Short- and Long-Term Biomarkers for Bacterial Robustness: A Framework for Quantifying Correlations between Cellular Indicators and Adaptive Behavior

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers—a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)—were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Our quantitative, systematic approach provides a framework to search for these biomarkers and to evaluate their predictive quality in order to select promising biomarkers that can serve to early detect and predict adaptive traits

Effect of sourdough fermentation on kamut prolamine

Introduction Kamut is frequently recommended as an excellent dietary substitute for wheat for wheat-hypersensitive people because of its presumed low allergenicity (http://www.kamut.com). However, in vitro and in vivo experiments showed no differences in allergic potential between wheat and kamut, probably due to the close phylogenetic relationship of these cereals (1). Allergenic impact depends on the content of immunotoxic sequences in flour. Nevertheless a long-time fermentation by selected sourdough lactic acid bacteria (LAB) may contribute to hydrolyse toxic peptides (2). This research focused on detailed analysis of the kamut proteins. Moreover, the potential of sourdough fermentation to hydrolyze prolamines giving rise to different peptide profiles was investigated with respect to yeast fermentation. Methods The protein composition in kamut and wheat flours (KF and WF) and doughs (K and W), and kamut sourdough (SK) was analysed. A subfractionation method was used to obtain different soluble protein fractions (3) which were analysed quantitatively by Bradford assay and qualitatively by SDS\u2013PAGE. The different proteolysis patterns have been studied in relation to the composition of sourdough LAB populations analysed by DGGE. Results Water soluble protein fraction was higher in KF than in WF; gliadin and glutenin fractions were quantitatively comparable. Albumin and globulin were dramatically reduced in doughs respect to their amount in flours. Moreover, a little gliadin reduction was observed in K and SK. Glutenin fractions were particularly higher in SK than KF. These results suggest that during fermentation process, water soluble proteins were similarly solubilised in the different flours, but glutenin proteins became more easily removable specifically from SK because of specific transformation processes mediated by LAB. About qualitative composition, water soluble protein fractions showed the same pattern in KF and WF; on the contrary gliadins and glutenins showed a different pattern of protein compositions. Moreover, an interesting proteolysis pattern in SK of glutenin fractions and particularly of gliadin fractions (100-20 kDa) was observed. Discussion The capacity of sourdough process to degrade gliadin proteins is related to the combination of peptidase profiles of sourdough LAB population. Even though the hydrolysis profile patterns of single LAB strains have non been analysed, the peptidase activity depends on concentration of sourdough and duration of the fermentation process. This is the first work on the proteolytic activity of kamut sourdough and on its ability to modify gliadin peptides involved in celiac disease. References 1. Simonato et al. Allergy 2002: 57:653\u2013654 2. Hurkman and Tanaka, Journal of Chromatography B, 849 (2007) 344\u2013350 3. Di Cagno et al. Appl.Environ.Microbiol 2002 68: 623-63

Effect of sourdough fermentation on kamut prolamine

Effect of sourdough fermentation on kamut prolamine Alessandra Russo, Diana I. Serrazanetti, M. Elisabetta Guerzoni, Andrea Gianotti Food Science Department, University of Bologna, Italy - [email protected] Introduction Kamut is frequently recommended as an excellent dietary substitute for wheat for wheat-hypersensitive people because of its presumed low allergenicity (http://www.kamut.com). However, in vitro and in vivo experiments showed no differences in allergic potential between wheat and kamut, probably due to the close phylogenetic relationship of these cereals (1). Allergenic impact depends on the content of immunotoxic sequences in flour. Nevertheless, a long-time fermentation by selected sourdough lactic acid bacteria (LAB) may contribute to hydrolyse toxic peptides (2). This research focused on detailed analysis of the kamut proteins. Moreover, the potential of sourdough fermentation to hydrolyze prolamines giving rise to different peptide profiles was investigated with respect to yeast fermentation. Methods The protein composition in kamut and wheat flours (KF and WF) and doughs (K and W), and kamut sourdough (SK) was analysed. A subfractionation method was used to obtain different soluble fractions (3) which were analysed quantitatively by Bradford assay and qualitatively by SDS–PAGE. The different proteolysis patterns have been studied in relation to the composition of sourdough LAB populations analysed by DGGE. Results Water soluble protein fraction was higher in KF than in WF; gliadin and glutenin fractions were quantitatively comparable. Albumin and globulin were dramatically reduced in doughs respect to their amount in flours. Moreover, a little gliadin reduction was observed in K and SK. Glutenin fractions were particularly higher in SK than KF. These results suggest that during fermentation process, water soluble proteins were similarly solubilised in the different flours, but glutenin proteins became more easily removable specifically from SK because of specific transformation processes mediated by LAB. Water soluble protein fractions showed the same qualitative pattern in KF and WF; on the contrary gliadins and glutenins showed a different pattern of protein compositions. Moreover, an interesting proteolysis pattern in SK of glutenin fractions and particularly of gliadin fractions (100-20 kDa) was observed. Discussion The capacity of sourdough process to degrade gliadin proteins is related to the combination of peptidase profiles of sourdough LAB population. Even though the hydrolysis profile patterns of single LAB strains have non been analysed, the peptidase activity depends on concentration of sourdough and duration of the fermentation process. This is the first work on the proteolytic activity of kamut sourdough and on its ability to modify gliadin peptides involved in celiac disease. References 1.Simonato et al. Allergy 2002: 57:653 2.Hurkman et al, Journal of Chromatography B, 849 (2007) 344 3.Di Cagno et al. Appl.Environ.Microbiol 2002 68: 62

Metabolic impact and potential exploitation of the stress reactions in lactobacilli

Lactic acid bacteria (LAB) are a functionally related group of organisms known primarily for their bioprocessing roles in food and beverages. The largest variety of metabolic properties is found in the group of lactobacilli the vast majority of which has been isolated in cereal environments, namely sourdoughs, in which their role ranges from sporadic contaminants to major fermentative flora. Growth or survival in each of these environmental niches depends on the ability of the organism to sense and respond to varying conditions such as temperature, pH, nutrients availability and cell population density. Fermentation process conditions, including temperature range, dough yield, oxygen, pH as well as the amount and composition of starter cultures, determine the cells' metabolic response. In fact, the exposure of microbial cells to stressful conditions during fermentation involves a broad transcriptional response with many induced or repressed genes. The complex network of such responses, involving several metabolic activities will reflect upon the metabolome of the fermentative flora, and thus on the composition and organoleptic properties of the final products. This review shall provide insight into stress response mechanisms and delineate the vast potential residing in the exploitation of the stress dependent metabolome of LAB focusing on bacteria of the sourdough environment as one of the richest sources of lactobacilli

Fermented tofu: Enhancement of keeping quality and sensorial properties

In this study a new way to produce tofu by means of soymilk fermentation by specific lactic acid bacteria, Lactobacillus casei and Lactobacillus acidophilus alone or in combination, and subsequent precipitation has been developed, in order to prevent undesired microbial and chemical spoilage, as well as improve the stability and the quality of the product. In particular, the combination L casei and L acidophilus generated tofu having shelf life exceeding 20 days and able to prevent the growth of the spoilage strains inoculated. This fermented tofu was characterized by the production of antimicrobial molecules, such as acetic acid, limonene, 2-nonen-1-ol, 1-nonanol, 2(5H)-furanone, benzyl alcohol, phenylethyl alcohol and heptanoic acid. Depending on the Lactobacillus species used, the fermentation process generated different metabolites profiles and sensorial properties. Another promising properties conferred by the lactic acid bacteria fermentation was the inhibition of unsaturated fatty acids (UFAs) peroxidation or reduction of the aldehydes originated to their corresponding alcohols

New insights in thermal resistance of staphylococcal strains belonging to the species Staphylococcus epidermidis, Staphylococcus lugdunensis and Staphylococcus aureus.

In this study the thermal resistance of seven strains of staphylococci was studied. Three of them (two Staphylococcus epidermidis and one Staphylococcus lugdunensis) were isolated from pasteurized tofu, while one Staphylococcus aureus from fermented sausages. Also the corresponding type strains were tested. Their heat resistance was assessed at 80\ub0C also in relation to different growth conditions (high NaCl concentrations and low pH). Moreover, the effect of two terpenes (carvacrol and citral) on their survival during thermal treatment was assessed. All the strains were able to survive a 20min thermal treatment and showed a similar behavior, i.e. an inactivation of about 6 log units in the first 2-4min of treatment, followed by constant survivor counts (2-4logCFU/ml). The pre-culture of staphylococci under stressing conditions (high NaCl concentration and low pH) increased the susceptibility to thermal treatment in three strains isolated from food matrices. The addition of carvacrol and citral during thermal treatment influenced the number of survived cells at the end of the treatment. This new insight in the thermal resistance of staphylococci needs to be better investigated, in order to study proper strategies to control their occurrence and behavior in real food systems

Effect of lactic acid bacteria fermentation on lipidic and phenolic profiles of tofu

Soy foods have long been a staple of the human diet in Asia and tofu may be the most popular food made of soy worldwide. Tofu is prepared from soymilk curd and it can also be further processed (grilled, frozen, or fermented) modifying taste, texture and end-uses compared to traditional tofu. Fermentation also prevent undesired microbial and chemical reactions improving the stability and the quality of the product. Moreover, in addition to be natural, nutritious and safe, epidemiological studies have shown that fermented soybean products exhibit healthy effects. In fact, soybean and its products contains biologically active compounds including isoflavones, essential fatty acids, sterols, phospholipids and tocopherols which may contribute to prevent chronic diseases such as hormone-dependent cancers, cardiovascular diseases and osteoporosis. Due to the influence of processing on the bioactive compounds content of soy food products, soybean, traditional tofu and tofu obtained by soymilk fermentation were studied by GC\u2013FID and HPLC coupled with fluorimetric, light scattering and electrospray ionization mass spectrometry detector, in order to analyse their lipidic profile (fatty acids, tocopherols and sterols) and isoflavones content and composition. In particular, for isoflavones analysis, a new method was set up. Fermentation was performed using specific strains of lactic acid bacteria (LAB), such as Lactobacillus casei and Lactobacillus acidophilus. Fatty acids profile of soybean and tofu samples were very similar, even in term of trans fatty acids and omega-3 fatty acids, so fermentation cannot differentiate tofu from soybean by fatty acid composition. The most abundant fatty acid was linoleic acid, followed by oleic acid, palmitic acid and \u3b1-linolenic acid, as reported by other authors. The processing conditions did not significantly affect neither tocopherol composition of tofu (traditional and fermented) compared to soybean and in all products the main tocopherol was \u3b3-tocopherol followed by \u3b4-tocopherol and \u3b1- tocopherol. As concerning phytosterol content, f-sitosterol was the main sterol found with the phytosterol composition that was similar in soybean and traditional and fermented tofu. The main effects of fermentation were on isoflavone profile: in fact, significantly differences were seen in fermented tofu isoflavones content compared to soybean and to traditional tofu. In particular, fifteen isoflavones were identified as aglycones and glycosides: the aglycone forms were significantly higher in fermented tofu compared to the traditional one. These results suggest that fermentation by LAB cannot discriminate the fatty acid, tocopherol and phytosterol composition of fermented tofu from the traditional one and from soybean, but it plays an essential role not only on shelf-life extend, but also in the increase of the content of aglycone isoflavones with important biologically implications on human health

Exploitation of starch industry liquid by-product to produce bioactive peptides from rice hydrolyzed proteins

Small peptides show higher antioxidant capacity than native proteins and may be absorbed in the intestine without further digestion. In our study, a protein by-product from rice starch industry was hydrolyzed with commercial proteolytic enzymes (Alcalase, Neutrase, Flavourzyme) and microbial whole cells of Bacillus spp. and the released peptides were tested for antioxidant activity. Among enzymes, Alcalase was the most performing, while microbial proteolytic activity was less efficient. Conversely, the antioxidant activity was higher in the samples obtained by microbial hydrolysis and particularly with Bacillus pumilus AG1. The sequences of low molecular weight antioxidant peptides were determined and analyzed for aminoacidic composition. The results obtained so far suggest that the hydrolytic treatment of this industrial by-product, with selected enzymes and microbial systems, can allow its exploitation for the production of functional additives and supplements rich in antioxidant peptides, to be used in new food formulas for human consumption