René Badertscher

Stable isotope ratios, major, trace and radioactive elements in emmental cheeses of different origins

Twenty Emmental cheeses from six European regions (Allgau (D), Bretagne (F), Finland, Savoie (F), Switzerland and Vorarlberg (A)) were analysed for stable isotope ratios such as 13C/12C, 15N/14N, 18O/16O, D/H and 87Sr/86Sr, as well as major (Ca, Mg, Na, K), trace (Cu, Mn, Mo, I) and radioactive elements (90Sr, 234U, 238U). The discriminating potential of these parameters was evaluated using difference tests of mean values and principal component analysis (PCA). “Finland”, “Bretagne” and “Savoie” cheeses were well separated using δ13C-, δ15N-, δ2H- and δ87Sr-values. Concentrations of molybdenum and sodium allowed the groups “Switzerland”, “Vorarlberg” and “Allgau” to be separated. 90Sr activity was highly correlated with the altitude of the production zone. Using this parameter, “Finland” and “Bretagne” were separated from “Vorarlberg” cheese. The results of the current screening test will help selecting the best tracers of origin for the remainder of the project.

Identification of Urinary Food Intake Biomarkers for Milk, Cheese, and Soy-Based Drink by Untargeted GC-MS and NMR in Healthy Humans

The measurement of food intake biomarkers (FIBs) in biofluids represents an objective tool for dietary assessment. FIBs of milk and cheese still need more investigation due to the absence of candidate markers. Thus, an acute intervention study has been performed to sensitively and specifically identify candidate FIBs. Eleven healthy male and female volunteers participated in the randomized, controlled crossover study that tested a single intake of milk and cheese as test products, and soy-based drink as a control. Urine samples were collected at baseline and up to 24 h at distinct time intervals (0-1, 1-2, 2-4, 4-6, 6-12, and 12-24 h) and were analyzed using an untargeted multiplatform approach (GC-MS and 1H NMR). Lactose, galactose, and galactonate were identified exclusively after milk intake while for other metabolites (allantoin, hippurate, galactitol, and galactono-1,5-lactone) a significant increase has been observed. Urinary 3-phenyllactic acid was the only compound specifically reflecting cheese intake although alanine, proline, and pyroglutamic acid were found at significantly higher levels after cheese consumption. In addition, several novel candidate markers for soy drink were identified, such as pinitol and trigonelline. Together, these candidate FIBs of dairy intake could serve as a basis for future validation studies under free-living conditions.

Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

ABSTRACT The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from l- and d-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.

GC-MS Based Metabolomics and NMR Spectroscopy Investigation of Food Intake Biomarkers for Milk and Cheese in Serum of Healthy Humans

The identification and validation of food intake biomarkers (FIBs) in human biofluids is a key objective for the evaluation of dietary intake. We report here the analysis of the GC-MS and 1H-NMR metabolomes of serum samples from a randomized cross-over study in 11 healthy volunteers having consumed isocaloric amounts of milk, cheese, and a soy drink as non-dairy alternative. Serum was collected at baseline, postprandially up to 6 h, and 24 h after consumption. A multivariate analysis of the untargeted serum metabolomes, combined with a targeted analysis of candidate FIBs previously reported in urine samples from the same study, identified galactitol, galactonate, and galactono-1,5-lactone (milk), 3-phenyllactic acid (cheese), and pinitol (soy drink) as candidate FIBs for these products. Serum metabolites not previously identified in the urine samples, e.g., 3-hydroxyisobutyrate after cheese intake, were detected. Finally, an analysis of the postprandial behavior of candidate FIBs, in particular the dairy fatty acids pentadecanoic acid and heptadecanoic acid, revealed specific kinetic patterns of relevance to their detection in future validation studies. Taken together, promising candidate FIBs for dairy intake appear to be lactose and metabolites thereof, for lactose-containing products, and microbial metabolites derived from amino acids, for fermented dairy products such as cheese.

Validated method for the determination of propane-1,2-diol, butane-2,3-diol, and propane-1,3-diol in cheese and bacterial cultures using phenylboronic esterification and GC–MS

A simple, fast, sensitive, and robust gas chromatography-mass spectrometry (GC-MS) method for the simultaneous determination of propane-1,2-diol, butane-2,3-diol, and propane-1,3-diol in cheese and bacterial cultures was developed. Target analytes were extracted and transformed into their phenylboronic esters prior to analysis. The method showed good sensitivity, without carryover between the samples. The detection limits for propane-1,2-diol, butane-2,3-diol, and propane-1,3-diol in cheese samples were 0.26, 0.02, and 0.11mgkg-1, respectively, and for bacterial culture samples were 1.32, 0.09, and 0.54mgkg-1, respectively. The Horwitz ratio showed good precision for all analytes (<0.45). The calibrated range in cheese for all analytes was very broad, from 0 to 1000mgkg-1, and in bacterial cultures was from 0 to 5000mgkg-1 with R2>0.9991. The results confirm excellent applicability of the proposed method for the determination of the target metabolites in cheese and bacterial culture samples.

Population Dynamics of Lactobacillus helveticus in Swiss Gruyère-Type Cheese Manufactured With Natural Whey Cultures

Lactobacillus helveticus, a ubiquitous bacterial species in natural whey cultures (NWCs) used for Swiss Gruyère cheese production, is considered to have crucial functions for cheese ripening such as enhancing proteolysis. We tracked the diversity and abundance of L. helveticus strains during 6 months of ripening in eight Swiss Gruyère-type cheeses using a culture-independent typing method. The study showed that the L. helveticus population present in NWCs persisted in cheese and demonstrated a stable multi-strain coexistence during cheese ripening. With regard to proteolysis, one of the eight L. helveticus populations exhibited less protein degradation during ripening.

Enlightening the Lactate Degradation Processes in Cheese and Bacterial Cultures Using Phenylboronic Esterification and GC-MS

Infoodproduction, fermentationplaysakeyroleforpreserving food. At the same time, the process can be used to achieve the desired qualities of aroma, taste and texture. For example, for various cheeses, the eye formation is important, which is caused by the release of carbon dioxide during the ripening process. The bacteria used for this have a multitude of metabolic activities that can also release other gases. Some of the degradation pathways lead to the formation of diols. Lactobacillus parabuchneri is an interesting example for such a pathway, because it canmetabolize lactate to propane-1,2-diol with simultaneous release of acetate and carbon dioxide. Lactobacillus buchneri, on the other hand, produces propane-1,3-diol during the co-fermentation of carbohydrates and glycerine. The 2,3-butanediol fermentation is a way of breaking down carbohydrates to produce energy under anoxic conditions. The pathways that convert lactate into acetate under anaerobic conditions are not well understood at the molecular level. A sensitive method was therefore developed and validated for the simultaneous quantitative measurement of the metabolites propane-1,2-diol, butane-2,3-diol, and propane1,3-diol in cheese and bacterial cultures. In a first step, the diols are extracted in water and esterified directly in the extract with the aid of phenylboronic acid. After extraction with toluene, the resulting phenylboronic esters are measured directly using GCMS in selected-ion monitoring (SIM) mode with an external calibration using butane-1,2-diol as the internal standard. The method is simple, fast, robust, allows precise measurements in the mg/kg range and can be used in complex matrices due to selective double extraction and is therefore also of interest for plant and non-dairy food fermentation.