Strains of the Lactobacillus casei group show diverse abilities for the production of flavor compounds in 2 model systems

peer-reviewedCheese flavor development is directly connected to the metabolic activity of microorganisms used during its manufacture, and the selection of metabolically diverse strains represents a potential tool for the production of cheese with novel and distinct flavor characteristics. Strains of Lactobacillus have been proven to promote the development of important cheese flavor compounds. As cheese production and ripening are long-lasting and expensive, model systems have been developed with the purpose of rapidly screening lactic acid bacteria for their flavor potential. The biodiversity of 10 strains of the Lactobacillus casei group was evaluated in 2 model systems and their volatile profiles were determined by gas chromatography-mass spectrometry. In model system 1, which represented a mixture of free AA, inoculated cells did not grow. In total, 66 compounds considered as flavor contributors were successfully identified, most of which were aldehydes, acids, and alcohols produced via AA metabolism by selected strains. Three strains (DPC2071, DPC3990, and DPC4206) had the most diverse metabolic capacities in model system 1. In model system 2, which was based on processed cheese curd, inoculated cells increased in numbers over incubation time. A total of 47 compounds were identified, and they originated not only from proteolysis, but also from glycolytic and lipolytic processes. Tested strains produced ketones, acids, and esters. Although strains produced different abundances of volatiles, diversity was less evident in model system 2, and only one strain (DPC4206) was distinguished from the others. Strains identified as the most dissimilar in both of the model systems could be more useful for cheese flavor diversification

Identification of the bacteria and their metabolic activities associated with the microbial spoilage of custard cream desserts

The famous French dessert “ile flottante” consists of a sweet egg white foam floating on a vanilla custard cream,which contains highly nutritive raw materials, including milk, sugar and egg. Spoilage issues are therefore a keyconcern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream dessertsmanufactured by 2 French companies. B. cereus group bacteria, coagulase negative Staphylococcus, Enterococcus and Leuconostoc spp. were isolated from spoiled products. Thirty-one bacterial isolates representative of the main spoilage species were tested for their spoilage abilities. Significant growth and pH decrease were observed regardless of species. While off-odours were detected with B. cereus group and staphylococci, yoghurt odours were detected with Enterococcus spp. and Leuconostoc spp. B. cereus group bacteria produced various esters and several compounds derived from amino acid and sugar metabolism. Most Staphylococci produced phenolic compounds. Enterococcus spp. and Leuconostoc spp. isolates produced high levels of compounds derived from sugar metabolism. Each type of spoilage bacteria was associated with a specific volatile profile and lactic acid was identified as a potential marker of spoilage of custard cream-based desserts. These findings provide valuable information for manufacturers to improve food spoilage detection and prevention of chilled desserts made with milk and egg

New insights about phenotypic heterogeneity within Propionibacterium freudenreichii argue against its division into subspecies

Propionibacterium freudenreichii is widely used in Swiss-type cheese manufacture, where it contributes to flavour and eye development. It is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit- and lac-/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac-/nit-) has also been reported. The aim of this study was to revisit the relevance of the subdivision of P. freudenreichii into subspecies, by confirming the existence of unclassifiable strains. Relevant conditions to test the ability of P. freudenreichii for lactose fermentation and nitrate reduction were first determined, by using 10 sequenced strains, in which the presence or absence of the lactose and nitrate genomic islands were known. We also determined whether the subdivision based on lac/nit phenotype was related to other phenotypic properties of interest in cheese manufacture, in this case, the production of aroma compounds, analysed by gas chromatography-mass spectrometry, for a total of 28 strains. The results showed that a too short incubation time can lead to false negative for lactose fermentation and nitrate reduction. They confirmed the existence of four lac/nit phenotypes instead of the two expected, thus leading to 13 unclassifiable strains out of the 28 characterized (7 lac+/nit+ and 6 lac-/nit-). The production of the 15 aroma compounds detected in all cultures varied more within a lac/nit phenotype (up to 20 times) than between them. Taken together, these results demonstrate that the division of P. freudenreichii into two subspecies does not appear to be relevant.Fil: de Freitas, Rosangela. Universidade Federal de Viçosa. Departamento de Tecnologia de Alimentos; Brasil. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Madec, Marie Noelle. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Chuat, Victoria. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Maillard, Marie Bernadette. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Falentin, Hélène. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Carvalho, Antonio Fernandes de. Universidade Federal de Viçosa. Departamento de Tecnologia de Alimentos; BrasilFil: Valence, Florence. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Thierry, Anne. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; Franci

Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix

International audienceIn complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS

A Temporal -omic Study of Propionibacterium freudenreichii CIRM-BIA1T Adaptation Strategies in Conditions Mimicking Cheese Ripening in the Cold

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It grows when cheeses are ripened in a warm room (about 24°C). Cheeses with an acceptable eye formation level are transferred to a cold room (about 4°C), inducing a marked slowdown of propionic fermentation, but P. freudenreichii remains active in the cold. To investigate the P. freudenreichii strategies of adaptation and survival in the cold, we performed the first global gene expression profile for this species. The time-course transcriptomic response of P. freudenreichii CIRM-BIA1T strain was analyzed at five times of incubation, during growth at 30°C then for 9 days at 4°C, under conditions preventing nutrient starvation. Gene expression was also confirmed by RT-qPCR for 28 genes. In addition, proteomic experiments were carried out and the main metabolites were quantified. Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from 30°C to 4°C (P<0.05 and |fold change|>1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1T strain over-expressed genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis, and in glycogen synthesis. Interestingly, the expression of different genes involved in the formation of important cheese flavor compounds, remained unchanged at 4°C. This could explain the contribution of P. freudenreichii to cheese ripening even in the cold. In conclusion, P. freudenreichii remains metabolically active at 4°C and induces pathways to maintain its long-term survival

The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications

Background: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings: The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters. Conclusions/Significance: With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity

Conversion of l-Leucine to Isovaleric Acid by Propionibacterium freudenreichii TL 34 and ITGP23

Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of l-leucine, α-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced α-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal norα-hydroxyisocaproic acid was detected. l-Isoleucine and l-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening

Detection of aminotransferase activity of Propionibacterium freudenreichii after SDS-PAGE

Aminotransferases (ATs) had previously been detected after native electrophoresis. We show now that aminotransferase(s) of Propionibacterium freudenreichii can be detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, it retained a high activity (84%) in the presence of 0.23% SDS, contrary to what was observed for aminotransferase(s) of Bifidobacterium bifidum (54%) and of six other cheese-related species (0–20%)

Efficient mechanical disruption of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity

Microbiological studies often involve bacterial cell fractionation, which is known to be difficult for Gram-positive as compared to Gram-negative bacteria. Our purpose was to test the breaking efficiency of a new high-pressure pilot homogenizer for three Gram-positive species involved in dairy technology and to assess theactivity of an intracellular aminotransferase. Varied pressures (50, 100 and 200MPa) were applied toconcentrated bacterial suspensions (1.2mg dry weight/ ml) of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii. Breaking efficiency was estimated by decreases in optical density at 650nm,cellular dry weight and viability. The proteins released were quantified and the residual intracellular aminotransferase activity was estimated using leucine as substrate. One run at 50MPa was sufficient to break 80% of lactobacilli cells whereas 200MPa were required for the same efficiency for L. lactis and P. freudenreichii. Whatever the pressure, leucine aminotransferase activity was recovered in the supernatant after cell breaking. This new high-pressure pilot homogenizer can allow rapid (20s/run), easy, continuous and highly efficient cellbreaking for intracellular enzyme recovery or other purposes. As the species tested were not phylogeneticallyrelated, and had different morphologies and cell wall compositions, we conclude that most Gram-positivebacteria may be broken efficiently by this new device

Production of branched-chain aroma compounds by Propionibacterium freudenreichii: links with the biosynthesis of membrane fatty acids

Aims: Short branched-chain fatty acids (BCFAs) are cheese flavour compounds, which result from the conversion of branched-chain amino acids (BCAAs). In Swiss cheese, the production of short BCFAs is mainly performed by Propionibacterium freudenreichii and is strain dependent. Our aim was to investigate the possible links between the biosynthesis of short BCFAs and membrane BCFAs in P. freudenreichii. Methods and Results: Short and membrane BCFAs were analysed by gas chromatography- mass spectrometry. Two strains differing in their capacities to release short BCFAs were selected. Tri-deuterated-labelled leucine was used in both strains as a precursor of short extracellular iso-BCFAs and of membrane iso-BCFAs. The proportions of anteiso : iso BCFAs synthesized varied as function of the BCAAs provided in the growth medium, from 72 : 28 to 100 : 0, with leucine and valine, and with isoleucine as sole BC precursors, respectively. The branching pattern of short BCFAs exactly matched that of membrane BCFAs, whatever the exogenous BCAAs provided. Conclusions: The biosynthesis of short BCFAs is closely related to that of membrane BCFAs in P. freudenreichii. Significance and Impact of the Study: The biosynthesis of short BCFAs in P. freudenreichii depends more on the strain than on the presence of exogenous BC precursors