Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate.

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.

Physiological and molecular changes of onion (Allium cepa L.) seeds under different aging conditions.

BACKGROUND: Onion seeds have limited storage capacity compared to other vegetable seeds. It is crucial to identify the mechanisms that induce tolerance to storage conditions and reduce seed deterioration. To address this goal, an experiment was conducted to evaluate changes in germination, biochemical, physiological, and molecular characteristics of onion seed landraces (Horand, Kazerun landraces and Zargan cultivar) at different aging levels (control, three-days and six-days accelerated aging, and natural aging for one year). RESULTS: The findings suggest that there was an increase in glucose, fructose, total sugar, and electrolyte leakage in the Horand (HOR), Kazerun (KAZ) landraces, and Zarghan (ZAR) cultivar, with Kazerun exhibiting the greatest increase. The percentage and rate of germination of Kazerun decreased by 54% and 33%, respectively, in six-day accelerated aging compared to the control, while it decreased by 12% and 14%, respectively, in Horand. Protein content decreased with increasing levels of aging, with a decrease of 26% in Kazerun landrace at six days of aging, while it was 16% in Horand landrace. The antioxidant activities of catalase, superoxide dismutase, and glutathione peroxidase decreased more intensively in Kazerun. The expression of AMY1, BMY1, CTR1, and NPR1 genes were lower in Kazerun landraces than in Horand and Zargan at different aging levels. CONCLUSIONS: The AMY1, BMY1, CTR1, and NPR1 genes play a pivotal role in onion seed germination, and their downregulation under stressful conditions has been shown to decrease germination rates. In addition, the activity of CAT, SOD, and GPx enzymes decreased by seed aging, and the amount of glucose, fructose, total sugar and electrolyte leakage increased, which ultimately led to seed deterioration. Based on the results of this experiment, it is recommended to conduct further studies into the molecular aspects involved in onion seed deterioration. More research on the genes related to this process is suggested, as well as investigating the impact of different priming treatments on the genes expression involved in the onion seed aging process.

Determination of Carbohydrate Composition in Lentils Using Near-Infrared Spectroscopy.

Carbohydrates are the main components of lentils, accounting for more than 60% of their composition. Their content is influenced by genetic factors, with different contents depending on the variety. These compounds have not only been linked to interesting health benefits, but they also have a significant influence on the techno-functional properties of lentil-derived products. In this study, the use of near-infrared spectroscopy (NIRS) to predict the concentration of total carbohydrate, fibre, starch, total sugars, fructose, sucrose and raffinose was investigated. For this purpose, six different cultivars of macrosperm (n = 37) and microsperm (n = 43) lentils have been analysed, the samples were recorded whole and ground and the suitability of both recording methods were compared. Different spectral and mathematical pre-treatments were evaluated before developing the calibration models using the Modified Partial Least Squares regression method, with a cross-validation and an external validation. The predictive models developed show excellent coefficients of determination (RSQ > 0.9) for the total sugars and fructose, sucrose, and raffinose. The recording of ground samples allowed for obtaining better models for the calibration of starch content (R > 0.8), total sugars and sucrose (R > 0.93), and raffinose (R > 0.91). The results obtained confirm that there is sufficient information in the NIRS spectral region for the development of predictive models for the quantification of the carbohydrate content in lentils.

Effect of Storage Conditions on the Volatilome, Biochemical Composition and Quality of Golden Delicious and Red Delicious Apple (Malus domestica) Varieties.

The effects of normal (NA) and controlled atmosphere (CA) storage and postharvest treatment with 1-methylcyclopropene (1-MCP) before CA storage for 5 months on the volatilome, biochemical composition and quality of 'Golden Delicious' (GD) and 'Red Delicious' (RD) apples were studied. Apples stored under NA and CA maintained and 1-MCP treatment increased firmness in both cultivars. NA storage resulted in a decrease of glucose, sucrose and fructose levels in both cultivars. When compared to CA storage, 1-MCP treatment caused a more significant decrease in sucrose levels and an increase in glucose levels. Additionally, 1-MCP-treated apples exhibited a significant decrease in malic acid content for both cultivars. All storage conditions led to significant changes in the abundance and composition of the volatilome in both cultivars. GD and RD apples responded differently to 1-MCP treatment compared to CA storage; higher abundance of hexanoate esters and (E,E)-α-farnesene was observed in RD apples treated with 1-MCP. While 1-MCP was effective in reducing (E,E)-α-farnesene abundance in GD apples, its impact on RD apples was more limited. However, for both cultivars, all storage conditions resulted in lower levels of 2-methylbutyl acetate, butyl acetate and hexyl acetate. The effectiveness of 1-MCP is cultivar dependent, with GD showing better results than RD.

FODMAP Content Like-by-like Comparison in Spanish Gluten-free and Gluten-containing Cereal-based Products.

Gluten-free foods (GF) availability on supermarket shelves is growing and it is expected to continue expanding in the years ahead. These foods have been linked to a lower content of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs), molecules that trigger gastrointestinal symptoms in sensitive persons. In this study, the FODMAP content of 25 cereal-based GF foods in Spain (breakfast cereals, pasta, bread, biscuits, bakery products, and dough and puff pastry) and 25 gluten-containing equivalents (GC) available in the same supermarket were analysed and compared. Lactose, fructose, glucose, sorbitol, mannitol, raffinose, stachyose and fructans were quantified. In a like-by-like analysis, GF foods were found to generally contain fewer FODMAPs than their GC counterparts. The ingredients used in the manufacture of GF cereal-based foods may contribute to this fact. When the individually wrapped size was considered, the proportion of samples classified as high-FODMAPs in GC and GF foods showed a trend towards fewer samples in the GF. However, not all the GF samples were low-FODMAP. Altogether, our findings provide essential information for FODMAP content databases of GF products in Spain.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

Arginine-fructose-glucose from red ginseng extract reduces stiffness of keratin fiber in corneocyte of skin.

PURPOSE: The moisture content of the stratum corneum of the skin changes depending on the external environment. The structure of keratinous fiber protein in corneocyte of the skin changes depending on the amount of moisture. As the moisture decreases, the population of the alpha-helix increases, the beta-sheet deceases, and the stiffness increases accordingly. Here, we investigated the effect of humectants from ginseng on the keratin structure. METHODS: Corneocyte was prepared from dry porcine skin with disc tape and measured through ATR-FT-IR. The signal from amide I of the keratin protein in corneocyte was detected, and the change in the ratio of alpha-helix and beta-sheet was calculated. The test samples were treated on the exfoliated corneocyte, and the degree of change was checked. RESULT: Arginine-fructose-glucose (AFG)-enriched extract of red ginseng was effective in changing the keratin structure and was superior to humectants such as glycerin. However, arginine, mono sugar were not effective, and the AFG form in which two sugars were bound to one amino acid could perform its function. CONCLUSION: The present study suggests that AFG, when applied to cosmetics, is expected to improve skin texture in a different way from existing moisturizers represented by glycerin by reducing the alpha-helix structure of corneocyte keratin.

Determination of Sugars and Acids in Grape Must Using Miniaturized Near-Infrared Spectroscopy.

An automatic determination of grape must ingredients during the harvesting process would support cellar logistics and enables an early termination of the harvest if quality parameters are not met. One of the most important quality-determining characteristics of grape must is its sugar and acid content. Among others, the sugars in particular determine the quality of the must and wine. Chiefly in wine cooperatives, in which a third of all German winegrowers are organized, these quality characteristics serve as the basis for payment. They are acquired upon delivery at the cellar of the cooperative or the winery and result in the acceptance or rejection of grapes and must. The whole process is very time-consuming and expensive, and sometimes grapes that do not meet the quality requirements for sweetness, acidity, or healthiness are destroyed or not used at all, which leads to economic loss. Near-infrared spectroscopy is now a widely used technique to detect a wide variety of ingredients in biological samples. In this study, a miniaturized semi-automated prototype apparatus with a near-infrared sensor and a flow cell was used to acquire spectra (1100 nm to 1350 nm) of grape must at defined temperatures. Data of must samples from four different red and white Vitis vinifera (L.) varieties were recorded throughout the whole growing season of 2021 in Rhineland Palatinate, Germany. Each sample consisted of 100 randomly sampled berries from the entire vineyard. The contents of the main sugars (glucose and fructose) and acids (malic acid and tartaric acid) were determined with high-performance liquid chromatography. Chemometric methods, using partial least-square regression and leave-one-out cross-validation, provided good estimates of both sugars (RMSEP = 6.06 g/L, R2 = 89.26%), as well as malic acid (RMSEP = 1.22 g/L, R2 = 91.10%). The coefficient of determination (R2) was comparable for glucose and fructose with 89.45% compared to 89.08%, respectively. Although tartaric acid was predictable for only two of the four varieties using near-infrared spectroscopy, calibration and validation for malic acid were accurate for all varieties in an equal extent like the sugars. These high prediction accuracies for the main quality determining grape must ingredients using this miniaturized prototype apparatus might enable an installation on a grape harvester in the future.

Validation of an Eco-Friendly Automated Method for the Determination of Glucose and Fructose in Wines.

Fermentable sugar dosage helps oenologists to establish a harvest's moment and control the fermentation process of the musts. The official analyses recommended for their determination are long, laborious, and must be carried out by specialized personnel. On the contrary, instrumental analysis automation limits human errors, increases precision, and reduces the time and cost of the analyses. In the food production sector, to use methods other than those recommended by supranational bodies in official reports, it is necessary to validate the analytical processes to establish the conformity of the results between the new methods and the reference ones. This work validated an automated enzymatic apparatus to determine the sum of glucose and fructose levels in wine samples. The validation was carried out on wine samples (dry red wine, dry white wine, moderately sweet wine, and sweet wine) containing different sugar concentrations by comparing data obtained using the OIV-MA-AS311-02 method performed by a specialized operator (reference method) and the same method performed by an automated apparatus. The difference between the results' means obtained with the two procedures was significant. Nevertheless, the automated procedure was considered suitable for the intended use since the differences between the averages were lower than the measurement uncertainty at the same concentration, and the repeatability results were better for the automated procedure than the reference method.

Authentication and Geographical Characterisation of Italian Grape Musts through Glucose and Fructose Carbon Isotopic Ratios Determined by LC-IRMS.

The authenticity of grape musts is normally checked through a time-consuming stable isotopic analysis of carbon (δ13C) after fermentation and distillation by following the official OIV MA AS-312-06 method. In this study, the alternative use of a technique based on δ13C isotopic analysis of the major sugars of the grape must by liquid chromatography coupled with isotope ratio mass spectrometry (LC-IRMS) is provided. It allows not only the detection of the fraudulent addition to grape must of exogenous glucose and fructose deriving from C4 plants but also the characterisation of it based on its geographical origin. In order to discriminate between musts from different areas of Italy, a preliminary dataset was considered; the δ13C isotopic ratios of glucose and fructose of around 100 authentic samples were analysed. The two analysed parameters, ranging from -29.8‱ to -21.9‱, are well correlated (R2 = 0.7802) and the northern regions showed significantly more negative δ13C values for both sugars than the rest of the dataset.

Differentiation and Quantitation of Coeluting Isomeric Amadori and Heyns Peptides Using Sugar-Specific Fragment Ion Ratios.

d-glucose and d-fructose present in blood, tissues, and organs of all mammals can react with amino groups, leading to glucated (Amadori) and fructated (Heyns) products, i.e., proteins glycated at lysine residues. While typically present at low concentration in humans, metabolic diseases including diabetes elevate sugar levels, favoring glycation and consecutive reactions leading to advanced glycation end products (AGEs) linked to diabetic complications and cardiovascular diseases. Analytical methods able to differentiate and to individually quantify Amadori- and Heyns-modified proteins in complex sample mixtures, e.g., serum, are still very limited. Here, we show that the reported and supposedly specific neutral losses displayed in tandem mass spectra of Heyns peptides cannot be used for a reliable differentiation as they were also observed for Amadori peptides. However, the combination of several neutral loss signals in fragment ion ratios at both precursor and fragment ion signals allowed the differentiation and relative quantitation of coeluting isomeric Amadori and Heyns peptides at different concentrations and peptide ratios. This was also true for digested human plasma. Thus, the presented strategy allows the quantitation of Amadori and Heyns peptides in complex samples, especially by spiking isotope-labeled peptides. This will allow searching for glucated and fructated biomarkers in clinical samples.

Comparative study of sugar extraction procedures for HPLC analysis and proposal of an ethanolic extraction method for plant-based high-protein ingredients.

BACKGROUND: The increasing importance of plant-based proteins in the food sector makes a reliable compositional analysis of plant-based high-protein ingredients a necessity. Specifically, the quantification of short-chain carbohydrates is relevant for multiple areas, including food product development, food labelling and fundamental food chemistry and food technology research. Commonly used extraction procedures for subsequent high-performance liquid chromatographic separation and quantification of short-chain carbohydrates have been discussed controversially regarding a range of complications that can potentially lead to inaccurate sugar determination. The present study compares the sugar levels in wheat flour and wholemeal wheat flour determined with different aqueous and ethanolic extraction procedures. These procedures included measures to prevent enzyme activity and microbial growth, which represent two of the most relevant challenges in sugar extraction from food samples. RESULTS: Differences in sugar levels (sum of sucrose/maltose, glucose and fructose) as high as 1.8% dry matter (wheat flour) were observed between the employed extraction procedures. Ethanolic extraction (80% ethanol in ultrapure water) with the use of the antimicrobial agent sodium azide but without Carrez clarification was identified as most promising for sugar determination in plant-based high-protein ingredients. CONCLUSION: A screening of high-protein ingredients derived from cereals (wheat gluten), pseudocereals (quinoa, amaranth, buckwheat) and legumes (soy, pea, lupin, lentil, carob, chickpea, faba bean) concerning their levels of sucrose, maltose, glucose and fructose confirmed the applicability of the chosen extraction procedure. © 2021 Society of Chemical Industry.

96-Well Microtiter Plate Made of Paper: A Printed Chemosensor Array for Quantitative Detection of Saccharides.

Simple, rapid, and accurate detection methods for saccharides are potentially applicable to various fields such as clinical and food chemistry. However, the practical applications of on-site analytical methods are still limited. To this end, herein, we propose a 96-well microtiter plate made of paper as a paper-based chemosensor array device (PCSAD) for the simultaneous classification of 12 saccharides and the quantification of fructose and glucose among 12 saccharides. The mechanism of the saccharide detection relied on an indicator displacement assay (IDA) on the PCSAD using four types of catechol dyes, 3-nitrophenylboronic acid, and the saccharides. The design of the PCSAD and the experimental conditions for the IDA were optimized using a central composite design. The chemosensors exhibited clear color changes upon the addition of saccharides on the paper because of the competitive boronate esterification. The color changes were employed for the subsequent qualitative, semiquantitative, and quantitative analyses using an automated algorithm combined with pattern recognition for digital images. A qualitative linear discrimination analysis offered discrimination of 12 saccharides with a 100% classification rate. The semiquantitative analysis of fructose in the presence of glucose was carried out from the viewpoint of food analysis utilizing a support vector machine, resulting in clear discrimination of the various concentrations of fructose. Most importantly, the quantitative detection of fructose in two types of commercial soft drinks was also successfully carried out without sample pretreatments. Thus, the proposed PCSAD can be a powerful method for on-site food analyses that can meet the increasing demand from consumers for sensors of saccharides.

Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1.

BACKGROUND: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. RESULTS: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. CONCLUSIONS: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated "metabolic burden". Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

Quality evaluation and geographical classification of immature rape and acacia honeys in China.

BACKGROUND: To reflect and understand the real situation regarding immature honey, the quality parameters of 141 honey samples from different regions of China were evaluated. The possibility of classifying Chinese honey from different geographical origins was also considered. RESULTS: The results revealed that the values of diastase, sucrose, proline and 5-hydroxymethylfurfural (HMF) met the international honey standard, although moisture, total glucose and fructose, and invertase content in most of the honey samples failed to meet the standards. In addition, honey from different geographical locations could be well separated based on the contents of maltose, sucrose, HMF and glucose oxidase. CONCLUSION: The maturity of honey has a great impact on its quality, and the geographical origin of immature honey could be classified according to the physicochemical parameters. © 2021 Society of Chemical Industry.

Carbohydrate composition in breast milk and its effect on infant health.

PURPOSE OF REVIEW: This narrative review presents the current state of available evidence regarding the role of breast milk carbohydrates on infant outcomes, with a primary focus on growth and body composition. RECENT FINDINGS: To date, there is a paucity of available data that exists in this realm. The current literature focuses on the role of two carbohydrate fractions in breast milk, and their relationships with infant outcomes in the first six months of life: oligosaccharides and fructose. A small but growing body of research indicates robust associations of both oligosaccharides and fructose in breast milk with infant weight and length, as well as bone, fat, and lean mass. There is also emerging evidence to support the role of these same carbohydrate fractions in breast milk in infant cognitive development. SUMMARY: The present state of the science suggests that oligosaccharides and fructose in breast milk play a role in infant growth and body composition and introduces intriguing associations of these two carbohydrate fractions with infant cognitive development as well.

Diagnostic Utility of Carbohydrate Breath Tests for SIBO, Fructose, and Lactose Intolerance.

BACKGROUND: Unexplained bloating, gas, and pain are common symptoms. If routine tests are negative, such patients are often labeled as irritable bowel syndrome. AIMS: To determine the diagnostic utility of breath tests that assess for small intestinal bacterial overgrowth (SIBO), fructose or lactose intolerance, and the predictive value of symptoms. METHODS: Patients with gas, bloating, diarrhea, abdominal pain (≥ 6 months), and negative endoscopy and radiology tests were assessed with symptom questionnaires, glucose (75 g), fructose (25 g), or lactose (25 g) breath tests. Breath tests were categorized as positive when H2 (≥ 20 ppm) or CH4 (≥ 15 ppm) increased above baseline values or as hypersensitive when symptoms changed significantly without rise in H2/CH4 or as negative. RESULTS: 1230 patients (females = 878) underwent 2236 breath tests. The prevalence of SIBO was 33% (294/883), fructose intolerance was 34% (262/763), and lactose intolerance was 44% (260/590). Hypersensitivity was found in 16% and 9%, respectively, during fructose and lactose breath tests. Although gas (89%), abdominal pain (82%), and bloating (82%) were highly prevalent, pretest symptoms or their severity did not predict an abnormal breath test, but symptoms during the breath test facilitated diagnosis of SIBO, fructose, and lactose intolerance and hypersensitivity. CONCLUSIONS: Approximately 45% of patients with unexplained gas and bloating had SIBO, fructose, or lactose intolerance; another 9-16% had visceral hypersensitivity. Pretest symptoms were poor predictors, but symptoms during the breath tests were useful. Breath tests are safe, provide significant diagnostic yield, and could be useful in routine gastroenterology practice.

Differential Sensing of Saccharides Based on an Array of Fluorinated Benzosiloxaborole Receptors.

Fluorinated benzosiloxaboroles-silicon congeners of benzoxaboroles, were synthesized and tested as molecular receptors for mono- and disaccharides. The receptors differed in the Lewis acidity of the boron center as well as in the number of potential binding sites. The calculated stability constants indicated different binding affinity of benzosiloxaborole derivatives towards selected saccharides, enabling their classification using a receptor array-based sensing. Unique fluorescence fingerprints were created on the basis of competitive interactions of the studied receptors with both Alizarin Red S (ARS) and tested saccharide molecules. Detailed chemometric analysis of the obtained fluorescence data (based on partial least squares-discriminant analysis and hierarchical clustering analysis) provided the differential sensing of common saccharides, in particular the differentiation between glucose and fructose. In addition, DFT calculations were carried out to shed light on the binding mechanism under different pH conditions.

Sugar Profiling of Honeys for Authentication and Detection of Adulterants Using High-Performance Thin Layer Chromatography.

Honey adulteration, where a range of sugar syrups is used to increase bulk volume, is a common problem that has significant negative impacts on the honey industry, both economically and from a consumer confidence perspective. This paper investigates High-Performance Thin Layer Chromatography (HPTLC) for the authentication and detection of sugar adulterants in honey. The sugar composition of various Australian honeys (Manuka, Jarrah, Marri, Karri, Peppermint and White Gum) was first determined to illustrate the variance depending on the floral origin. Two of the honeys (Manuka and Jarrah) were then artificially adulterated with six different sugar syrups (rice, corn, golden, treacle, glucose and maple syrup). The findings demonstrate that HPTLC sugar profiles, in combination with organic extract profiles, can easily detect the sugar adulterants. As major sugars found in honey, the quantification of fructose and glucose, and their concentration ratio can be used to authenticate the honeys. Quantifications of sucrose and maltose can be used to identify the type of syrup adulterant, in particular when used in combination with HPTLC fingerprinting of the organic honey extracts.

Nectar composition in moth-pollinated Platanthera bifolia and P. chlorantha and its importance for reproductive success.

MAIN CONCLUSION: Sugars (glucose, fructose and sucrose), as well as proteogenic and non-proteogenic amino acids, are present in the nectar of Platanthera bifolia and P. chlorantha. Nectar quantity and quality are floral traits that are subjected to pollinator-mediated selection. Nectar sugar and amino acid (AA) composition in two sister species, P. bifolia and P. chlorantha, was analysed and the interspecies differences in nectar and the importance of these nectar characteristics for reproductive success were investigated. Nectar was collected from four P. bifolia and three P. chlorantha populations that exist in different habitats in three regions of NE Poland. Nectar from about 30 flowers (from each population) was sampled and analysed using high-performance liquid chromatography. We found the same primary sugars and AA components in the nectar of both species, although their content varied between the populations according to habitat properties. The nectar of P. bifolia and P. chlorantha both had low sugar concentrations (9.04-20.68%) and were dominated by hexoses, with sucrose:hexoses ratios between 0.03 and 0.31 across the different populations (the average for the P. bifolia populations was 0.17 and the average for the P. chlorantha populations was - 0.05). Total sugar content did not influence reproductive success and we found positive selection on fructose content. In general, 23 different AAs were detected in both Platanthera species. Cysteine and γ-aminobutyric acid were present in only one population of P. chlorantha. Sarcosine dominated among the non-proteogenic AAs. To our knowledge, this is the first report that characterizes the sugar and AA profiles in the nectar of P. bifolia and P. chlorantha in natural populations in the context of effectiveness of reproduction. Total AAs negatively influenced male reproductive success (r = - 0.79). Pollinators of the investigated species were found to be sensitive to the AAs' taste, from taste classes I and IV. Correlation between male reproductive success and the content of AAs from these groups was 0.79 in both cases. In this manuscript, we investigated the characteristics of P. bifolia and P. chlorantha nectar, and compared these characteristics to the available data in the context of their adaptations to the requirements of pollinators and with regard to the importance of nectar quality for reproductive success of the studied species.