Proteomic Profile of Flaxseed (Linum usitatissimum L.) Products as Influenced by Protein Concentration Method and Cultivar.

The research is focused on the quantitative evaluation of the flaxseed (Linum usitatissimum L.) proteome at the level of seed cake (SC), fine flour-sieved a fraction below 250 µm (FF)-and protein concentrate (PC). The evaluation was performed on three oilseed flax cultivars (Agriol, Raciol, and Libra) with different levels of α-linolenic acid content using LC-MS/MS (shotgun proteomics) analysis, which was finalized by database searching using the NCBI protein database for Linum usitatissimum and related species. A total of 2560 protein groups (PGs) were identified, and their relative abundance was calculated. A set of 33 quantitatively most significant PGs was selected for further characterization. The selected PGs were divided into four classes-seed storage proteins (11S globulins and conlinins), oleosins, defense- and stress-related proteins, and other major proteins (mainly including enzymes). Seed storage proteins were found to be the most abundant proteins. Specifically, 11S globulins accounted for 41-44% of SC proteins, 40-46% of FF proteins, and 72-84% of PC proteins, depending on the cultivar. Conlinins (2S albumins) were the most abundant in FF, ranging from 10 to 13% (depending on cultivar). The second most important class from the point of relative abundance was oleosins, which were represented in SC and FF in the range of 2.1-3.8%, but only 0.36-1.20% in PC. Surprisingly, a relatively high abundance of chitinase was found in flax products as a protein related to defence and stress reactions.

Comparison of Yield Characteristics, Chemical Composition, Lignans Content and Antioxidant Potential of Experimentally Grown Six Linseed (Linum usitatissimum L.) Cultivars.

Linseed represents a rich source of nutritional, functional and health-beneficial compounds. Nevertheless, the chemical composition and content of bioactive compounds may be quite variable and potentially affected by various factors, including genotype and the environment. In this study, the proximate chemical composition, lignans content and antioxidant potential of six experimentally grown linseed cultivars were assessed and compared. A diagonal cultivation trial in the University of South Bohemia Experimental Station in Ceské Budejovice, Czech Republic, was established in three subsequent growing seasons (2018, 2019 and 2020). The results showed that the cultivar and growing conditions influenced most studied parameters. The lack of precipitation in May and June 2019 negatively affected the seed yield and the level of secoisolariciresinol diglucoside but did not decrease the crude protein content, which was negatively related to the oil content. The newly developed method for lignans analysis allowed the identification and quantification of secoisolariciresinol diglucoside and matairesinol. Their content correlated positively with the total polyphenol content and antioxidant assays (DPPH and ABTS radical scavenging activity), indicating the significant contribution to the biofunctional properties of linseed. On the other hand, we did not detect minor linseed lignans, pinoresinol and lariciresinol. The results of this study showed the importance of cultivar and growing conditions factors on the linseed chemical composition and the lignans content, determining its nutritional and medicinal properties.

Novel Approaches for Elongation of Fish Oils into Very-Long-Chain Polyunsaturated Fatty Acids and Their Enzymatic Interesterification into Glycerolipids.

Elongation of the Very-Long-Chain Fatty Acids-4 (ELOVL4) enzyme that is expressed in neuronal tissues, sperm, and testes mediates biosynthesis of very-long-chain polyunsaturated fatty acids (VLC-PUFAs) from dietary long chain PUFAs (LC-PUFAs). The VLC-PUFAs are critical for neuronal and reproductive function. Therefore, mutations in ELOVL4 that affect VLC-PUFA biosynthesis contribute to retinal degenerative diseases including Autosomal Dominant Stargardt-like Macular Dystrophy (STGD3). Recent studies have also shown not only a depletion of retinal VLC-PUFAs with normal aging but also a more significant loss of VLC-PUFAs in donor eyes of patients with age-related macular degeneration (AMD). However, currently, there are no natural sources of VLC-PUFAs to be evaluated as dietary supplements for the attenuation of retinal degeneration in animal models of STGD3. Here, we report the development of a novel chemical approach for elongation of eicosapentaenoic (C20:5 n-3) and docosahexaenoic (C22:6 n-3) acids from fish oils by 6 carbon atoms to make a unique group of VLC-PUFAs, namely all-cis-hexacosa-11,14,17,20,23-pentaenoic acids (C26:5 n-3) and all-cis-octacosa-10,13,16,19,22,25-hexaenoic acids (C28:6 n-3). The three-step elongation approach that we report herein resulted in a good overall yield of up to 20.2%. This more sustainable approach also resulted in improved functional group compatibility and minimal impact on the geometrical integrity of the all-cis double bond system of the VLC-PUFAs. In addition, we also successfully used commercial deep-sea fish oil concentrate as an inexpensive material for the C6 elongation of fish oil LC-PUFAs into VLC-PUFAs, which resulted in the making of gram scales of VLC-PUFAs with an even higher isolation yield of 31.0%. The quality of fish oils and the content of oxidized lipids were key since both strongly affected the activity of the PEPPSI-IPr catalyst and ultimately the yield of coupling reactions. Downstream enzymatic interesterification was used for the first time to prepare structured glycerolipids enriched with VLC-PUFAs that could be evaluated in vivo to determine absorption and transport to target tissues relative to those of the free fatty acid forms. It turned out that in the synthesis of structured triacylglycerols and glycerophospholipids with VLC-PUFAs, the polarity of the immobilized lipase carrier and its humidity were essential.

Value-Added Squalene in Single-Cell Oil Produced with Cutaneotrichosporon oleaginosus for Food Applications.

Single-cell oil (SCO) produced by oleaginous microorganisms is potentially a more land-efficient and sustainable alternative to vegetable oil. The cost of SCO production can be reduced by value-added co-products like squalene, a highly relevant compound for the food, cosmetic, and pharmaceutical industry. For the first time, squalene in the oleaginous yeast Cutaneotrichosporon oleaginosus was analyzed, reaching 172.95 ± 61.31 mg/100 g oil in a lab-scale bioreactor. Using the squalene monooxygenase inhibitor terbinafine, cellular squalene was significantly increased to 2169 ± 262 mg/100 g SCO, while the yeast remained highly oleaginous. Further, SCO from a 1000 L scale production was chemically refined. The squalene content in the deodorizer distillate (DD) was found to be higher than that in DD from typical vegetable oils. Overall, this study demonstrates squalene as a value-added compound in SCO from C. oleaginosus for application in food and cosmetics without the use of genetic modifications.

Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars.

As a source of nutritionally important components, hemp seeds are often dehulled for consumption and food applications by removing the hard hulls, which increases their nutritional value. The hulls thus become waste, although they may contain valuable protein items, about which there is a lack of information. The present work is therefore aimed at evaluating the proteome of hemp (Cannabis sativa L.) at the whole-seed, dehulled seed, and hull levels. The evaluation was performed on two cultivars, Santhica 27 and Uso-31, using LC-MS/MS analysis. In total, 2833 protein groups (PGs) were identified, and their relative abundances were determined. A set of 88 PGs whose abundance exceeded 1000 ppm (MP88 set) was considered for further evaluation. The PGs of the MP88 set were divided into ten protein classes. Seed storage proteins were found to be the most abundant protein class: the averages of the cultivars were 65.5%, 71.3%, and 57.5% for whole seeds, dehulled seeds, and hulls, respectively. In particular, 11S globulins representing edestin (three PGs) were found, followed by 7S vicilin-like proteins (four PGs) and 2S albumins (two PGs). The storage 11S globulins in Santhica 27 and Uso-31 were found to have a higher relative abundance in the dehulled seed proteome (summing to 58.6 and 63.2%) than in the hull proteome (50.5 and 54%), respectively. The second most abundant class of proteins was oleosins, which are part of oil-body membranes. PGs belonging to metabolic proteins (e.g., energy metabolism, nucleic acid metabolism, and protein synthesis) and proteins related to the defence and stress responses were more abundant in the hulls than in the dehulled seeds. The hulls can, therefore, be an essential source of proteins, especially for medical and biotechnological applications. Proteomic analysis has proven to be a valuable tool for studying differences in the relative abundance of proteins between dehulled hemp seeds and their hulls among different cultivars.

Milk Thistle Oilseed Cake Flour Fractions: A Source of Silymarin and Macronutrients for Gluten-Free Bread.

The utilization of plant by-products as functional food ingredients has received increasing attention in the last decade. One such by-product generated during milk thistle oil pressing is oilseed cakes, which could be used as a novel food ingredient. Therefore, the study aimed at investigating the effects of the addition of milk thistle oilseed cake (MTOC) flour fractions obtained via dry sieving, differing in particle size (unsieved; coarse: >710 µm; medium: 315−710 µm; and fine: <315 µm), on the quality of gluten-free bread and stability of silymarin during breadmaking. The 10% addition of the fractions into gluten-free bread increased the protein, fibre, fat, ash and silymarin content. The breads with the coarse fraction had the highest content of fibre, whereas the breads with the fine fraction excelled in protein, fat and ash content. The medium fraction was characterized as the richest source of silymarin, whilst the fine fraction was the poorest. Silymarin constituents were slightly released during dough rising but also partially decomposed during baking; moreover, silydianin was the most susceptible and degraded the most. The enriched breads had better sensory and textural properties compared to the control bread. The results suggest that MTOC flour fractions can improve the potential health benefits and nutritional profile of gluten-free bread.

Formation of Previously Undescribed Δ7-Phytosterol Oxidation Products and Tocopherylquinone Adducts in Pumpkin Seed Oil during Roasting, Screw-Pressing, and Simulated Culinary Processing at Elevated Temperatures.

The influence of pumpkin seed roasting conditions (110-140 °C) and screw-pressing on the formation of previously undescribed Δ7-phytosterol oxidation products and tocopherylquinone adducts with nucleophilic phosphatidylethanolamine species was investigated. The roasting process of pumpkin seed paste at a temperature above 120 °C for 30 min considerably enhanced the formation of Δ7-oxysterols. Targeted analysis [electron impact mass spectrometry (MS), 1D-nuclear magnetic resonance] led to the identification of five novel markers of pumpkin paste roasting, among which (3ß,5α,22E,24S)-stigmasta-7,22-dien-6-one-3-ol (6-oxo-α-spinasterol), stereoisomers of (3ß,5α,22E)-7,8-epoxystigmast-22-en-3-ol (7,8-epoxy-α-spinasterol), and (3ß,5α)-22,23-epoxystigmast-7-en-3-ol (7,8-epoxy-α-spinasterol) were reported in edible oils for the first time. Simulated culinary processing provided novel stereoisomers of (3ß,5α,22E)-stigmasta-7,22-dien-3,6-diol, unusual (3ß,5α,22E)-stigmasta-7,22-dien-6,15-dione-3-ol, and (5α,22E)-stigmasta-7,22-dien-3-one accompanied by minor stereoisomers of (3ß,5α)-7,8;22,23-diepoxystigmastan-3-ol. Moreover, a clear relationship between the pumpkin seed oil stability index and synergistic effect of glycerophospholipids with present tocochromanols was found. High-resolution atmospheric pressure chemical ionization-MS experiments clearly demonstrated the formation of various γ-tocopherylquinone adducts with primary amines, namely, octylamine. The mitigation strategy of potentially detrimental oxysterols from pumpkin seed oil included optimization of processing parameters while maintaining the formation of desirable sensory-active compounds.

Oilseed Cake Flour Composition, Functional Properties and Antioxidant Potential as Effects of Sieving and Species Differences.

Oilseed cakes are produced as a by-product of oil pressing and are mostly used as feed. Their use for human consumption is due to the functional properties and benefits for human health. Herein, oilseed cake flours of eight species (flax, hemp, milk thistle, poppy, pumpkin, rapeseed, safflower, sunflower) were sieved into fractions above (A250) and below (B250) 250 µm. The chemical composition, SDS-PAGE profiles, colour, functional properties and antioxidant activities of these flours were evaluated. The B250 fractions were evaluated as being protein and ash rich, reaching crude protein and ash content ranging from 31.78% (milk thistle) to 57.47% (pumpkin) and from 5.0% (flax) to 11.19% (poppy), respectively. A high content of carbohydrates was found in the flours of hemp, milk thistle and safflower with a significant increase for the A250 fraction, with a subsequent relation to a high water holding capacity (WHC) for the A250 fraction (flax, poppy, pumpkin and sunflower). The A250 milk thistle flour was found to have the richest in polyphenols content (TPC) (40.89 mg GAE/g), with the highest antioxidant activity using an ABTS•+ assay (101.95 mg AAE/g). The A250 fraction for all the species exhibited lower lightness than the B250 fraction. The obtained results indicate that sieving oilseed flour with the aim to prepare flours with specific functional characteristics and composition is efficient only in combination with a particular species.

Influence of dough composition on the formation of processing contaminants in yeast-leavened wheat toasted bread.

The influence of dough composition on acrylamide, 3-monochloropropane-1,2-diol (3-MCPD) esters, and glycidyl esters (GE) formation during bread toasting was investigated. The doughs differed in added amounts of soy lecithin, salt, and reducing agents (l-cysteine and glutathione). The toasting of bread for 2.5 min considerably enhanced the formation of acrylamide and 3-MCPD esters. The addition of lecithin (1%, w/w) resulted in four times higher content of 3-MCPD esters in toasted bread slices. No distinct relationship between dough composition and GE formation in untoasted and toasted bread was found. The addition of reducing agents (0.05%, w/w) mitigated during toasting not only the formation of 3-MCPD esters (more than six times) but also the extent of Maillard reaction that resulted in three times lower amounts of acrylamide and predominant formation of alcohol-like compounds. Toasted bread without reducing agents contained typical Maillard reaction compounds such as aldehydes, alkyl pyrazines, and derivatives of furan.

Buckwheat Secondary Metabolites: Potential Antifungal Agents.

Research groups have put significant emphasis on the evaluation of nutritional, health-promoting, and other biological activities of secondary metabolites from buckwheat. Among these phytochemicals, phenolic and lipophilic antioxidants, particularly, phenolic acids, flavonoids, and tocopherols, have been the focus of the latest studies since antioxidant activity has recently been associated with the possibility of inhibiting fungal growth and mycotoxin biosynthesis. The mycotoxin contamination of cereal and pseudocereal grains caused primarily by Fusarium, Penicillium, and Aspergillus species poses a significant hazard to human health. Therefore, efforts to examine the involvement of plant antioxidants in the biosynthesis of mycotoxins at the transcriptional level have emerged. In addition, hydrophobic interactions of buckwheat phenolics with cell membranes could also explain their capacity to reduce fungal development. Eventually, possibilities of enhancing the biological activity of cereal and pseudocereal phytochemicals have been studied, and sourdough fermentation has been proposed as an efficient method to increase antioxidant activities. This effect could result in an increased antifungal effects of sourdough and bakery products. This review reports the main advances in research on buckwheat phenolics and other antioxidant phytochemicals, highlighting possible mechanisms of action and processes that could improve their biological activities.

Formation of 5α-Sitostan-3-one, 5α-Campestan-3-one, and Steroidal Hydrocarbons in Edible Oils during Catalytic Hydrogenation.

Targeted analysis confirmed the presence of new phytosterol degradation products in fully hydrogenated commercial samples. EI-MS, APCI-MS, and 1D-NMR experiments led to the identification of 10 novel markers of catalytic hydrogenation, among which 5α-sitostan-3-one and 5α-campestan-3-one, isomers of saturated and monounsaturated steroidal hydrocarbons, were reported in edible oils for the first time. Examination of the phytosterol degradation mechanism was done by the catalytic transfer deuteration technique. The mitigation strategy of potentially detrimental compounds included optimization of processing parameters. The effect of catalyst dosage (≤0.1% based on Ni basis) and temperature region (>180 °C) were the most crucial factors in phytosterol degradation control.

Antifungal Polyamides of Hydroxycinnamic Acids from Sunflower Bee Pollen.

The aim of the bioassay-guided fractionation was the selection of the most potent group of compounds responsible for the protection of sunflower bee pollen grains. Synthesis of prospective antifungal polyamides of hydroxycinnamic acids was based on previous structural elucidation of ethanol soluble fraction by 1H,1H-PFG-COSY, 1H,13C-HSQC, FT-IR, FT-Raman, and LC-MS experiments. The main compounds found were tri- p-coumaroylspermidines accompanied by other HCAA of spermidine and putrescine. Several model HCAA derivatives were prepared to test their antifungal activity against widespread spoilage fungi ( A. niger 42 CCM 8189, F. culmorum DMF 0103, and P. verrucosum DMF 0023). A. niger CCM 8189 and F. culmorum DMF 0103 exhibited higher resistance to the antifungal effects of hydroxycinnamic acid amides, whereas P. verrucosum DMF 0023 was the most sensitive strain. It has been discovered the effect of HCAA polarity on the role of secondary metabolites in the microbial protection of pollen grains. The combination of bioassay-guided fractionation, structural elucidation, selection of prospective compounds, and their synthesis to determine their antifungal properties could be considered as an original approach.

Antioxidant effect of mono- and dihydroxyphenols in sunflower oil with different levels of naturally present tocopherols.

Antioxidant properties of mono- and dihydroxyphenolic acids and their alkyl esters were examined, with emphasis on the relationship between their molecular structure and antioxidant activity. Test media with different tocopherol level were used for determining the oxidative stability: original refined sunflower oil (total tocopherols 149.0 mg/kg), partially tocopherol-stripped sunflower oil (total tocopherols 8.7 mg/kg) and distilled fatty acid methyl esters (FAME) as a tocopherol-free medium. The chemical reaction of tocopherols with diazomethane tested for the purpose to eliminate their antioxidant activity failed due to the negligible degree of methylation of hydroxyl group in the tocopherol molecule. Caffeic acid and protocatechuic acid (3,4-dihydroxyphenolic acids) and their alkyl esters were found to be more active antioxidants than monohydroxyphenolic acid (p-hydroxybenzoic acid), 2,5-dihydroxyphenolic acid (gentisic acid), 3-methoxy-4-hydroxyphenolic acids (vanillic and ferulic acids) and their corresponding alkyl esters. Naturally present tocopherols in refined sunflower oil proved to have a synergistic effect on gentisic acid but not on its alkyl esters. In contrast, tocopherols showed an antagonistic effect on alkyl esters of caffeic acid, because their protection factors decreased with increasing level of tocopherols in the test medium. Moreover, the antioxidant activity of these alkyl esters decreased with increasing length of their alkyl chain in conformity with the polar paradox hypothesis. PRACTICAL APPLICATIONS: Tocopherols as naturally present antioxidants influence considerably the antioxidant activity of other antioxidants added to plant oils used as a test medium. Distilled fatty acid methyl esters prepared from refined sunflower oil may serve as an optimal tocopherol-free test medium. Some alkyl esters of phenolic acids were evaluated to be applicable as natural more lipophilic antioxidants in comparison with phenolic acids.