Table_1_Antifungal Activity of Lactic Acid Bacteria Combinations in Dairy Mimicking Models and Their Potential as Bioprotective Cultures in Pilot Scale Applications.docx

<p>Consumer's demand for naturally preserved food products is growing and the use of bioprotective cultures is an alternative to chemical preservatives or a complementary tool to hurdle technologies to avoid or delay fungal spoilage of dairy products. To develop antifungal cultures for the dairy product biopreservation, experiments were conducted both in vitro and in situ. Firstly, the antifungal activity of 32 strains of lactic acid bacteria (LAB) and propionibacteria was screened alone, and then on combinations based on 5 selected lactobacilli strains. This screening was performed in yogurt and cheese models against four major spoilage fungi previously isolated from contaminated dairy products (Penicillium commune, Mucor racemosus, Galactomyces geotrichum, and Yarrowia lipolytica). Selected combinations were then tested as adjunct cultures in sour cream and semi-hard cheeses produced at a pilot scale to evaluate their antifungal activity during challenge tests against selected fungal targets (P. commune, M. racemosus, and Rhodotorula mucilaginosa) and shelf life tests; and their impact on product organoleptic properties. The screening step allowed selecting two binary combinations, A1 and A3 composed of Lactobacillus plantarum L244 and either Lactobacillus harbinensis L172 or Lactobacillus rhamnosus CIRM-BIA1113, respectively. In situ assays showed that the A1 combination delayed the growth of P. commune, M. racemosus and R. mucilaginosa for 2–24 days on sour cream depending of the antifungal culture inoculum, without effect on organoleptic properties at low inoculum (10⁶ colony-forming units (CFU)/mL). Moreover, the A1 and A3 combinations also delayed the growth of P. commune in semi-hard cheese for 1–6 days and 1 day, respectively. Antifungal cultures neither impacted the growth of starter cultures in both sour cream and cheese nor the products' pH, although post acidification was observed in sour cream supplemented with these combinations at the highest concentrations (2.10⁷ CFU/mL). The combination of both in vitro and in situ screening assays allowed developing 2 antifungal combinations exhibiting significant antifungal activity and providing future prospects for use as bioprotective cultures in dairy products.</p

Time-course comparison of transcriptome and proteome changes in <i>P. freudenreichii</i> CIRM-BIA1^T strain, for genes involved in metabolic categories CD, CE, DNA, E, L, Mi, Nt, and P.

a<p>Spot number (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone.0029083.s006" target="_blank">Table S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone-0029083-g002" target="_blank">Fig. 2</a>).</p><p>Bold characters indicate genes that are differentially expressed according to microarray experiments (<i>P</i><0.05 and |fold change|>1 for at least one sampling time) and RT-qPCR (<i>P</i><0.05) experiments.</p

Time-course comparison of transcriptome and proteome changes in <i>P. freudenreichii</i> CIRM-BIA1^T strain, for genes involved in metabolic category CH.

a<p>Spot number (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone.0029083.s006" target="_blank">Table S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone-0029083-g002" target="_blank">Fig. 2</a>).</p><p>Bold characters indicate genes that are differentially expressed according to microarray experiments (<i>P</i><0.05 and |fold change|>1 for at least one sampling time) and RT-qPCR (<i>P</i><0.05) experiments.</p

Time-course of <i>P. freudenreichii</i> CIRM-BIA1^T metabolic activity over a 40 h-incubation at 30°C followed by a further 9 days at 4°C.

<p>Lactate was added at 64 h (<b>↓</b>) to mimic cheese ripening conditions. <b>A</b>, growth monitored by optical density (650 nm) measurements (black circle), lactate consumption (cross), production of acetate (white triangle) and propionate (black triangle); <b>B</b>, production of pyruvate (white rhombus) and succinate (black rhombus), consumption of aspartate (white triangle) and asparagine (black triangle); <b>C</b>, production of methylbutanoate (sum of 2-methyl- and 3-methylbutanoate acids). ¥: sampling times for microarray experiments.</p

Time-course comparison of transcriptome and proteome changes in <i>P. freudenreichii</i> CIRM-BIA1^T strain, for genes involved in metabolic category Ph and PM.

a<p>Spot number (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone.0029083.s006" target="_blank">Table S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone-0029083-g002" target="_blank">Fig. 2</a>).</p><p>Bold characters indicate genes that are differentially expressed according to microarray experiments (<i>P</i><0.05).</p

Schematic representation of carbon metabolism in <i>P. freudenreichii</i> CIRM-BIA1^T during storage at 4°C and relevant for this study.

<p>Protein and gene names are written in plain and in italic fonts, respectively. The color indicates the change in expression at 4°C, compared to reference time (exponential growth at 30°C, 20 h): in red, genes up-regulated at 4°C using microarray and RT-qPCR data and protein present at larger amounts; in pink, genes up-regulated at 4°C using microarray results only; in blue, genes down-regulated at 4°C using microarray and RT-qPCR data; in green, genes up-regulated at 4°C using microarray data only; in black, genes not being differentially expressed at 4°C using microarray data. * genes up-regulated using RT-qPCR data, but not significantly affected using microarray experiments. Red lines emphasize the metabolic pathways that seem to be favored at 4°C in <i>P. freudenreichii</i> considering the proteomic and transcriptomic results. Plain lines are used to symbolize one reaction, dotted lines several reactions (intermediary products not detailed). Gene and protein abbreviations are as follow: <i>acn</i> (aconitase), Ald/<i>ald</i> (alanine dehydrogenase), AspA2/<i>aspA2</i> (aspartate ammonia-lyase), Cat/<i>cat</i> (coenzyme A transferase), <i>dld</i> (D-lactate dehydrogenase), Eno1/e<i>no1</i> (enolase 1), <i>fba2</i> (fructose-bisphosphate aldolase class I), FumC/<i>fumC</i> (fumarate hydratase class-II), <i>glgA</i> (glycosyltransferase), <i>glgC</i> (glucose-1-P adenylyltransferase), <i>gltA1,2</i> (citrate synthases), <i>icd</i> (putative isocitrate/isopropylmalate dehydrogenase), <i>ldh</i> (L-lactate dehydrogenase), <i>mdh</i> (malate dehydrogenase), MmdA/<i>mmdA</i> (methylmalonyl-CoA carboxytransferase 12S subunit), MutA/<i>mutA</i> (methylmalonyl-CoA mutase small subunit), MutB/<i>mutB</i> (methylmalonyl-CoA mutase large subunit), <i>pccB</i> (propionyl-CoA carboxylase β-chain), Pgi/<i>pgi</i> (glucose-6-P isomerase), Pgm1/<i>pgm1</i> (phosphoglucomutase), PpdK/<i>ppdk</i> (pyruvate phosphate dikinase), <i>ptsI</i> (PTS enzyme I), <i>sdaA</i> (L-serine dehydratase), <i>sdhA</i> (succinate dehydrogenase, subunit A), <i>sdhA3</i> (succinate dehydrogenase, flavoprotein subunit), <i>sdhB</i> (succinate dehydrogenase, subunit B), <i>sdhB3</i> (succinate dehydrogenase), <i>sdhC1</i> (succinate dehydrogenase, subunit C), <i>sdhC2</i> (succinate dehydrogenase, cytochrome B-558 subunit), ?: undefined protein (probable phosphoenolpyruvic carboxylase).</p

Time-course comparison of transcriptome and proteome changes in <i>P. freudenreichii</i> CIRM-BIA1^T strain, for genes involved in metabolic categories A, AA and C.

a<p>Spot number (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone.0029083.s006" target="_blank">Table S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone-0029083-g002" target="_blank">Fig. 2</a>).</p><p>Bold characters indicate genes that are differentially expressed according to microarray experiments (<i>P</i><0.05 and |fold change|>1 for at least one sampling time) and RT-qPCR (<i>P</i><0.05) experiments.</p

Indicators of <i>P. freudenreichii</i> CIRM-BIA1^T strain metabolism during a 40 h-incubation at 30°C followed by 9 days at 4°C.

a<p>Methylbutanoate is sum of 2-methylbutanoate and 3-methylbutanoate.</p>b<p>Not detected.</p>c<p>Values at 30°C and 4°C significantly differed at <i>P</i> = 0.09.</p>d<p>Values at 30°C and 4°C significantly differed at P = 0.12.</p><p>Values are means and standard deviations of three independent experiments.</p

Figure 2

<p>Two-dimensional analysis of proteins produced during <i>P. freudenreichii</i> CIRM-BIA1^T growth (<b>A</b>) at 30°C (reference time 20 h) and then (<b>B</b>) at 4°C (3 days). Numbers identify spots which volume decreased at 4°C (<b>A</b>), or increased at 4°C (<b>B</b>). The identification by MS/MS of each spot can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029083#pone.0029083.s006" target="_blank">Table S4</a>.</p

Additional file 2: Table S2. of Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage

List of genes induced in the stationary phase (3d post-inoculation) compared to the exponential phase (1d post-inoculation) (P value adjusted ≤0.05). (XLSX 98 kb