Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods

Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102107 pfu.mL1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). Franklin L. Nobrega and Ana Rita Costa acknowledge FCT for grants SFRH/BD/86462/2012 and SFRH/BPD/94648/2013, respectively. Melvin F. Siliakus acknowledges funding from the Biobased Ecologically Balanced Sustainable Industrial Chemistry (BE-BASIC) foundation. Electron microscopy work was performed at the Wageningen Electron Microscopy Centre (WEMC) of Wageningen University

Prospects for Food Fermentation in South-East Asia, Topics From the Tropical Fermentation and Biotechnology Network at the End of the AsiFood Erasmus+Project

Fermentation has been used for centuries to produce food in South-East Asia and some foods of this region are famous in the whole world. However, in the twenty first century, issues like food safety and quality must be addressed in a world changing from local business to globalization. In Western countries, the answer to these questions has been made through hygienisation, generalization of the use of starters, specialization of agriculture and use of long-distance transportation. This may have resulted in a loss in the taste and typicity of the products, in an extensive use of antibiotics and other chemicals and eventually, in a loss in the confidence of consumers to the products. The challenges awaiting fermentation in South-East Asia are thus to improve safety and quality in a sustainable system producing tasty and typical fermented products and valorising by-products. At the end of the “AsiFood Erasmus+ project” (www.asifood.org), the goal of this paper is to present and discuss these challenges as addressed by the Tropical Fermentation Network, a group of researchers from universities, research centers and companies in Asia and Europe. This paper presents current actions and prospects on hygienic, environmental, sensorial and nutritional qualities of traditional fermented food including screening of functional bacteria and starters, food safety strategies, research for new antimicrobial compounds, development of more sustainable fermentations and valorisation of by-products. A specificity of this network is also the multidisciplinary approach dealing with microbiology, food, chemical, sensorial, and genetic analyses, biotechnology, food supply chain, consumers and ethnology

Non-DLVO adhesion of F-specific RNA bacteriophages to abiotic surfaces: Importance of surface roughness, hydrophobic and electrostatic interactions

International audienceWe report on the adhesion features of three F-specific RNA-phages (MS2, GA and Q beta) onto surfaces (1-dodecanethiol gold-coated surface, glass, polypropylene and stainless steel) that significantly differ with regard to their surface roughness and hydrophobic/hydrophilic balance. The number of adhered phages on the surfaces is quantified using RT-PCR. Adhesion experiments are performed under static conditions in the absence of bulk phages aggregation (1 mM and 100 mM NaNO3 electrolyte, pH 7). The nanoscale surface features and the hydrophobicity of the deposition substrates are quantitatively addressed using Atomic Force Microcopy and Chemical Force Microscopy. The hydrophobicity of the phages is evaluated from their propensity to adhere onto flat and highly hydrophobic surface. Results show that, regardless of electrolyte concentration and surface roughness, the adhesion capacity of phages systematically follows the hydrophobicity sequence MS2 < Q beta < GA. The capacity of each phage to adhere onto the surfaces increases with increase in the degree of hydrophobicity and/or the roughness of the deposition substrate. Increasing the electrostatic interactions between phages and deposition surface by decreasing solution ionic strength leads to a reduction in surface concentration of adhered phages except for cases where the roughness of the deposition surface is significant. Altogether, the results obtained confirm the limited ability of the classical DLVO theory to predict the adhesion of complex viral (nano)particles to surfaces and emphasize the necessity to take into account the roughness of the deposition substrate and the hydrophobicity degree of both viruses and surfaces

Direct Quantitative Detection and Identification of Lactococcal Bacteriophages from Milk and Whey by Real-Time PCR: Application for the Detection of Lactococcal Bacteriophages in Goat's Raw Milk Whey in France

The presence of Lactococcus bacteriophages in milk can partly or completely inhibit milk fermentation. To prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups of Lactococcus bacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 102 PFU/mL. The curve slopes were −3.49, −3.69, and −3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient (2) of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. This method was then used to detect the bacteriophages in whey and goat's raw milk coming from three farms located in the Rhône-Alpes region (France)

Antiviral effect of cationic compounds on bacteriophages

The antiviral activity of several cationic compounds - cetytrimethylammonium (CTAB), chitosan, nisin and lysozyme - was investigated on the bacteriophage c2 (DNA head and non-contractile tail) infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA) infecting E.coli. Firstly, these activities were evaluated in a phosphate buffer pH 7- 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 log(pfu)/mL to 1,5 log(pfu)/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min). These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds

Influence of the Backslopping Practice on the Microbial Diversity of the &lt;i&gt;Lactococcus&lt;/i&gt; Population in a Model Cheesemaking

International audienc