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This work reports on monoamines (MA), diamines (DA) and polyamines (PA) as well as free amino acids (fAA) in nutritional supplements and infusions derived from green coffee beans. Samples were investigated using a ultra-high performance liquid chromatography UV/FLR method, which was validated regarding specificity, linearity, range, precision, accuracy and limits of detection and quantification. Nutritional supplements based on green coffee beans showed large amounts of total amines ranging from 1090 to 2593 mg/kg, with exceptional high levels of spermidine up to 724 mg/kg, and a content of fAA from 4004 to 12,389 mg/kg. Infusions brewed from green coffee beans showed much lower contents of amines (14–17 mg/L) and fAA (78–100 mg/L). However, if the customary edible portion was considered, infusions from green coffee were a mainly better source for MA and PA as well for fAA, compared to nutritional supplements. Tryptamine and tyramine were predominant as MA putrescine as DA, and spermidine as PA, respectively. Graphic abstract

Insects are rich in major nutrients, such as protein and fat. Recently, minor nutrients like vitamins have become the subjects of interest in insects. Hence, this study reports on the development and validation of a method for the determination of vitamin B12 in mealworm (Tenebrio molitor larvae), cricket (Gryllus assimilis), grasshopper (Locusta migratoria) and cockroach (Shelfordella lateralis), using an ultra-high performance liquid chromatography approach with preliminary immunoaffinity chromatography sample preparation. The method was validated regarding linearity, specificity, accuracy and precision, as well as limits of detection/quantification, and was found to be satisfactory for the desired application. Found levels of vitamin B12 were 1.08 µg/100 g for mealworm, 2.88 µg/100 g for cricket, 0.84 µg/100 g for grasshopper, and 13.2 µg/100 g dry weight for cockroach, representing the first validated report on the content of vitamin B12 in edible insects. Observed interferences are likely caused by the presence of pseudovitamin B12.

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an "A1/A2 hypothesis" was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common β-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of β-casein A1 were also found in commercial "A2 milk" in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous β-casein A2A2) and by screening commercial products on the market (to confirm the absence of β-casein variants A1, B, and C in dairy products labelled "A2 milk") to protect consumers from this unexpected fraud.

Biogenic amines (BA) in various commercial cheese samples (n = 151) representing most common cheese varieties were determined using UHPLC. In general, cumulative BA levels varied to a great extent with exceptional samples having amounts up to 150–313 mg/100 g cheese, whereas only 5% of the analysed cheeses showed total concentrations higher than 90 mg/100 g (median 5.7 mg/100 g). Histamine was found in 79% of all samples, with maximum concentrations up to 116 mg/100 g, but only 5% of the cheeses had a histamine level above 17 mg/100 g (median 0.9 mg/100 g). For tyramine (72% occurrence), highest values were found for some acid-curd cheeses (up to 160 mg/100 g) and certain “mountain cheeses” (median 1.0 mg/100 g). Putrescine was detected in 70% of the cheeses (up to 84 mg/100 g for some acid-curd cheeses; median 0.6 mg/100 g). Cadaverine was found in 48% of the samples, with highest concentrations for some acid-curd cheeses (median 0.2 mg/100 g). Tryptamine had the lowest occurrence (15%) and a median concentration of 0.3 mg/100 g.

As raw cow's milk possesses a limited shelf-life and can also pose a threat to the consumer's health, thermal preservation techniques are customary nowadays. A range of compounds can be used for the assessment of successfulness of such treatments, so-called time temperature integrators (TTIs). Although most water-soluble vitamins are known to be depleted during heat exposure in milk, their application as TTI hasn't been investigated yet. Especially vitamin B6 with its various possible vitamers (chemical different forms that exhibit vitamin activity) might be a suitable system for a new vitamin-based TTI. Therefore, the applicability of B6 vitamers in cow's milk as a TTI system was to be evaluated in this study. A recently developed ultra-high performance liquid chromatography method for the simultaneous assessment of all relevant B6 vitamers in cow's milk was used to characterize the status and composition of B6 vitamers in a market survey (n = 78) from Austria, Germany and Czech Republic, comprising milk produced by various thermal and non-thermal preservation techniques such as pasteurization, extended shelf life, ultra-high temperature (UHT). Examining generated data using classification and regression decision trees, discrimination of UHT milk from other preservation techniques, based on the ratio of B6 vitamers was possible. Hence, representing the first report for process authentication of cow's milk based on the composition of minor nutrients (B6 vitamers).

Awassi dairy sheep farmers in the Middle East are facing high feed costs, particularly during the milk production season, along with large fluctuation in grain and straw prices. Incorporating agro-industrial byproducts into the diet of Awassi dairy sheep can help to balance diets and decrease costs. But it may affect milk quality through changes in the fatty acid profile. Six experimental diets were compared to a control diet in the research station of the International Center for Agricultural Research in the Dry Areas (ICARDA) in Tal Hadya, Syria. Milking Awassi ewes (n = 56) were used to test six experimental diets, in which supplements comprised molasses, sugar beet pulp, or cotton seed cake to a traditional control diet contained barley, wheat bran, and barley straw. Milk samples were collected on a weekly basis from April to June. The daily milk production and fat content were enhanced in diets containing urea-treated wheat straw (P

This work shows the influences of free amino acids on the formation of related biogenic amines by thermal decarboxylation in an entire commercial roasting process. Two different C. arabica bean samples were investigated, which differed in terms of origin, variety, altitude and post-harvest process. The formation and degradation pattern of investigated amino acids and biogenic amines was comparable in both different bean samples. For the first time, the intermediate and thermogenic driven formation of histamine and agmatine were shown in the roasting process. Agmatine was formed in two different intensities. Histidine degradation and histamine formation showed molar conversion rates of 90% (Brazilian sample, semi-washed) and 95.6% (Kenyan sample, wet processed), whereas arginine was decarboxylated to agmatine in two steps, with rates of I: 23.5% and 29.8%, and II: 47.3% and 46.6%, respectively. No significant formation of putrescine, ethylamine, ethanolamine, tyramine, tryptamine, cadaverine, phenylethylamine, octopamine and ornithine could be found. The comparison to lab-scale roasting trials in the past offered varying results in regard to the changes of spermidine and histamine in the commercial batches of this study.

The aim of this study was to investigate the changes of individual chlorogenic acids during the coffee roasting process under authentic commercial conditions. Unique chemical reactions of caffeoylquinic acid (CQA) and dicaffeoylquinic acid (diCQA) isomers were shown in different Coffea species (C. arabica vs. C. canephora), batch sizes (1 kg vs. 20 kg) and roaster designs. Temperature determination setups of different roaster machines appeared gave varying results. Isomerization reactions of CQA in C. arabica beans were observed between the end of the drying phase and the crack reaction. Regarding C. canephora beans, the slower degradation rate of 3-CQA and 4-CQA isomers after the crack reaction in contrast to C. arabica species was described for the first time.

Adequate heat treatment of raw milk is required to guarantee microbiological safety, but there is a huge variation in the heat load of different categories of heat-treated milk due to different thermal treatment temperatures when using different processes. However, uncontrolled "over-processing” of milk leads to certain undesirable losses of nutritional and organoleptic quality. To evaluate the actual intensity of heating, numerous analytical tools have been established to determine several associated time‐temperature integrators. Enzymes are appropriate indicators for the assessment of lower heat treatment; higher heat load can be evaluated using indicators such as β-lactoglobulin, α-lactalbumin, lactulose, furosine, and hydroxymethylfurfural. Analytical methods are mainly based on photometric/fluorimetric methods for determination of enzymatic activity, and high-performance liquid chromatography for β-lactoglobulin, α-lactalbumin, lactulose, furosine, and hydroxymethylfurfural. Capillary electrophoresis and other analytical tools (e.g., gas chromatography, infrared methods) are also appropriate to evaluate the intensity of heat treatment of milk and dairy products.

The aim of this study was to develop a high-throughput UHPLC method for the determination vitamin B1 active compounds; thiamin, thiamin monophosphate and thiamin diphosphate in bovine milk. In order to sustain the native vitamin B1 phosphorus esters, sample preparation is crucial. Various acids as well as commonly used enzymes and their enzyme mixtures were compared. Method accuracy was confirmed using certified reference material as well as comparison with the corresponding CEN method, and was found to be satisfactory. Studied milk samples showed significant amounts of thiamin monophosphate, which can make up to 53.9% of the total vitamin B1 content in commercial milk, and up to 78% in raw milk. Moreover, a tremendous variation of the total content of vitamin B1 was observed between single cows, which ranged from 0.24 mg/L up to 0.54 mg/L of total vitamin B1.

Besides sensorial assays for quantifying pungent components in chilies, nowadays mainly HPLC methods focus on the determination of individual capsaicinoids. Compared to HPLC, ultra-highperformance chromatography (UHPLC) enables shorter runtimes and enhanced resolution. Thus, the aim of this work was to establish a rapid UHPLC method for the determination of capsaicin and dihydrocapsaicin, the most powerful pungent capsaicinoids found in chili fruits. The developed UHPLC method features a significantly reduced separation time (1.7 min) and high chromatographic resolution, hence presenting a feasible approach for high-throughput pungency profiling. Compared to UV, fluorescence detection showed superior sensitivity with a limit of quantification for capsaicin of 0.160 μg g −1 and 0.175 μg g −1 for dihydrocapsaicin and proving suitable for analysing even very low pungency chili products. Moreover, to investigate the massive variability between chilies, typical varieties that were grown in Austria were characterized for their intrinsic pungency, with total capsaicinoids amounts ranging from 213 μg g −1 for Jalapeno to 28200 μg g −1 dry weight for ‘Bhut Jolokia’. Moreover, analyses of different fruit compartments showed tremendous deviation between the pericarp and the placenta with seeds.

Thermal time integrators (TTIs) are useful tools to describe the heat load of pasteurized, Ultra-high temperature (UHT) and emerging Extended Shelf Life (ESL) milk, which can be produced using filtration or direct/indirect heat treatment. However, threshold limits are mandatory for β-lactoglobulin in pasteurized and UHT milk, but currently not for ESL milk, for which the Austrian Food Codex grants a transition period till 2018 for dairies using indirect heating technologies. The objective of this study was to compare two sample sets of bovine milk retailed in Austria, purchased in 2012 (n = 71) and 2015 (n = 80) including pasteurized, UHT and differently processed ESL milk samples. Furosine, acid-soluble α-lactalbumin and β-lactoglobulin were determined with RP-HPLC, and lactulose enzymatically. Results were not significantly different comparing both years, but rather highlight the significance and suitability of these TTIs to distinguish between varying heat load applied on milk. Using furosine, lactulose and α-lactalbumin, a differentiation of pasteurized, filtered and directly heated ESL milk from indirectly heated ESL milk as well as from UHT milk is possible, whereas β-lactoglobulin allowed the discrimination of directly heated ESL milk from filtered ESL milk and pasteurized milk, respectively.

The concentration of the artificial compound furosine in liquid milk can be used as an indicator to assess the heat load applied onto the product, which allows discrimination between differently processed milk (pasteurized, Extended Shelf Life (ESL) milk, Ultra-High Temperature (UHT) treated milk). In this work, a new UHPLC method for the rapid determination of furosine in 2 min only (total runtime 6 min) is presented, using PDA detection at 280 nm. The method was validated for the analysis of liquid milk samples regarding linearity, instrumentation precision, limits of detection and quantification, repeatability of sample work-up and recovery. Moreover, the method was compared to an existing RP-HPLC method. Selected commercial liquid milk samples (n = 36) were analyzed, showing lowest furosine amounts for pasteurized milk, whereas highest amounts were found in UHT milk. For ESL milk, quite diverse concentrations were analyzed ranging from 11.70 up to 115.16 mg furosine 100 g−1 protein, illustrating the different processing technologies of this category with lowest amounts for filtered ESL milk, followed by directly heated, and indirectly heated ESL milk samples, respectively.

Soybean seed is one of the richest food sources of spermidine and other polyamines. Recent findings from human and animal models have confirmed spermidine as a potential anti-aging substance acting through the initiation of autophagy pathways as well as through antioxidant and anti-inflammatory properties. As this might be of relevance for selecting soybeans for soy food production, the present research addresses the natural variation of spermidine concentration of soybean to determine the influences of genotype and environmental factors on spermidine and other polyamines, and to study possible relationships between spermidine and major seed quality traits. Sixteen early maturity soybean genotypes were grown near Vienna, Austria for three seasons, and harvest samples were subject to ultra-high performance liquid chromatography (UHPLC) for determining concentrations of polyamines and free amino acids. Based on individual samples, spermidine concentration ranged between 167 and 291 mg kg−1 dry seed, and both genotype and growing season significantly affected spermidine level. Spermidine concentration was closely correlated to putrescine but was not related to seed protein content or other major seed constituents determined by NIRS analysis. These results demonstrate the feasibility of plant breeding approaches to modify the spermidine level of soybean which might support the future development of functional soy foods.

Analysis of bovine milk proteins is crucial in many food and non-food industrial applications, nevertheless labour-intensive wet-chemical, low-throughput methods are still routinely used. In this work, external cavity-quantum cascade laser (EC-QCL) mid-infrared spectroscopy is employed as a rapid method for protein analysis of commercial bovine milk. Combined analysis of the amide I and II bands enabled quantitation of individual proteins (casein, β-lactoglobulin, α-lactalbumin) and total protein content. IR spectra of spiked and diluted milk samples were employed for calibration of the target analytes in the presence of a complex matrix by partial least squares (PLS) regression modelling. A sample set of different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed, and results agreed well with reference methods. Quantitation of temperature sensitive proteins enables detailed distinction between milk types experiencing different heat loads during processing, and discrimination between diverse bovine milk types is successfully demonstrated. Fil: Montemurro, Milagros. Vienna University of Technology; Austria. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Desarrollo Analítico y Quimiometría; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schwaighofer, Andreas. Vienna University of Technology; Austria Fil: Schmidt, Anatol. University of Natural Resources and Life Sciences; Austria Fil: Culzoni, Maria Julia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Desarrollo Analítico y Quimiometría; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mayer, Helmut K.. University of Natural Resources and Life Sciences; Austria Fil: Lendl, Bernhard. Vienna University of Technology; Austria

This study provides a direct comparability between milk from cows fed hay or silage produced from the same parent plant material; the influence of feeding either hay or silage on amounts of various milk components was investigated. To give an overview regarding differences in milk composition, protein fractions, free amino acids, polyamines and biogenic amines as well as fatty acid profile and sensory properties were analysed. Moreover, the content of vitamin E and four different B-vitamins (B1, B2, B6 and B12) was determined. Results showed that there are some significant differences between hay milk and silo milk, especially regarding certain key nutrients. Linoleic acid, α-linolenic acid, lysine and putrescine showed higher concentrations in hay milk, while vitamin B12 and spermine were increased in silo milk. Sensory analysis showed that feeding of silage had a significant influence on colour (yellow), consistency and odour intensity, which were more pronounced in silo milk.

The aim of this study was to develop a multi-analyte UHPLC method for furans and to apply it to commercial coffee samples as well as commercial roasting trials. Furans, as rising time–temperature indicators (TTIs), promised to be an alternative to unsatisfactory roasting temperature measurements. Consequently, a UHPLC–UV method for the determination of 5-hydroxymethyl-2-furfural (HMF), 5-hydroxymethyl-2-furoic acid (HMFA), 2-furfural (F), 5-methylfurfural (MF), 2-furyl methyl ketone (FMC), 2-furoic acid (FA), and for 3-caffeoylquinic acid (3-CQA) was developed and validated. Commercial roasted coffee beans contained 77.7–322 mg/kg HMF, 73.3–158 mg/kg HMFA, 109–200 mg/kg 2-F, 157–209 mg/kg MF, 12.3–32.8 mg/kg FMC, and 137–205 mg/kg FA. Roasting trial samples showed strong rising HMF contents (max.: Arabica: 769 mg/kg, Robusta: 364 mg/kg) followed by a distinct decline. Only MF and FA appeared as steady rising TTIs in the roasting process in Arabica and Robusta beans. 3-CQA fitted well as a decreasing TTI as expected.

Insects are rich in major nutrients, such as protein and fat. Recently, minor nutrients like vitamins have become the subjects of interest in insects. Hence, this study reports on the development and validation of a method for the determination of vitamin B

Three heat load indicators, i.e., furosine, lactulose and acid-soluble β-lactoglobulin, were determined in whipping cream samples purchased from Austrian market ( n = 33), as well as commercial samples obtained directly from one Austrian dairy company (one brand, n = 25). Furosine contents were 47.8 ± 14.0, 72.2 ± 36.6, and 172.5 ± 17.7 mg 100 g −1 protein, and β-lactoglobulin contents were 143 ± 91, 195 ± 150, and 89 ± 31 mg L −1 for retailed pasteurised, extended shelf life (ESL), and ultra-high temperature (UHT) cream samples, respectively. Lactulose concentrations (analysed enzymatically) were 29 ± 10, 56 ± 41, and 201 ± 24 mg L −1 for pasteurised, ESL and UHT cream samples, respectively. Linear correlation obtained for furosine and lactulose concentrations indicated that these indicators can reliably assess the heat load of pasteurised, ESL and UHT cream samples, whereas β-lactoglobulin was definitely not appropriate to discriminate between these heating categories.

Analysis of proteins in bovine milk is usually tackled by time-consuming analytical approaches involving wet-chemical, multi-step sample clean-up procedures. The use of external cavity-quantum cascade laser (EC-QCL) based IR spectroscopy was evaluated as an alternative screening tool for direct and simultaneous quantification of individual proteins (i.e. casein and beta-lactoglobulin) and total protein content in commercial bovine milk samples. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. A sample set comprising different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed and results were compared to reference methods. Concentration values of the QCL-IR spectroscopy approach obtained within several minutes are in good agreement with reference methods involving multiple sample preparation steps. The potential application as a fast screening method for estimating the heat load applied to liquid milk is demonstrated.

Green coffee infusion is a novel and emerging food. This is the first study to report on caffeine and chlorogenic acid content in green coffee infusion as ingested by the consumer. Caffeine was determined by adapting the corresponding DIN/ISO HPLC method. Chlorogenic acids were measured by AOAC standardized photometric method. Both methods were adapted and validated for the green coffee beans and resulting infusions. Results for caffeine in green coffee infusion reached from 113 to 188 mg/L in C. arabica infusions, and from 186 to 293 mg/L in C. canephora infusions. A decaffeinated green coffee infusion sample contained 6.2 mg/L. Results of chlorogenic acid ranged from 628 to 1040 mg/L in C. arabica infusions, and from 682 and 1210 mg/L in C. canephora infusions. Decaffeinated Arabica green coffee infusion sample had a concentration of 767 mg/L. Infusions from Robusta exceeded Arabica samples by far regarding caffeine and chlorogenic acid content. Results are discussed with numerous references in detail.

Since only isoflavone aglycones are considered to be bioactive, the determination of total aglycones that are released from conjugated isoflavones after hydrolytic treatment may facilitate an objective alternative for quantifying isoflavone contents in soy products. Given this major benefit, a new ultra-high pressure liquid chromatography (UV-UPLC™) method was developed for the fast and reliable determination of total aglycones in soybeans (daidzein, glycitein, and genistein) after enzymatic hydrolysis applying helix pomatia digestive juice. Capitalizing on the enhanced performance of UPLC™, aglycones were separated within 3 min only, with a total runtime of 8 min till the next injection. Thus, especially compared to HPLC protocols, UPLC™ proved to be superior due to significantly shorter runtimes and accordingly increasing sample throughput. Additionally, regarding the performed validation (linearity, precision, recovery, selectivity, and robustness), the established method proved to be suitable for quantifying total aglycones in soybeans. Moreover, method applicability was demonstrated by analyzing 23 commercial soybean cultivars for their isoflavone contents. Cumulative aglycone levels ranged from 100 to 255 mg per 100 g, hence implying an average ratio of 52%, 41%, and 7% of total isoflavones for genistein, daidzein and glycitein, respectively. However, for some soybeans, other distinct aglycone distributions were observed as well.

The analytical determination of bovine milk proteins is important in food and non-food industrial applications and yet, rather labour-intensive wet-chemical, low-throughput methods have been employed since decades. This work proposes the use of external cavity-quantum cascade laser (EC-QCL) spectroscopy for the simultaneous quantification of the most abundant bovine milk proteins and the total protein content based on the chemical information contained in mid-infrared (IR) spectral features of the amide I band. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. Protein concentrations in commercial bovine milk samples were calculated after chemometric compensation of the matrix contribution employing science-based calibration (SBC) without sample pre-processing. The use of EC-QCL spectroscopy together with advanced multivariate data analysis allowed the determination of casein, α-lactalbumin, β-lactoglobulin and total protein content within several minutes. Fil: Kuligowski, Julia. Health Research Institute La Fe; España. Vienna University of Technology; Austria Fil: Schwaighofer, Andreas. Vienna University of Technology; Austria Fil: Alcaraz, Mirta Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Quintás, Guillermo. Leitat Technological Center; España. Health Research Institute La Fe; España Fil: Mayer, Helmut. University of Natural Resources and Life Science; Austria Fil: Vento, Máximo. Health Research Institute La Fe; España. University & Polytechnic Hospital La Fe; España Fil: Lendl, Bernhard. Vienna University of Technology; Austria

The aim of this study was to develop a high-throughput UHPLC method for the determination vitamin B

Physico-chemical characteristics of sheep and goat milk in Austria as influenced by seasonal effects and regional differences were investigated. Considerable seasonal variations were observed regarding most constituents. Sheep milk from three different dairy plants showed very similar chemical composition and physical properties, whereas average means of sheep milk were significantly different from goat milk except for freezing point, pH, and a few fatty acids (C12:0, C18:0, C18:1). The mean values obtained for sheep and goat milk during the whole season were: pH 6.59/6.59, freezing point −0.544/−0.542 °C, ash 0.853/0.813%, total solids 15.78/11.70%, crude protein 5.21/3.15%, casein 3.98/2.39%, whey protein 0.92/0.52%, urea 0.432/0.335 g L−1, fat 5.75/3.74%, lactose 4.64/4.32%, citric acid 1.535/1.018 g L−1, phosphorus 1.454/1.009 g L−1, chloride 1.196/1.755 g L−1, sodium 0.442/0.317 g L−1, potassium 1.248/2.015 g L−1, calcium 1.846/1.288 g L−1, magnesium 0.192/0.138 g L−1, orotic acid 17.02/12.09 mg kg−1,and cholesterol 11.6/9.8 mg 100 g−1 milk, respectively.

Fifty samples consisted of 24 maize kernels and 26 maize based-food products from retail market in Yogyakarta, Indonesia were analysed for deoxynivalenol (DON) using high performance liquid chromatography (HPLC) combined with ultraviolet detection after immunoaffinity column (IAC) clean-up process. Prior to use, performance of the analytical method was evaluated in term of recovery, repeatability, and detection limit. The recoveries of DON in spiked samples (50–360 μg DON/kg sample) were obtained in a range of 70.9–97.3% with relative standard deviation (RSD) for repeatability within a day less than 16%. Limit of detection based on the response ratio of signal to noise (3/1) was noted being 20 μg DON/kg sample. All analysed samples contained DON ranging between 47 and 348 μg/kg with median and means of 111.0 and 124.6 μg/kg, respectively. The contamination of DON in the studied samples may not be taken into account as hazard for human and animal health since the concentrations were lower than maximum limit of DON contamination in foodstuffs (500 μg/kg) established by European Commission (2006).

Despite the increasing economic importance of goat’s milk in Europe, no current data on the chemical composition and physical properties of this milk species are available in Austria. Thus, milk samples of six dairy goat breeds in Austria (Bunte Deutsche Edelziege, Pinzgauer Ziege, Saanenziege, Strahlenziege, Toggenburger Ziege, Weise Deutsche Edelziege) were analysed for physicochemical characteristics for 8 months from March to October. Considerable seasonal variations, but nearly no statistically significant differences between milk samples from these six goat breeds were observed regarding most parameters. The mean values obtained for all breeds during the whole season were as follows: pH 6.55, freezing point depression 0.549 °C, ash 0.828%, total solids 12.24% (w/w), crude protein 3.35% (w/w), casein 2.40% (w/w), whey protein 0.63% (w/w), urea 0.325 g⋅L−1, fat 3.67%, lactose 4.23%, citric acid 0.913 g⋅L−1, phosphorus 1.088 g⋅L−1, chloride 1.708 g⋅L−1, sodium 0.304 g⋅L−1, potassium 1.759 g⋅L−1, calcium 1.256 g⋅L−1, magnesium 0.128 g⋅L−1, orotic acid 13.59 mg⋅kg−1, β-carotene 0.419 mg⋅L−1, retinol 0.502 mg⋅L−1, riboflavin 1.050 mg⋅L−1, and cholesterol 12.4 mg⋅100 g−1 milk, respectively. The studies on fatty acid profiles revealed the following mean values: C4:0 2.73%, C6:0 2.10%, C8:0 1.86%, C10:0 5.80%, C12:0 2.52%, C14:0 7.07%, C16:0 23.73%, C18:0 11.84%, and C18:1 28.14%. Unexpected high levels were obtained for C18:2 (5.06%) and C18:3 (0.59 %), respectively. This work provides an updated information on the current physicochemical characteristics of goat’s milk in Austria, which can be of great importance in the fields of dairy and food technology, nutrition science, and food analysis, respectively.

Probiotic enterococci are frequently encountered as additives in animal nutrition, but no fast standardized methods for their unambiguous identification at strain level exist. Six polymerase chain reaction (PCR)-based methods previously described for similar tasks but other genera were evaluated concerning their suitability for the rapid, stringent and reliable identification of Enterococcus at strain level. The most appropriate method should facilitate the instant identification of isolates from animal feeds and should be applicable in routine quality control. Amplified ribosomal DNA analysis (ARDRA), internally transcribed spacer region (ITS)-PCR, randomly amplified polymorphic DNA (RAPD)-PCR, repetitive element sequence based (rep)-PCR, tRNA intergenic spacer PCR and 16S rRNA gene typing were investigated with 74 Enterococcus, mainly E. faecium, strains representing 11 species. RAPD-PCR proved superior by discriminating at the strain level. Depending on the enzyme, ARDRA yielded fingerprints specific for a species or even single strains. The remaining methods successfully identified enterococci at the genus level.

Recent trends to extend the shelf life of pasteurized milk, without the negative flavour normally associated with ultra-high-temperature (UHT) milk, have resulted in the development of extended shelf life (ESL) milk. Acid-soluble β-lactoglobulin (β-Lg) and furosine contents were chosen as relevant indicators for heat load of ESL milk products. RP-HPLC methods were developed using the same column (Symmetry 300™, Waters), which enabled the separation of whey proteins within 22 min; furosine was analysed using ion-pair RP-HPLC within 8 min. Electrophoresis was used as a high-throughput and cost-effective screening method to assess the impact of thermal processes on milk and to distinguish different categories of heat-treated milk samples. Liquid milk samples (n = 128; including 7 raw, 33 pasteurized, 71 ESL, and 17 UHT milk samples) were obtained from retail outlets in Austria and analysed. Only 45% of the analysed samples designated as ESL milk showed furosine contents 1800 mg·L−1 milk, which had been discussed as threshold levels for ESL milk. A further 55% of the analysed ESL milk samples had low acid-soluble β-Lg ( 40 mg·100 g−1 protein), levels comparable to the excessive heat load of UHT milk. Thus, there is an urgent need for an EU regulation to define legal limits for the tolerable heat load of ESL milk as soon as possible.

Determination of antimicrobial wild-type minimum inhibitory concentration (MIC) distributions is a prerequisite before differentiating susceptible bacteria from bacteria with acquired resistance. The antimicrobial susceptibility was determined for 190 strains of eight species of the Lactobacillus delbrueckii group. These were L. acidophilus, L. amylovorus, L. crispatus, L. delbrueckii, L. helveticus, L. gallinarum, L. gasseri and L. johnsonii. For most antimicrobial agents a clear distinction between susceptible and resistant bacteria was observed. Tetracycline resistance was abundant among L. johnsonii and L. amylovorus strains isolated from animals, while the L. delbrueckii and L. helveticus strains isolated from dairy products, and L. gasseri isolated from humans, rarely contained acquired resistance.

Time temperature integrators (TTIs) are useful tools in estimating the heat load applied on differently processed dairy products. The objective of this study was to analyze and assess three TTIs – lactulose, furosine, and acid-soluble β-lactoglobulin (β-Lg) – in 70 high heated dairy products at retail in Austria and Germany comprising whipping cream, coffee cream/milk, and condensed milk products. While β-Lg was not appropriate to evaluate the heat load of these products, furosine and especially lactulose increased with rising intensity of heat treatment, and are appropriate to distinguish between several heating categories analyzed. Pasteurized (n = 8) and “heat treated” (n = 5) whipping cream samples showed lowest furosine (48 ± 14/ 45 ± 19 mg.100 g−1 protein) and low lactulose (29 ± 10/57 ± 28 mg.L−1) concentrations, followed by ESL whipping cream (n = 10), ESL coffee cream (n = 1), and UHT whipping cream (n = 10) (furosine = 72 ± 37/71/161 ± 30 mg.100 g−1 protein; lactulose = 56 ± 41/161/195 ± 39 mg.L−1), respectively. Sterilized condensed milk samples (n = 14) showed the highest concentrations of both TTIs and could be clearly separated from UHT treated samples (n = 5) (furosine = 491 ± 196/216 ± 46 mg.100 g−1 protein; lactulose = 1997 ± 658/409 ± 161 mg.L−1), whereas the so-called heat-treated samples (n = 9) had a heat load in between showing an extreme range of variation for both TTIs.

There is urgent need for having available suitable methods and data regarding the susceptibility levels of antibiotic resistant and sensitive strains of bifidobacteria. Based on a defined standard operation procedure, agar disc diffusion and broth microdilution were compared in order to evaluate the antimicrobial susceptibility profiles of 82 B. pseudolongum and 80 B. thermophilum strains mainly originating from the meat production chain. The methods that were assessed showed interpretable agreement within this study. The disc diffusion zone diameters are highly reproducible making the method a useful alternative to broth microdilution for antimicrobial susceptibility screening of bifidobacteria.

Summary Kefirs were produced under laboratory conditions using 10 different kefir grain cultures from different households, and reconstituted cow, ewe, goat and mare milk as substrates. The base milks and corresponding kefirs were examined for their content of water-soluble vitamins and orotic acid. On average, enrichment of vitamin concentration of>20% was observed with thiamine (only ewe milk kefirs), pyridoxine (kefirs from all milk species except cow milk), and folic acid (kefirs from all milk species except mare milk). Orotic acid content was reduced during fermentation throughout.

Fifteen new isolates from pig faeces were identified as Bifidobacterium animalis subsp. lactis by polymerase chain reaction (PCR) using primers specific for this subspecies. Ten of the isolates could be differentiated at strain level by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). These new strains were different from the type strain and other reference strains of this subspecies, and from all commercial dairy strains with probiotic functionality. Thus, possibly some of the isolates are potential candidates for new probiotic Bifidobacterium strains that can be unambiguously identified by RAPD-PCR and PFGE, which could be of interest for the food industry. In contrast, reference strains and commercial dairy strains revealed great genetic homogeneity, showing almost identical DNA fingerprints using RAPD-PCR and PFGE. Some reference strains and all commercial probiotic strains that had been originally designated as B. animalis or B. lactis have to be assigned to B. animalis subsp. lactis to be correctly labelled.

Different methods were applied to standard mixtures of milk from different species and to model cheeses of different ages to study their applicability for the detection of cows’ milk and determination of the percentage of cows’ milk in mixed milk cheeses. Urea–polyacrylamide gel electrophoresis (urea–PAGE) and anion-exchange high performance liquid chromatography (HPLC) of caseins was restricted to the control of the adulteration of milk. Isoelectric focusing (IEF) of γ-caseins according the EU reference method was well suited for detection of cows’ milk even in matured cheese, but could not discriminate ewes’ milk from goats’ milk. IEF and cation-exchange HPLC of para-κ-caseins were appropriate for differentiation of bovine, ovine and caprine milk. Urea–PAGE and IEF of whey proteins was useful for the species authentication of whey cheeses. Polymerase chain reaction using species–specific primers was a highly sensitive technique for the qualitative detection of cows’ milk even in overripe mixed cheese.

Forty potentially probiotic Lactobacillus strains as well as reference strains of different genera were grown under standardised conditions. Cell masses were harvested and DNA was isolated. For identification, all strains were subjected to genus-specific polymerase chain reaction (PCR), and the affiliation with the genus Lactobacillus was confirmed for all isolates. Using two species-specific primer-pairs for Lactobacillus reuteri, specific amplicons were observed for eight of the forty investigated strains. For differentiation, these eight strains as well as the reference strains of the species L. reuteri and closely related species were subjected to randomly amplified polymorphic DNA (RAPD)-PCR using fourteen arbitrary primers. Two selected strains as well as probiotic and common reference strains were further investigated applying pulsed field gel electrophoresis (PFGE). With the latter two methods, individual profiles were found for most strains, but no difference between probiotic and common strains could be made out.

This paper reviews the methodology applied for the identification and characterisation of enterococci and covers phenotypic, genotypic and phylogenetic techniques. Although conventional phenotypic typing schemes are useful for rapid and simple identification of enterococcal species for routine applications, other methods like standardised sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), multilocus enzyme electrophoresis (MLEE), antimicrobial susceptibility testing, serotyping, pyrolysis mass spectrometry (pyMS) and vibrational spectroscopic methods allow a more in-depth characterisation of enterococci. Many of the recently described enterococcal species exhibit deviations from hitherto so-called classical enterococci with regard to their phenotypical properties. Therefore, genotypic methods have to be used to clarify their possible assignment to the genus Enterococcus. In this review, special emphasis is given on recently developed polymerase chain reaction (PCR)-based typing methods such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), specific and random amplification (SARA) and modifications of PCR-ribotyping as well as pulsed-field gel electrophoresis (PFGE) and partial sequence analysis. The use of PCR and probes for genus and species identification of enterococci is also considered like the application of sequence data of conserved DNA regions (e.g., ribosomal ribonucleic acid (rRNA) genes) in the case of species identification.

Aims: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. Methods and Results: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. Conclusions: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. Significance and Impact of the Study: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.

The level of undenatured acid-soluble β-lactoglobulin can be used as an indicator to assess the heat load applied to liquid milk, thus further allowing the discrimination between milk originating from different thermal production processes. In this work, a new UHPLC method for the rapid determination of bovine β-lactoglobulin in 1.8min only (total runtime 3min) is presented using simple UV detection at 205nm. Separation selectivity for possibly co-eluting other major whey proteins (bovine serum albumin, lactoferrin, α-lactalbumin, immunoglobulin G) was verified, and the method validated for the analysis of liquid milk samples regarding linearity (20-560μg/mL, R(2)>0.99), instrumentation precision (RSDs

In Austria, a big manufacturer of processed cheese was faced with a mysterious problem of bitterness and a texture defect in processed cheese slices occurring in distinct batches every once in a while and unexpectedly. To clear up this phenomenon, reference samples of good quality as well as problem cheeses were analysed for chemical composition. Proteolytic changes were studied by urea-polyacrylamide gel electrophoresis (PAGE), SDS-PAGE and isoelectric focusing of caseins as well as of soluble nitrogen fractions. Peptides of the water-soluble nitrogen fractions were analysed by reversed phase-high performance liquid chromatography (RP-HPLC). Surprisingly, bitter cheese samples showed very weak or even no bands in the α s1 - and β -casein region, but only γ -caseins and mainly low molecular weight peptides, which was also confirmed by Kjeldahl analysis. In comparison with the respective reference samples, RP-HPLC profiles of bitter cheese samples showed extremely high concentrations of hydrophilic and hydrophobic peptides. Since significantly higher concentrations of ash and phosphorus were detected in bitter cheese samples, it was noticed that the respective batches of processed cheese slices had been produced apparently using an overdose of a specific emulsifying agent (of high phosphorus content).

The effects of the indigenous raw milk flora on the chemical composition of Austrian Bergkâse was evaluated by means of repeated cheesemaking experiments from raw or pasteurised milk. GeneraIly, changes caused by the pasteurisation of raw milk did not significantly affect the gross com- ( position of the resulting cheeses but, as monitored by sampIing during maturation, systematically showed effects on proteolysis, Whereas degradation of ast-casein proved to be delayed in cheeses made from pasteurised milk due to effects caused by heat treatment and partial eIimination of the raw milk flora, an enhanced cIeavage of l3-casein was found in cheeses from pasteurised milk. These differences were also evident in the water-soluble fraction and, partly, in fractions containing smaller breakdown products and were presumably caused by heat-induced changes in the plasmin/plas- minogen complex. Additionally, effects of heat treatment and of indigenous raw milk flora on alka- line phosphatase and Iipolysis were observed. © InralElsevier, Paris. Bergkâse / raw milk flora / pasteurisation / composition / proteolysis

The proteolytic changes occurring during the ripening of Parmesan cheese were studied using urea-polyacrylamide gel electrophoresis (urea-PAGE) of caseins, HPLC analysis offree amino acids (FAA) and Kjeldahl determination of soluble nitrogen fractions. An electrophoretic ripening index for the evaluation of proteolysis in Parmesan cheese was established. The separation of caseins by alkaline PAGE (12 % T, 2.6 % C, pH 8.9, 5 M urea) was followed by the densitometric analysis of the y- and ~-casein fractions. The relationship between the resulting coefficients (y-Cn/~-Cn) and the age of reference samples of Original ltalian Grana Padano (6-22 months) was linear up to 15 months, allowing an evaluation of the extent of proteolysis and therefore a deduction of the age of the Parmesan samples analysed. Threshold levels (y-Cn/~-Cn) were proposed for the verification of the required age of Parmesan cheese. The coefficients (y-Cn/~-Cn) as weil as the 13-casein content of two addition al series of references and of 117 commercial Parmesan sampI es are presented. Commercial Parmesan samples retailed as a loaf or as prepacked slices were generally found to fulfill the requirements concerning endoproteolytic changes during ripening. However, man y grated Parmesan samples taken from retail outlets in Austria showed poor quality, which was probably due to the adulteration with products with low proteolysis (e.g., cheese rind, very young cheese). HPLC analysis ofFAA was also used to characterize the ripening process. Although FAA content ofreference samples of Grana Padano showed a very high degree of variability, a distinct increase of FAA content could be obser- ved during the ripening period. However, two series of reference samples, which had completely different electrophoretic casein patterns, could not be distinguished by HPLC analysis of the total amount of FAA. As an experiment, two loaves of Parmesan cheese were removed from the ripe- ning room at an age of 4 and 2 months and were subsequently stored at 6 "C for 18 months. During the storage period, endoproteolytic changes were slowed down and breakdown of C:XS1-and ~-casein as weil as the accumulation of degradation products proceeded at a reduced rate, and could be detected using urea-PAGE casein patterns, but not by HPLC analysis of FAA content. In conse- quence of these findings, no accurate evaluation of the age of commercial Parmesan samples was pos- sible by means of the total amount of FAA. Since Kjeldahl determination of WSN and HPLC analysis of FAA content give insufficient information about the distinct endoproteolytic changes, which were found to be typical for Parmesan cheese, urea-PAGE of the casein fraction has to be do ne to enable * Oral communication at the IDF symposium 'Ripening and quality of cheeses', Besancon, France

Using standard mixtures of milk as well as model Camembert cheeses of different ages, quantitative determination of the percentage of cows' milk in mixed-milk cheese was performed by isoelectric focusing (IEF) and subsequent densitometric evaluation of γ-caseins (Cn). Because ewes' and goats' milk cannot be distinguished by this method, bovine, ovine and caprine milks were differentiated using IEF and cation-exchange high performance liquid chromatography (HPLC) of para-κ-Cn. As they were not substantially affected by the degree of ripening, these methods were well suited for determination of the percentages of cows', ewes' and goats' milk in mixed cheese. Obviously, because of the different casein content of ewes' and goats' milk, different calibration curves had to be used for cheeses made either from cows' and ewes' milk or from cows' and goats' milk. As the estimated percentage of cows' milk in mixed cheese is greatly affected by the casein content of the milks used for cheesemaking, quantitative results in adulteration control have to be regarded as approximate values.

The effects of composite milk protein phenotypes on the composition and cheesemaking properties of milk were studied. Pooled milks from 12 selected groups of Brown cattle with particular phenotype combinations of β-casein (A2A2, A2B), κ-casein (AA, AB, BB) and β-lactoglobulin (AA, BB) were investigated. αs1-Cn BB was common to all animals. Milk collection and cheesemaking trials on a pilot scale were repeated four times during winter 1993. The interaction between all three gene loci (β-Cn, κ-Cn, β-Lg) was found to be highly significant (P < 0.001) for the contents of total protein, casein, whey protein and fat. The casein number was 0–4% (absolute) higher for β-Lg BB milk than for β-Lg AA milk. The relative amount of κ-casein consistently increased from κ-Cn AA to κ-Cn BB. The losses of fat and curd fines in the whey and moisture-adjusted cheese yield were also affected by the genotype combinations. Recovery of milk solids was 3% higher for β-Cn A2B milk than for β-Cn A2A2 milk (P = 0.010).

Dans un grand nombre d'experimentations faites a grande echelle, des emmentals ont ete fabriques a partir de lait cru, avec addition de 0, 10 et 20 % d'eau au melange caille/lactoserum apres decoupage du caille. Pendant l'affinage de 16 semaines dans des conditions standards, des echantillons appropries ont ete preleves dans les meules de fromages et soumis a des analyses. Les resultats montrent que les parametres de composition, c'est-a-dire les teneurs en matiere grasse, matiere seche et calcium, etaient en partie influences par l'affinage, mais restaient totalement inaffectes par le pourcentage d'eau ajoutee. Toutefois, la dilution du lactoserum reduisait la quantite de lactose fermentescible des fromages non affines et entrainait des differences dans le pH et la teneur en acide lactique. Du fait de l'acceleration du developpement des bacteries propioniques induite par le pH, la degradation des lactates a ete trouvee plus prononcee dans les emmentals fabriques avec une addition de 20 % d'eau. En consequence, les taux eleves d'acide propionique et l'acceleration de la formation de l'ouverture etaient evidents dans de tels fromages. D'un autre cote, la quantite croissante d'eau additionnee au melange caille/lactoserum entrainait une reduction de la degradation de la caseine α s1 et une baisse des parametres de proteolyse secondaire. De plus, la contrainte et la deformation a la fracture apparente etaient tres affectees par l'addition d'eau. Les emmentals fabriques avec 20 % d'eau montrent un taux significativement plus eleve de contrainte a la fracture, qui peut etre percu comme une mesure instrumentale de la fermete sensorielle. Comme de plus grands taux de deformation a la fracture impliquent generalement une elasticite sensorielle croissante du fromage, la technologie de l'addition d'eau semble etre un outil approprie pour minimiser les defectuosites sensorielles (fromage a « pâte courte » de consistance), ameliorer la capacite de stockage et diminuer le risque d'une fermentation tardive.

A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag™ method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence (λ(Ex) 250 nm/λ(Em) 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200-600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.