Identifying Plasma and Urinary Biomarkers of Fermented Food Intake and Their Associations with Cardiometabolic Health in a Dutch Observational Cohort.

Identification of food intake biomarkers (FIBs) for fermented foods could help improve their dietary assessment and clarify their associations with cardiometabolic health. We aimed to identify novel FIBs for fermented foods in the plasma and urine metabolomes of 246 free-living Dutch adults using nontargeted LC-MS and GC-MS. Furthermore, associations between identified metabolites and several cardiometabolic risk factors were explored. In total, 37 metabolites were identified corresponding to the intakes of coffee, wine, and beer (none were identified for cocoa, bread, cheese, or yoghurt intake). While some of these metabolites appeared to originate from raw food (e.g., niacin and trigonelline for coffee), others overlapped different fermented foods (e.g., 4-hydroxybenzeneacetic acid for both wine and beer). In addition, several fermentation-dependent metabolites were identified (erythritol and citramalate). Associations between these identified metabolites with cardiometabolic parameters were weak and inconclusive. Further evaluation is warranted to confirm their relationships with cardiometabolic disease risk.

Associations between dairy fat intake, milk-derived free fatty acids, and cardiometabolic risk in Dutch adults.

PURPOSE: Milk-derived free fatty acids (FFAs) may act as both biomarkers of intake and metabolic effect. In this study we explored associations between different types of dairy consumption, a selection of milk-derived free fatty acids, and cardiometabolic disease (CMD) risk factors. METHODS: Sixty-seven FFAs were quantified in the plasma of 131 free-living Dutch adults (median 60 years) using gas chromatography-flame ionization detector. Intakes of different dairy foods and groups were assessed using a food frequency questionnaire. Twelve different CMD risk factors were analyzed. Multiple linear regressions were used to evaluate the associations under study. RESULTS: Based on the fully adjusted models, 5 long-chain unsaturated FFAs (C18:1 t13 + c6 + c7 + u, C18:2 c9t11 + u, C20:1 c11, C20:3 c8c11c14, and C20:4 c5c8c11c14), 2 medium-chain saturated FFAs (C15, C15 iso), and a trans FFA (C16:1 t9) were positively associated with at least one variable of dairy intake, as well as plasma total and LDL cholesterol, blood pressure, and SCORE (p ≤ 0.05). A long-chain PUFA associated with high-fat fermented dairy intake (C18:2 t9t12), was negatively associated with serum triglyceride levels, and a long-chain saturated FFA associated with cheese intake (C18:1 u1) was negatively associated with plasma LDL cholesterol and serum triglyceride levels. No clear associations were observed between dairy intake and CMD risk factors. CONCLUSION: Milk-derived FFAs could act as sensitive biomarkers for dairy intake and metabolism, allowing the association between dairy and CMD risk to be more precisely evaluated.

Validated method for the determination of free volatile carboxylic acids in cheese and bacterial cultures by GC-FID after esterification in aqueous extract and headspace injection.

A simple, rapid, sensitive and robust gas chromatographic method was developed for the simultaneous determination of free volatile carboxylic acids (FVCA) in cheese and bacterial cultures. The target analytes were extracted and converted directly from the aqueous phase to their ethyl esters using headspace. The lower detection limits for the volatile carboxylic acids in the cheese samples were less than 0.3 and less than 0.6 µmol kg-1 in the bacterial culture samples. The lower limits of quantitation in cheese were better than 0.001 mmol kg-1 for all analytes. The upper limits of quantitation varied from 39 to 136 mmol kg-1 in cheese and 78 to 272 mmol kg-1 in bacterial cultures depending on the analyte. The Horwitz ratio showed good precision for all analytes (less than 0.77). The proposed method is suitable for the determination of target metabolites directly from aqueous extracts and can also be validated for other matrices.

Serum and Urine Metabolites in Healthy Men after Consumption of Acidified Milk and Yogurt.

The identification of molecular biomarkers that can be used to quantitatively link dietary intake to phenotypic traits in humans is a key theme in modern nutritional research. Although dairy products (with and without fermentation) represent a major food group, the identification of markers of their intake lags behind that of other food groups. Here, we report the results from an analysis of the metabolites in postprandial serum and urine samples from a randomized crossover study with 14 healthy men who ingested acidified milk, yogurt, and a non-dairy meal. Our study confirms the potential of lactose and its metabolites as markers of lactose-containing dairy foods and the dependence of their combined profiles on the fermentation status of the consumed products. Furthermore, indole-3-lactic acid and 3-phenyllactic acid are two products of fermentation whose postprandial behaviour strongly discriminates yogurt from milk intake. Our study also provides evidence of the ability of milk fermentation to increase the acute delivery of free amino acids to humans. Notably, 3,5-dimethyloctan-2-one also proves to be a specific marker for milk and yogurt consumption, as well as for cheese consumption (previously published data). These molecules deserve future characterisation in human interventional and observational studies.

Comparison of nutritional composition between plant-based drinks and cow's milk.

The high decline in liquid milk consumption in Western countries has been compensated by the increased consumption of processed dairy products and the rapidly increasing number of new plant-based beverages constantly introduced in the market, advertised as milk substitutes and placed on shelves near milk products. To provide better understanding about the nutritional value of these drinks compared with cow's milk, 27 plant-based drinks of 8 different species and two milk samples were purchased from two big retailers in Switzerland, and their composition regarding protein, carbohydrate, fat, vitamin, and mineral contents and residue load [glyphosate, aminomethylphosphonic acid (AMPA), and arsenic] was analyzed quantitatively and qualitatively. Energy and nutrient intakes were calculated and compared with the dietary reference values for Germany, Austria and Switzerland (D-A-CH). In addition, the digestible indispensable amino acid score (DIAAS) was calculated to estimate the quality of the proteins. Milk contained more energy; fat; carbohydrate; vitamins C, B2, B12, and A; biotin; pantothenic acid; calcium; phosphorus; and iodine than most plant-based drinks. Soy drinks provided slightly more protein and markedly more vitamins B1 and B6, folic acid, and vitamins E and D2 (with supplemented vitamin D2) and K1, magnesium, manganese, iron, and copper than milk and the other plant-based drinks. However, with the exception of cow's milk and soy drinks, which had > 3% protein, most milk alternatives contained ≤ 1% protein; therefore, they cannot be considered good protein sources. In regard to protein quality, milk was outstanding compared with all plant-based drinks and exhibited higher calculated DIAASs. Our results show that the analyzed plant-based drinks are not real alternatives to milk in terms of nutrient composition, even if the actual fortification is taken into account. Improved fortification is still an issue and can be optimized using the most bioavailable and soluble derivatives. Complete replacement of milk with plant-based drinks without adjusting the overall diet can lead to deficiencies of certain important nutrients in the long term.

Molecular effects of the consumption of margarine and butter varying in trans fat composition: a parallel human intervention study.

BACKGROUND: Whereas the dietary intake of industrial trans fatty acids (iTFA) has been specifically associated with inflammation, cardiovascular disease, and type 2 diabetes, understanding the impact of dietary fats on human health remains challenging owing to their complex composition and individual effects of their lipid components on metabolism. The aim of this study is to profile the composition of blood, measured by the fatty acid (FAs) profile and untargeted metabolome of serum and the transcriptome of blood cells, in order to identify molecular signatures that discriminate dietary fat intakes. METHODS: In a parallel study, the molecular effects of consuming dairy fat containing ruminant TFA (rTFA) or margarine containing iTFA were investigated. Healthy volunteers (n = 42; 45-69 y) were randomly assigned to diets containing margarine without TFA as major source of fat (wTFA control group with 0.4 g TFA per 100 g margarine), margarine with iTFA (iTFA group with 4.1 g TFA per 100 g margarine), or butter with rTFA (rTFA group with 6.3 g TFA per 100 g butter) for 4 weeks. The amounts of test products were individually selected so that fat intake contributed to 30-33% of energy requirements and TFA in the rTFA and iTFA groups contributed to up to 2% of energy intake. Changes in fasting blood values of lipid profiles (GC with flame-ionization detection), metabolome profiles (LC-MS, GC-MS), and gene expression (microarray) were measured. RESULTS: Eighteen FAs, as well as 242 additional features measured by LC-MS (185) and GC-MS (54) showed significantly different responses to the diets (PFDR-adjusted < 0.05), mainly distinguishing butter from the margarine diets while gene expression was not differentially affected. The most abundant TFA in the butter, i.e. TFA containing (E)-octadec-11-enoic acid (C18:1 t11; trans vaccenic acid), and margarines, i.e. TFA containing (E)-octadec-9-enoic acid (C18:1 t9; elaidic acid) were reflected in the significantly different serum levels of TFAs measured after the dietary interventions. CONCLUSIONS: The untargeted serum metabolome differentiates margarine from butter intake although the identification of the discriminating features remains a bottleneck. The targeted serum FA profile provides detailed information on specific molecules differentiating not only butter from margarine intake but also diets with different content of iTFAs in margarine. TRIAL REGISTRATION: ClinicalTrials.gov NCT00933322.

Postprandial Responses on Serum Metabolome to Milk and Yogurt Intake in Young and Older Men.

The identification and validation of biomarkers of food intake (BFIs) is a promising approach to develop more objective and complementary tools to the traditional dietary assessment methods. Concerning dairy, their evaluation in terms of intake is not simple, given the variety of existing foods, making it difficult to establish the association between specific dairy products consumption and the effects on human health, which is also dependent on the study population. Here, we aimed at identifying BFI of both milk (M) and yogurt (Y) in 14 healthy young (20-35 years) and 14 older (65-80 years). After a 3-week run-in period of dairy exclusion from the diet, the subjects acutely consumed 600 ml of M or Y. Metabolomics analyses were conducted on serum samples during the following 6 h (LC-MS and GC-MS). Several metabolites showing increased iAUC after milk or yogurt intake were considered as potential BFI, including lactose (M > Y, 2-fold), galactitol (M > Y, 1.5-fold), galactonate (M > Y, 1.2-fold), sphingosine-1-phosphate (M > Y from 2.1-fold), as well as an annotated disaccharide (Y > M, 3.6-fold). Delayed serum kinetics were also observed after Y compared to M intake lysine (+22 min), phenylalanine (+45 min), tyrosine (+30min), threonine (+38 min) 3-phenyllactic acid (+30 min), lactose (+30 min), galactitol (+45min) and galactonate (+30 min). The statistical significance of certain discriminant metabolites, such as sphingosine-1-phosphate and several free fatty acids, was not maintained in the older group. This could be related to the physiological modifications induced by aging, like dysregulated lipid metabolism, including delayed appearance of dodecanoic acid (+60 min) or altered postprandial appearance of myristic acid (+70% Cmax), 3-dehydroxycarnitine (-26% Cmin), decanoylcarnitine (-51% Cmin) and dodecanoylcarnitine (-40% Cmin). In conclusion, candidate BFI of milk or yogurt could be identified based on the modified postprandial response resulting from the fermentation of milk to yogurt. Moreover, population specificities (e.g., aging) should also be considered in future studies to obtain more accurate and specific BFI.

Evaluating the Robustness of Biomarkers of Dairy Food Intake in a Free-Living Population Using Single- and Multi-Marker Approaches.

Studies examining associations between self-reported dairy intake and health are inconclusive, but biomarkers hold promise for elucidating such relationships by offering objective measures of dietary intake. Previous human intervention studies identified several biomarkers for dairy foods in blood and urine using non-targeted metabolomics. We evaluated the robustness of these biomarkers in a free-living cohort in the Netherlands using both single- and multi-marker approaches. Plasma and urine from 246 participants (54 ± 13 years) who completed a food frequency questionnaire were analyzed using liquid and gas chromatography-mass spectrometry. The targeted metabolite panel included 37 previously-identified candidate biomarkers of milk, cheese, and/or yoghurt consumption. Associations between biomarkers and energy-adjusted dairy food intakes were assessed by a 'single-marker' generalized linear model, and stepwise regression was used to select the best 'multi-marker' panel. Multi-marker models that also accounted for common covariates better captured the subtle differences for milk (urinary galactose, galactitol; sex, body mass index, age) and cheese (plasma pentadecanoic acid, isoleucine, glutamic acid) over single-marker models. No significant associations were observed for yogurt. Further examination of other facets of validity of these biomarkers may improve estimates of dairy food intake in conjunction with self-reported methods, and help reach a clearer consensus on their health impacts.

Serum Metabolites Responding in a Dose-Dependent Manner to the Intake of a High-Fat Meal in Normal Weight Healthy Men Are Associated with Obesity.

Although the composition of the human blood metabolome is influenced both by the health status of the organism and its dietary behavior, the interaction between these two factors has been poorly characterized. This study makes use of a previously published randomized controlled crossover acute intervention to investigate whether the blood metabolome of 15 healthy normal weight (NW) and 17 obese (OB) men having ingested three doses (500, 1000, 1500 kcal) of a high-fat (HF) meal can be used to identify metabolites differentiating these two groups. Among the 1024 features showing a postprandial response, measured between 0 h and 6 h, in the NW group, 135 were dose-dependent. Among these 135 features, 52 had fasting values that were significantly different between NW and OB men, and, strikingly, they were all significantly higher in OB men. A subset of the 52 features was identified as amino acids (e.g., branched-chain amino acids) and amino acid derivatives. As the fasting concentration of most of these metabolites has already been associated with metabolic dysfunction, we propose that challenging normal weight healthy subjects with increasing caloric doses of test meals might allow for the identification of new fasting markers associated with obesity.

Microbiota and Metabolite Modifications after Dietary Exclusion of Dairy Products and Reduced Consumption of Fermented Food in Young and Older Men.

The gut microbiota adapts to age-related changes in host physiology but is also affected by environmental stimuli, like diet. As a source of both pre- and probiotics, dairy and fermented foods modulate the gut microbiota composition, which makes them interesting food groups to use for the investigation of interactions between diet and ageing. Here we present the effects of excluding dairy products and limiting fermented food consumption for 19 days on gut microbiota composition and circulating metabolites of 28 healthy, young (YA) and older (OA) adult men. The intervention affected gut microbial composition in both groups, with significant increases in Akkermansia muciniphila and decreases in bacteria of the Clostridiales order. Lower fasting levels of glucose and insulin, as well as dairy-associated metabolites like lactose and pentadecanoic acid, were observed after the intervention, with no effect of age. The intervention also decreased HDL and LDL cholesterol levels. Dairy fat intake was positively associated with the HDL cholesterol changes but not with the LDL/HDL ratio. In conclusion, restricting the intake of dairy and fermented foods in men modified their gut microbiota and blood metabolites, while the impact of the dietary restrictions on these outcomes was more marked than the effect of age.

Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.

Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI®) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

Development and performance evaluation of a novel dynamic headspace vacuum transfer "In Trap" extraction method for volatile compounds and comparison with headspace solid-phase microextraction and headspace in-tube extraction.

Headspace in-tube extraction (HS-ITEX) and solid phase microextraction (HS-SPME) sampling, followed by gas chromatography-mass spectrometry (GC-MS), are widely used to analyze volatile compounds in various food matrices. While the extraction efficiency of volatile compounds from foodstuffs is crucial for obtaining relevant results, these efficiency of these extraction methods limited by their long extraction times and requirements for large sample quantity. This study reports on the development and application of a new extraction technique based on HS-ITEX hardware, which improves the extraction rate and capacity by operating under reduced pressure, called Dynamic Headspace Vacuum Transfer In-Trap Extraction (DHS-VTT). The results of the study indicate that DHS-VTT improves the extraction of the target compounds. The area of the mass spectrometer signal for each compound can be up to 450 times more intense than the HS-SPME and HS-ITEX techniques performed in the same experimental conditions of extraction temperature and time. DHS-VTT runs in automated mode, making it possible to work with smaller sample quantity and also favors the HS extraction of all volatile compounds. In addition, the necessary modifications to the installation were cheap and the life of an ITEX trap is up to 10 times longer than an SPME fibre.

Assessment of lactase activity in humans by measurement of galactitol and galactonate in serum and urine after milk intake.

Background: Lactase is an enzyme that hydrolyzes lactose into glucose and galactose in the small intestine, where they are absorbed. Hypolactasia is a common condition, primarily caused by genetic programming, that leads to lactose maldigestion and, in certain cases, lactose intolerance. Galactitol and galactonate are 2 products of hepatic galactose metabolism that are candidate markers for the intake of lactose-containing foods. Objectives: The primary objective of the study was to explore the changes in serum and urine metabolomes during postprandial dairy product tests through the association between lactase persistence genotype and the postprandial dynamics of lactose-derived metabolites. Methods: We characterized the 6-h postprandial serum kinetics and urinary excretion of lactose, galactose, galactitol, and galactonate in 14 healthy men who had consumed a single dose of acidified milk (800 g) which contained 38.8 g lactose. Genotyping of LCT-13910 C/T (rs4988235) was performed to assess primary lactase persistence. Results: There were 2 distinct postprandial responses, classified as high and low metabolite responses, observed for galactose, and its metabolites galactitol and galactonate, in serum and urine. In all but 1 subject, there was a concordance between the high metabolite responses and genetic lactase persistence and between the low metabolite responses and genetic lactase nonpersistence (accuracy 0.92), galactitol and galactonate being more discriminative than galactose. Conclusions: Postprandial galactitol and galactonate after lactose overload appear to be good proxies for genetically determined lactase activity. The development of a noninvasive lactose digestion test based on the measurement of these metabolites in urine could be clinically useful. This trial was registered at clinicaltrials.gov as NCT02230345.

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies.

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state-of-the-art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

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.

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.

Blood lactose after dairy product intake in healthy men.

The absence of a dedicated transport for disaccharides in the intestine implicates that the metabolic use of dietary lactose relies on its prior hydrolysis at the intestinal brush border. Consequently, lactose in blood or urine has mostly been associated with specific cases in which the gastrointestinal barrier is damaged. On the other hand, lactose appears in the blood of lactating women and has been detected in the blood and urine of healthy men, indicating that the presence of lactose in the circulation of healthy subjects is not incompatible with normal physiology. In this cross-over study we have characterised the postprandial kinetics of lactose, and its major constituent, galactose, in the serum of fourteen healthy men who consumed a unique dose of 800 g milk or yogurt. Genetic testing for lactase persistence and microbiota profiling of the subjects were also performed. Data revealed that lactose does appear in serum after dairy intake, although with delayed kinetics compared with galactose. Median serum concentrations of approximately 0·02 mmol/l lactose and approximately 0·2 mmol/l galactose were observed after the ingestion of milk and yogurt respectively. The serum concentrations of lactose were inversely correlated with the concentrations of galactose, and the variability observed between the subjects' responses could not be explained by the presence of the lactase persistence allele. Finally, lactose levels have been associated with the abundance of the Veillonella genus in faecal microbiota. The measurement of systemic lactose following dietary intake could provide information about lactose metabolism and nutrient transport processes under normal or pathological conditions.

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.

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.