early stage of growth. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. RESULTS: Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L. lactis MG1363 strain. Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. lactis MG1363 strain was supported by a coherent and early adaptation to oxygen after 1 hour of culture at both gene expression and metabolic levels. In oxygen metabolism, the over-expression of all the genes of the nrd (ribonucleotide reductases) operon or fhu (ferrichrome ABC transports) genes was particularly significant. In carbon metabolism, the presence of oxygen led to an early shift at the gene level in the pyruvate pathway towards the acetate/2,3-butanediol pathway confirmed by the kinetics of metabolite production. Finally, the MG1363 strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage. CONCLUSIONS: Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. lactis to initial oxidative stress. An early and transitional adaptation to oxidative stress was revealed for L. lactis subsp. cremoris MG1363 in the presence of initially high levels of oxygen. This enables the cells to maintain key traits that are of great importance for industry, such as rapid acidification and reduction of the redox potential of the growth media.

Contribution of Lactococcus lactis reducing properties to the downregulation of a major virulence regulator in Staphylococcus aureus, the agr system.

Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.

Microgradients of pH do not occur around Lactococcus colonies in a model cheese.

Lactococci inoculated into cheese grow as colonies producing lactic acid. The pH microgradients were investigated around colonies in a complex food such as cheese. The results, obtained using a nondestructive technique, demonstrated that pH microgradients did not occur regardless of the acidification kinetics and the size of the colony.

Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712.

The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, d-lactate dehydrogenase and α-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus.

The efficacy of nisin can drastically vary when produced in situ in model cheeses.

Nisin, a bacteriocin produced by strains of Lactococcus lactis, has a broad inhibitory effect against Gram-positive bacteria. This study investigated the efficacy of nisin Z against Lactobacillus sakei when produced by a nisin-producing strain L. lactis in model cheeses manufactured with ultrafiltrated milk. These cheeses, containing 0, 4 or 10% of gelatin in their dry matter, were inoculated with both strains. Measurement of Lb. sakei loss of viability was an indirect indicator of nisin in situ efficacy. After 24 h, the loss of viability of Lb. sakei was from 0.73 ± 0.14 to 3.30 ± 0.60 log(10) cfu g(-1) in the cheeses with 0 and 10% of gelatin, respectively, indicating a better in situ efficacy of nisin when gelatin was incorporated. However, the concentration of nisin produced by Lactococcus was similar (3.5 µg g(-1)) in all model cheeses when measured using an enzyme-linked immune sorbent assay (ELISA). The growth of Lactococcus was slightly improved when gelatin was incorporated, leading to a higher lactate concentration, which is one of the factors explaining the increased nisin efficacy. These results reinforced previous observations that prediction of nisin efficacy in complex food systems remains difficult.

Dynamic analysis of the Lactococcus lactis transcriptome in cheeses made from milk concentrated by ultrafiltration reveals multiple strategies of adaptation to stresses.

Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic- and oxidative-stress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions.

Colonial vs. planktonic type of growth: mathematical modeling of microbial dynamics on surfaces and in liquid, semi-liquid and solid foods.

Predictive models are mathematical expressions that describe the growth, survival, inactivation, or biochemical processes of foodborne bacteria. During processing of contaminated raw materials and food preparation, bacteria are entrapped into the food residues, potentially transferred to the equipment surfaces (abiotic or inert surfaces) or cross-contaminate other foods (biotic surfaces). Growth of bacterial cells can either occur planktonically in liquid or immobilized as colonies. Colonies are on the surface or confined in the interior (submerged colonies) of structured foods. For low initial levels of bacterial population leading to large colonies, the immobilized growth differs from planktonic growth due to physical constrains and to diffusion limitations within the structured foods. Indeed, cells in colonies experience substrate starvation and/or stresses from the accumulation of toxic metabolites such as lactic acid. Furthermore, the micro-architecture of foods also influences the rate and extent of growth. The micro-architecture is determined by (i) the non-aqueous phase with the distribution and size of oil particles and the pore size of the network when proteins or gelling agent are solidified, and by (ii) the available aqueous phase within which bacteria may swarm or swim. As a consequence, the micro-environment of bacterial cells when they grow in colonies might greatly differs from that when they grow planktonically. The broth-based data used for modeling (lag time and generation time, the growth rate, and population level) are poorly transferable to solid foods. It may lead to an over-estimation or under-estimation of the predicted population compared to the observed population in food. If the growth prediction concerns pathogen bacteria, it is a major importance for the safety of foods to improve the knowledge on immobilized growth. In this review, the different types of models are presented taking into account the stochastic behavior of single cells in the growth of a bacterial population. Finally, the recent advances in the rules controlling different modes of growth, as well as the methodological approaches for monitoring and modeling such growth are detailed.

Bacterial Colonies in Solid Media and Foods: A Review on Their Growth and Interactions with the Micro-Environment.

Bacteria, either indigenous or added, are immobilized in solid foods where they grow as colonies. Since the 80's, relatively few research groups have explored the implications of bacteria growing as colonies and mostly focused on pathogens in large colonies on agar/gelatine media. It is only recently that high resolution imaging techniques and biophysical characterization techniques increased the understanding of the growth of bacterial colonies, for different sizes of colonies, at the microscopic level and even down to the molecular level. This review covers the studies on bacterial colony growth in agar or gelatine media mimicking the food environment and in model cheese. The following conclusions have been brought to light. Firstly, under unfavorable conditions, mimicking food conditions, the immobilization of bacteria always constrains their growth in comparison with planktonic growth and increases the sensibility of bacteria to environmental stresses. Secondly, the spatial distribution describes both the distance between colonies and the size of the colonies as a function of the initial level of population. By studying the literature, we concluded that there systematically exists a threshold that distinguishes micro-colonies (radius < 100-200 µm) from macro-colonies (radius >200 µm). Micro-colonies growth resembles planktonic growth and no pH microgradients could be observed. Macro-colonies growth is slower than planktonic growth and pH microgradients could be observed in and around them due to diffusion limitations which occur around, but also inside the macro-colonies. Diffusion limitations of milk proteins have been demonstrated in a model cheese around and in the bacterial colonies. In conclusion, the impact of immobilization is predominant for macro-colonies in comparison with micro-colonies. However, the interaction between the colonies and the food matrix itself remains to be further investigated at the microscopic scale.

Diffusion of solutes inside bacterial colonies immobilized in model cheese depends on their physicochemical properties: a time-lapse microscopy study.

During cheese processing and ripening, bacteria develop as colonies. Substrates and metabolites must then diffuse either from or into the colonies. Exploring how the inner cells of the colony access the substrates or get rid of the products leads to study the diffusion of solutes inside bacterial colonies immobilized in cheese. Diffusion limitations of substrates within the bacterial colony could lead to starvation for the cells in the center of the colony. This study aimed at better understands ripening at the colony level, by investigating how diffusion phenomena inside colonies vary depending on both the physicochemical properties of the solutes and Lactococcus lactis strain. Dextrans (4, 70, and 155 kDa) and milk proteins (BSA, lactoferrin and αS1-casein) of different sizes and physicochemical properties were chosen as model of diffusing solutes, and two L. lactis strains presenting different surface properties were immobilized as colonies in a model cheese. Diffusion of solutes inside and around colonies was experimentally followed by time-lapse confocal microscopy. Dextran solutes diffused inside both lactococci colonies with a non-significantly different effective diffusion coefficient, which depended mainly on size of the solute. However, whereas flexible and neutral hydrophilic polymers such as dextran can diffuse inside colonies whatever its size, none of the three proteins investigated in this study could penetrate inside lactococci colonies. Therefore, the diffusion behavior of macromolecules through bacterial colonies immobilized in a model cheese did not only depends on the size of the diffusing solutes, but also and mainly on their physicochemical properties. Milk caseins are probably first hydrolyzed by the cell wall proteases of L. lactis and/or other proteases present in the cheese, and then the generated peptides diffuse inside colonies to be further metabolized into smaller peptides and amino acids by all the cells located inside the colonies.

Nisin quantification by ELISA allows the modeling of its apparent diffusion coefficient in model cheeses.

The diffusion of small solutes in cheese is of key importance for most enzymatic reactions involved in the ripening process. However, only a limited amount of data is available on salt diffusion and practically none on peptide diffusion. Nisin, a bacteriocin peptide, migrated in model cheeses made from ultrafiltered (UF) retentate. A profile concentration device and an enzyme-linked immunosorbent assay (ELISA), specifically developed for nisin quantification in cheese, were used to model the apparent diffusion coefficients for nisin according to Fick's law. This average coefficient was 49.5 µm(2)/s in UF cheese (n = 2). When 10% gelatin was added to the retentate, this value decreased to 34.4 µm(2)/s (n = 2). The two cheeses differed in their macrostructure (rheology) and microstructure (confocal microscopy). This study provides the first apparent diffusion coefficients for a peptide in cheese and supports the hypothesis that composition and structure influence the diffusion of small solutes such as peptides.

Milk acidification by Lactococcus lactis is improved by decreasing the level of dissolved oxygen rather than decreasing redox potential in the milk prior to inoculation.

Although redox potential is very rarely taken into account in food fermentation it could be as influential as pH on bacterial activities. Lactococcus lactis is already known to exhibit a powerful reducing activity in milk but its reduction activity was shown to occur prior to its acidification activity with a potential interaction between these two lactococcal activities. Therefore, acidification lag-type phase could be shortened by decreasing the redox potential of milk before inoculation. As the redox potential is highly dependent on the dissolved oxygen level, our objective was to study their separate and combined influences on acidification and growth kinetics of pure L. lactis strains in milk. Results showed that high level of dissolved oxygen is significantly more influential on growth, and even more on acidification kinetics, than initial decreased redox potential of milk. Reduction of milk was drastic and mostly due to bacterial activity. The redox potential of milk only dropped when dissolved oxygen was entirely consumed. When there was no dissolved oxygen from the beginning, L. lactis immediately decreased the redox potential of milk and acidified afterwards. When the level of dissolved oxygen was initially high, acidification and reduction of milk occurred at the same time. Acidification kinetics was then biphasic with a slower rate during the aerobic stage and a faster rate during the anaerobic stage. The seven strains tested demonstrated diversity in both their acidification kinetics and their adaptation to high level of dissolved oxygen, independent of their growth kinetics. To conclude, we have shown that the level of dissolved oxygen in milk has a dramatic influence on acidification kinetics and could be used to control acidification kinetics in dairy industries.