Integrated Biorefinery for a Next-Generation Methanization Process Focusing on Volatile Fatty Acid Valorization: A Critical Review.

This review addresses the critical issue of a rapidly increasing worldwide waste stream and the need for sustainable management. The paper proposes an integrated transformation toward a next-generation methanization process, which leads not only to treating waste but also to converting it into higher value compounds and greener energy. Although the current and commonly used anaerobic digestion process is useful for biogas production, it presents limitations of resource exploitation and some negative environmental impacts. Focusing on the acidogenic stage in waste stream processing, the paper discusses the recent strategies to enhance the recovery of volatile fatty acids (VFAs). These acids serve as precursors for synthesizing a variety of biochemicals and biofuels, offering higher value products than solely energy recovery and soil fertilizers. Additionally, the importance of recycling the fermentation residues back into the biorefinery process is highlighted. This recycling not only generates additional VFAs but also contributes to generating clean energy, thereby enhancing the overall sustainability and efficiency of the waste management system. Moreover, the review discusses the necessity to integrate life cycle assessment (LCA) and techno-economic analysis (TEA) to evaluate the environmental impacts, sustainability, and processing costs of the proposed biorefinery.

Purification of Natural Pigments Violacein and Deoxyviolacein Produced by Fermentation Using Yarrowia lipolytica.

Violacein and deoxyviolacein are bis-indole pigments synthesized by a number of microorganisms. The present study describes the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis, the subsequent extraction of the intracellular pigments, and ultimately their purification using column chromatography. The results show that the optimal separation between the pigments occurs using an ethyl acetate/cyclohexane mixture with different ratios, first 65:35 until both pigments were clearly visible and distinguishable, then 40:60 to create a noticeable separation between them and recover the deoxyviolacein, and finally 80:20, which allows the recovery of the violacein. The purified pigments were then analyzed by thin-layer chromatography and nuclear magnetic resonance.

Phenolic Compounds Recovery from Blood Orange Peels Using a Novel Green Infrared Technology Ired-Irrad®, and Their Effect on the Inhibition of Aspergillus flavus Proliferation and Aflatoxin B1 Production.

The intensification of total phenolic compound (TPC) extraction from blood orange peels was optimized using a novel green infrared-assisted extraction technique (IRAE, Ired-Irrad®) and compared to the conventional extraction using a water bath (WB). Response surface methodology (RSM) allowed for the optimization of ethanol concentration (E), time (t), and temperature (T) in terms of extracted TPC and their antiradical activity, for both WB extraction and IRAE. Using WB extraction, the multiple response optimums as obtained after 4 h at 73 °C and using 79% ethanol/water were 1.67 g GAE/100 g for TPC and 59% as DPPH inhibition percentage. IRAE increased the extraction of TPC by 18% using 52% ethanol/water after less than 1 h at 79 °C. This novel technology has the advantage of being easily scalable for industrial usage. HPLC analysis showed that IRAE enhanced the recovery of gallic acid, resveratrol, quercetin, caffeic acid, and hesperidin. IR extracts exhibited high bioactivity by inhibiting the production of Aflatoxin B1 by 98.9%.

A Combined Metabolomics and Fluxomics Analysis Identifies Steps Limiting Oil Synthesis in Maize Embryos.

Enhancing fatty acid synthesis (FAS) in maize (Zea mays) has tremendous potential nutritional and economic benefits due to the rapidly growing demand for vegetable oil. In maize kernels, the endosperm and the embryo are the main sites for synthesis and accumulation of starch and oil, respectively. So far, breeding efforts to achieve elevated oil content in maize have resulted in smaller endosperms and therefore lower yield. Directly changing their carbon metabolism may be the key to increasing oil content in maize kernels without affecting yield. To test this hypothesis, the intracellular metabolite levels were compared in maize embryos from two different maize lines, ALEXHO S K SYNTHETIC (Alex) and LH59, which accumulate 48% and 34% of oil, respectively. Comparative metabolomics highlighted the metabolites and pathways that were active in the embryos and important for oil production. The contribution of each pathway to FAS in terms of carbon, reductant, and energy provision was assessed by measuring the carbon flow through the metabolic network (13C-metabolic flux analysis) in developing Alex embryos to build a map of carbon flow through the central metabolism. This approach combined mathematical modeling with biochemical quantification to identify metabolic bottlenecks in FAS in maize embryos. This study describes a combination of innovative tools that will pave the way for controlling seed composition in important food crops.

Impact of the Physicochemical Composition and Microbial Diversity in Apple Juice Fermentation Process: A Review.

Fermented apple beverages are produced all over the world with diverse characteristics associated with each country. Despite the diversifications, cider producers are confronted with similar issues and risks. The nature of the raw material, also known as the fermentation medium, plays a key role in fermentation. A well-defined composition of apples is, therefore, required to produce cider with good quality. In addition, ferment and its metabolism are important factors in the fermentation process. The producers of cider and other alcoholic beverages are looking in general for novel yeast strains or for the use of native strains to produce "authentic" and diversified beverages that are distinct from each other, and that attract more and more consumers. Research articles on cider production are infrequent compared to wine production, especially on the impact of the chemical composition and microbial diversity of apples on fermentation. Even though the processing of fermented beverages is close in terms of microbial interactions and production, the study of the specific properties of apples and the production challenges of cider production is advantageous and meaningful for cider producers. This review summarizes the current knowledge on apple composition and the impact of the must composition on fermentation and yeast growth. In addition, the microbial diversity of cider, activities, and its influence on fermentation are reviewed.

Water-Soluble Polysaccharides from Ephedra alata Stems: Structural Characterization, Functional Properties, and Antioxidant Activity.

In this study, the physicochemical characterization, functional properties, and antioxidant activity of polysaccharides extracted from Ephedra alata (EAP) were investigated. EAP were extracted in water during 3 h with a liquid/solid ratio of 5 in a water bath at 90 °C. The structure of the extracted EAP was examined by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and gas chromatography-mass spectrometry (GC-MS). The functional properties and biochemical activities of EAP were determined. The chemical analysis revealed that the contents of carbohydrates, uronic acid, and proteins were 73.24% ± 1.24%, 6.82% ± 0.57%, and 6.56% ± 0.36%, respectively. The results showed that the extracted EAP essentially contain three functional groups: C=O, C-H, and O-H. SEM images showed that EAP present numerous high porosity particles. The monosaccharide composition revealed a polymer composed of glucose (43.1%), galactose (36.4%), mannose (14.9%), arabinose (3.7%), and gluconic acid (1.7%). EAP showed interesting functional properties (solubility, oil holding capacity, foaming and emulsion properties). Finally, the results revealed that EAP displayed excellent antihypertensive and antioxidant activities. Overall, EAP present a promising natural source of food additives, antioxidants, and antihypertensive agents.

Fermentation at non-conventional conditions in food- and bio-sciences by the application of advanced processing technologies.

The interest in improving the yield and productivity values of relevant microbial fermentations is an increasingly important issue for the scientific community. Therefore, several strategies have been tested for the stimulation of microbial growth and manipulation of their metabolic behavior. One promising approach involves the performance of fermentative processes during non-conventional conditions, which includes high pressure (HP), electric fields (EF) and ultrasound (US). These advanced technologies are usually applied for microbial inactivation in the context of food processing. However, the approach described in this study focuses on the use of these technologies at sub-lethal levels, since the aim is microbial growth and fermentation under these stress conditions. During these sub-lethal conditions, microbial strains develop specific genetic, physiologic and metabolic stress responses, possibly leading to fermentation products and processes with novel characteristics. In some cases, these modifications can represent considerable improvements, such as increased yields, productivities and fermentation rates, lower accumulation of by-products and/or production of different compounds. Although several studies report the successful application of these technologies during the fermentation processes, information on this subject is still scarce and poorly understood. For that reason, the present review paper intends to assemble and discuss the main findings reported in the literature to date, and aims to stimulate interest and encourage further developments in this field.

Effect of high hydrostatic pressure on background microflora and furan formation in fruit purée based baby foods.

The baby foods industry is currently seeking technologies to pasteurize products without formation of processing contaminants such as furan. This work demonstrates the applicability of high hydrostatic pressure (HHP) as a non-thermal decontamination intervention for fruit purée based baby foods. HHP processing was evaluated at 200, 300, and 400 MPa pressures, for 5, 10 and 15 min of treatment times at 25, 35 and 45 °C. HHP application at 400 MPa, 45 °C for 15 min ensured complete inactivation (about 6 log10) of total mesophilic aerophiles, as well as yeasts and molds. No furan was detected in HHP processed products. Thus, the key advantage of HHP over thermal processing is the ability to achieve commercially acceptable microbiological inactivation while avoiding the formation of processing contaminants such as furan.

Current advances in biological production of propionic acid.

Propionic acid and its derivatives are considered "Generally Recognized As Safe" food additives and are generally used as an anti-microbial and anti-inflammatory agent, herbicide, and artificial flavor in diverse industrial applications. It is produced via biological pathways using Propionibacterium and some anaerobic bacteria. However, its commercial chemical synthesis from the petroleum-based feedstock is the conventional production process bit results in some environmental issues. Novel biological approaches using microorganisms and renewable biomass have attracted considerable recent attention due to economic advantages as well as great adaptation with the green technology. This review provides a comprehensive overview of important biotechnological aspects of propionic acid production using recent technologies such as employment of co-culture, genetic and metabolic engineering, immobilization technique and efficient bioreactor systems.

Recent advances in γ-aminobutyric acid (GABA) properties in pulses: an overview.

Beans, peas, and lentils are all types of pulses that are extensively used as foods around the world due to their beneficial effects on human health including their low glycaemic index, cholesterol lowering effects, ability to decrease the risk of heart diseases and their protective effects against some cancers. These health benefits are a result of their components such as bioactive proteins, dietary fibre, slowly digested starches, minerals and vitamins, and bioactive compounds. Among these bioactive compounds, γ-aminobutyric acid (GABA), a non-proteinogenic amino acid with numerous reported health benefits (e.g. anti-diabetic and hypotensive effects, depression and anxiety reduction) is of particular interest. GABA is primarily synthesised in plant tissues by the decarboxylation of l-glutamic acid in the presence of glutamate decarboxylase (GAD). It is widely reported that during various processes including enzymatic treatment, gaseous treatment (e.g. with carbon dioxide), and fermentation (with lactic acid bacteria), GABA content increases in the plant matrix. The objective of this review paper is to highlight the current state of knowledge on the occurrence of GABA in pulses with special focus on mechanisms by which GABA levels are increased and the analytical extraction and estimation methods for this bioactive phytochemical. © 2017 Society of Chemical Industry.

Seed oil extraction from red prickly pear using hexane and supercritical CO2 : assessment of phenolic compound composition, antioxidant and antibacterial activities.

BACKGROUND: Investigating Opuntia species for their seed oil content is of much importance owing to their potential use for food and in cosmetic applications. These oils have an important content in unsaturated fatty acids as well as antioxidant compounds (e.g. polyphenols, vitamin E), which have been associated with the prevention of some chronic diseases. Moreover, Opuntia stricta oils possess important antimicrobial activities. For instance, the main focus of this study was to compare the effectiveness of conventional (hexane extraction) and novel (supercritical (SC)-CO2 ) extraction methods for the recovery of oil and phenolic compounds from O. stricta seeds. The oil yield of both extracts was then compared and the polyphenol content and composition of both extracts were determined by liquid chromatography-high-resolution mass spectrometry. Additionally, antioxidant (DPPH assay) and antimicrobial activities (disc diffusion method) of O. stricta seed oils were determined. RESULTS: The oil yield (based on Soxhlet's method) of O. stricta seeds was determined using SC-CO2 (49.9 ± 2.2%), and hexane (49.0 ± 1.5%). Although obtaining similar oil extraction yields using the two methods, the extracted oil using SC-CO2 was more enriched in polyphenols (172.2 ± 11.9 µg gallic acid equivalents (GAE) g-1 oil) than that extracted using hexane (76.0 ± 6.9 µg GAE g-1 of oil). Polyphenol profiles showed that the SC-CO2 process led to the yield of more compounds (45) than that using hexane extraction (11). Moreover, the antioxidant and antimicrobial activities of SC-CO2 extract showed a high percentage of inhibition. CONCLUSION: SC-CO2 extraction of O. stricta seed oil led to extraction of oil with a similar yield to that with hexane extraction, but with higher polyphenol content. The extract containing polyphenols exhibited high antioxidant and antibacterial properties, demonstrating their great potential as feedstock for high-oil quality. © 2016 Society of Chemical Industry.

Influence of Innovative Processing on γ-Aminobutyric Acid (GABA) Contents in Plant Food Materials.

Over the last several decades, γ-aminobutyric acid (GABA) has attracted much attention due to its diverse physiological implications in plants, animals, and microorganisms. GABA naturally occurs in plant materials and its concentrations may vary considerably, from traces up to µmol/g (dry basis) depending on plant matrix, germination stage, and processing conditions, among other factors. However, due to its important biological activities, considerable interest has been shown by both food and pharmaceutical industries to improve its concentration in plants. Natural and conventional treatments such as mechanical and cold stimulation, anoxia, germination, enzyme treatment, adding exogenous glutamic acid (Glu) or gibberellins, and bacterial fermentation have been shown effective to increase the GABA concentration in several plant materials. However, some of these treatments can modify the nutritional, organoleptic, and/or functional properties of plants. Recent consumer demand for food products which are "healthy," safe and, having added benefits (nutraceuticals/functional components) has led to explore new ways to improve the content of bioactive compounds while maintaining desirable organoleptic and physicochemical properties. Along this line, nonthermal processing technologies (such as high-pressure processing, pulsed electric fields, and ultrasound, among others) have been shown as means to induce the biosynthesis and accumulation of GABA in plant foods; and the main findings so far reported are presented in this review. Moreover, the most novel tools for the identification of metabolic response in plant materials based on GABA analysis will be also described.

Nitraria retusa fruit prevents penconazole-induced kidney injury in adult rats through modulation of oxidative stress and histopathological changes.

CONTEXT: Nitraria retusa (Forssk.) Asch. (Nitrariaceae) is a medicinal plant which produces edible fruits whose antioxidant activity has been demonstrated. OBJECTIVE: The current study elucidates the potential protective effect of N. retusa fruit aqueous extract against nephrotoxicity induced by penconazole, a triazole fungicide, in the kidney of adult rats. MATERIALS AND METHODS: Adult Wistar rats were exposed either to penconazole (67 mg/kg body weight), or to N. retusa extract (300 mg/kg body weight) or to their combination. Penconazole was administered by intra-peritoneal injection every 2 days from day 7 until day 15, the sacrifice day, while N. retusa extract was administered daily by gavage during 15 days. Oxidative stress parameters, kidney biomarkers and histopathological examination were determined. RESULTS: Nitraria retusa extract administration to penconazole treated rats decreased kidney levels of malondialdehyde (-10%), hydrogen peroxide (-12%), protein carbonyls (PCOs, -11%) and advanced oxidation protein products (AOPP, -16%); antioxidant enzyme activities: catalase (-13%), superoxide dismutase (-8%) and glutathione peroxidase (GPx, -14%), and the levels of non-enzymatic antioxidants: non-protein thiols (-9%), glutathione (-7%) and metallothionein (-12%). Furthermore, this plant extract prevented kidney biomarker changes by reducing plasma levels of creatinine, urea, uric acid and LDH and increasing those of ALP and GGT. Histopathological alterations induced by penconazole (glomeruli fragmentation, Bowman's space enlargement, tubular epithelial cells necrosis and infiltration of inflammatory leucocytes) were attenuated following N. retusa administration. DISCUSSION AND CONCLUSION: Our results indicated that N. retusa fruit extract had protective effects against penconazole-induced kidney injury, which could be attributed to its phenolic compounds.

Ultrasound-Assisted Extraction, Centrifugation and Ultrafiltration: Multistage Process for Polyphenol Recovery from Purple Sweet Potatoes.

This work provides an evaluation of an ultrasound-assisted, combined extraction, centrifugation and ultrafiltration process for the optimal recovery of polyphenols. A purple sweet potato (PSP) extract has been obtained using ultrasonic circulating extraction equipment at a power of 840 W, a frequency of 59 kHz and using water as solvent. Extract ultrafiltration, using polyethersulfone (PES), was carried out for the recovery of polyphenol, protein and anthocyanin. Pre-treatment, via the centrifugation of purple sweet potato extract at 2500 rpm over 6 min, led to better polyphenol recovery, with satisfactory protein removal (reused for future purposes), than PSP extract filtration without centrifugation. Results showed that anthocyanin was efficiently recovered (99%) from permeate. The exponential model fit well with the experimental ultrafiltration data and led to the calculation of the membrane's fouling coefficient. The optimization of centrifugation conditions showed that, at a centrifugation speed of 4000 rpm (1195× g) and duration of 7.74 min, the optimized polyphenol recovery and fouling coefficient were 34.5% and 29.5 m-1, respectively. The removal of proteins in the centrifugation process means that most of the anthocyanin content (90%) remained after filtration. No significant differences in the intensities of the HPLC-DAD-ESI-MS² peaks were found in the samples taken before and after centrifugation for the main anthocyanins; peonidin-3-feruloylsophoroside-5-glucoside, peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside, and peonidin-3-caffeoyl-feruloyl sophoroside-5-glucoside. This proves that centrifugation is an efficient method for protein removal without anthocyanin loss. This study considers this process an ultrasound-assisted extraction-centrifugation-ultrafiltration for purple sweet potato valorization in "green" technology.

Seed oil polyphenols: rapid and sensitive extraction method and high resolution-mass spectrometry identification.

Phenolic content is a primary parameter for vegetables oil quality evaluation, and directly involved in the prevention of oxidation and oil preservation. Several methods have been reported in the literature for polyphenols extraction from seed oil but the approaches commonly used remain manually handled. In this work, we propose a rapid and sensitive method for seed oil polyphenols extraction and identification. For this purpose, polyphenols were extracted from Opuntia stricta Haw seed oil, using high frequency agitation, separated, and then identified using a liquid chromatography-high resolution mass spectrometry method. Our results showed good sensitivity and reproducibility of the developed methods.

Healing efficiency of oligosaccharides generated from almond gum (Prunus amygdalus) on dermal wounds of adult rats.

Almond gum is a naturally occurring polymer produced by almond trees and shrubs. Its abundance, as well as its low cost production makes it a potential feedstock for use in food and pharmaceuticals. In this regard, almond gum oligosaccharides were enzymatically generated, purified and their monosaccharide composition assessed using gas chromatography-flame ionization detector. Oligosaccharide analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography - mass spectrometry showing a main chain composed of galactose units [→3)-Gal-(1→] branched mainly with arabinose residues [Ara-(1→]. The potent role of the generated oligosaccharides on rats wound healing was investigated. They have been applied either alone or supplemented, as active substance, with cream formulation, on full-thickness wound created on the dorsum of the rats. The effect of oligosaccharides was assessed by measuring the wound closure percentage, reaching an average of around 100% when applied alone or supplemented to cream formulation. The healing percentage for the control group was only 74.3% at the same day. The histological evaluation of skin sections visualized by light microscopy revealed an improved collagen deposition and an increased fibroblast and vascular densities.

Optimization of Aqueous Extraction of Polyphenols from Cuminum cyminum Seeds Using Response Surface Methodology and Assessment of Biological Activity.

(1) Background: Cumin seeds, extracted from the plant Cuminum cyminum, are abundant in phenolic compounds and have been extensively researched for their chemical makeup and biological effects. The objective of this research is to enhance the water extraction of polyphenols through the water bath (WB) technique and to evaluate the antiradical, antibacterial, and anticancer effects of the extract. (2) Methods: Response Surface Methodology was used to find the best parameters to extract polyphenols. Three experimental parameters, time, temperature, and solid-liquid ratio, were tested. The disc diffusion method has been used to determine the antimicrobial activities against Salmonella Typhimurium, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans. The antiradical activity was performed using the DPPH method, while total phenolic content was performed using Folin-Ciocalteu. High-Performance Liquid Chromatography (HPLC) was conducted to analyze the phytochemical profile of WB extracts. The anticancer activity of the lyophilized extract was assessed against three cancer cell lines (colon (HT29), lung (A549), and breast (MCF7) cancer cell lines).; (3) Results: The optimal conditions for water extraction were 130 min at 72 °C. The total phenolic compounds yield (14.7 mg GAE/g DM) and antioxidant activity (0.52 mg trolox eq./mL) were obtained using a 1:40 solid-liquid ratio. The primary polyphenols identified were the flavonoids rutin (0.1 ppm) and ellagic acid (3.78 ppm). The extract had no antibacterial or antifungal activities against the microorganisms tested. The extract showed anticancer activity of about 98% against MCF7 (breast cancer cell line), about 81% against HT29 (colon cancer cell line), and 85% against A549 (lung cancer cell line) at high doses. (4) Conclusions: Extraction time and a high solid-liquid ratio had a positive impact on polyphenol recovery and in maintaining their quantity and quality. Furthermore, the optimal aqueous extract exhibited strong antiradical activity reflected by the inhibition of free radicals in addition to a significant specificity against the tested cancer cell lines.

Highlighting the tricarboxylic acid cycle: liquid and gas chromatography-mass spectrometry analyses of (13)C-labeled organic acids.

The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography-mass spectrometry methods to determine (13)C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [(13)C]glucose or [(13)C]glutamine. Once steady-state labeling was reached, (13)C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.

Peanut Allergenicity: An Insight into Its Mitigation Using Thermomechanical Processing.

Peanuts are the seeds of a legume crop grown for nuts and oil production. Peanut allergy has gained significant attention as a public health issue due to its increasing prevalence, high rate of sensitization, severity of the corresponding allergic symptoms, cross-reactivity with other food allergens, and lifelong persistence. Given the importance of peanuts in several sectors, and taking into consideration the criticality of their high allergic potential, strategies aiming at mitigating their allergenicity are urgently needed. In this regard, most of the processing methods used to treat peanuts are categorized as either thermal or thermomechanical techniques. The purpose of this review is to provide the reader with an updated outlook of the peanut's allergens, their mechanisms of action, the processing methods as applied to whole peanuts, as well as a critical insight on their impact on the allergenicity. The methods discussed include boiling, roasting/baking, microwaving, ultrasonication, frying, and high-pressure steaming/autoclaving. Their effectiveness in alleviating the allergenicity, and their capacity in preserving the structural integrity of the treated peanuts, were thoroughly explored. Research data on this matter may open further perspectives for future relevant investigation ultimately aiming at producing hypoallergenic peanuts.

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis.

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.