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.

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.

Antifungal and antimicrobial proteins and peptides of potato (Solanum tuberosum L.) tubers and their applications.

Potato proteins are well known for their nutritional, emulsifying, foaming, gel forming or antioxidant properties that all make from them valuable protein source for food industry. Antifungal, antimicrobial and also antiviral properties, described for potato proteins in the review, enrich the possibilities of potato protein usage. Potato proteins were divided into patatin, protease inhibitors and fraction of other proteins that also included, besides others, proteins involved in potato defence physiology. All these proteins groups provide proteins and peptides with antifungal and/or antimicrobial actions. Patatins, obtained from cultivars with resistance to Phytophthora infestans, were able to inhibit spore germination of this pathogen. Protease inhibitors represent the structurally heterogeneous group with broad range of antifungal and antimicrobial activities. Potato protease inhibitors I and II reduced the growth of Phytophthora infestans, Rhizoctonia solani and Botrytis cinerea or of the fungi of Fusarium genus. Members of Kunitz family (proteins Potide-G, AFP-J, Potamin-1 or PG-2) were able to inhibit serious pathogens such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli or Candida albicans. Potato snakins, defensins and pseudothionins are discussed for their ability to inhibit serious potato fungi as well as bacterial pathogens. Potato proteins with the ability to inhibit growth of pathogens were used for developing of pathogen-resistant transgenic plants for crop improvement. Incorporation of potato antifungal and antimicrobial proteins in feed and food products or food packages for elimination of hygienically risk pathogens brings new possibility of potato protein usage.

Proteomic characterization and antifungal activity of potato tuber proteins isolated from starch production waste under different temperature regimes.

Proteins were obtained from effluent of a starch manufacture by using different isolation temperatures (40, 60, 80, and 100 °C). The proteins, remaining in effluent after treatment of potato juice at 80 and 100 °C differed significantly in composition and in structural stability as well as in trypsin inhibitory and antifungal activities in comparison with the variants of 40 and 60 °C. The protein samples of 80 °C exhibited the highest antifungal activity and its average value of IC50 against five strains of two Fusarium species was determined in average at 0.18 mg ml-1. The 80 °C protein samples consisted predominantly of low-molecular proteins (7-17 kDa) identified as potato tuber protease inhibitors I and II. Predominantly, protease inhibitors II were identified for the protein samples obtained by 100 °C and here we identified 7 spots in comparison with 12 identified for the 80 °C samples. Samples of 40 and 60 °C with low antifungal activities represent high variability of detected and identified proteins. We identified various representatives of aspartic, cysteine, and serine protease inhibitors in both types of samples. These samples also contained Kunitz-type protease inhibitors that were not found in the 80 and 100 °C samples which documented thermal unstableness of Kunitz-type protease inhibitors. Functional stability at high temperatures and antifungal activity of isolated potato protease inhibitors I and II support the potential of this fraction usage in food, feed, pharmaceutical, or agricultural industry and offer new products for starch manufactures. At the same time, utilization of the stable protein fraction of waste deproteinized potato water promotes exploitation of potato starch production resources.

N-glycome profiling of patatins from different potato species of Solanum genus.

It is hypothesized that oligosaccharides are another potential source of immunological cross-reaction between different plant allergens. Patatin is the most abundant glycoprotein in potato and has been described to have an oligosaccharide of composition Man3(Xyl)GlcNAc2(Fuc). In this work, N-glycosylation profiles of patatin proteins isolated from tubers of different potato species were investigated and compared. Oligosaccharides were released by enzymatic digestion with PNAGase A and analyzed primarily by matrix-assisted laser desorption ionization mass spectrometry. For glycan labeling, a modified version of on-target derivatization with phenylhydrazine was applied. This study found the presence of glycan structures not described previously in patatins of potato tubers, and their glycan profiles significantly differed. This knowledge about the glycosylation of potato patatins may be helpful for correct choice of potato species to decrease the presence of specific glycan epitopes causing food allergy as well as for utilization of potatoes for the manufacture of therapeutic proteins.

Cultivar variability of patatin biochemical characteristics: table versus processing potatoes (Solanum tuberosum L.).

Biochemical characteristics of patatin proteins purified by ion-exchange and affinity chromatography from tubers of 20 potato cultivars were studied to evaluate their genotype differences with respect to utility groups, table potato cultivars (TPCs) and processing potato cultivars (PPCs). Both groups of cultivars showed similar values of protein content in dry matter (3.98-7.39%) and of patatin relative abundance (5.40-35.40%). Three mass levels (∼40.6, 41.8, and 42.9 kDa) of purified patatins were found by MALDI-TOF MS within all cultivars. Differences among mass levels corresponding with the mass of sugar antenna (∼1.2 kDa) confirmed the previous concept of different glycosylation extentsin patatin proteins. It was showed that the individual types of patatin varying in their masses occur in the patatin family in a ratio specific for each of the cultivars, with the lowest mass type being the major one. Electrophoretic analyses demonstrated wide cultivar variability in number of patatin forms. Especially 2D-PAGE showed 17-23 detected protein spots independently on the utility group. Specific lipid acyl hydrolase (LAH) activity of purified patatins from the individual tested cultivars varied between 0.92 and 5.46 µmol/(min mg). Patatin samples within most of the TPCs exhibited higher values of specific LAH activity than samples of PPCs. It may be supposed that individual patatin forms do not have similar physiological roles.

Chemical composition and nutritional value of protein concentrates isolated from potato (Solanum tuberosum L.) fruit juice by precipitation with ethanol or ferric chloride.

Effects of protein precipitators, ethanol and ferric chloride, on yield, resolubility, chemical composition and nutritional value of protein concentrates isolated from industrial potato fruit juice (PFJ) were studied. Optimum precipitating concentrations of ethanol and ferric chloride in PFJ were 4 M (23.1% v/v) and 20 mM (2% w/v), resulting in yield of 69% and 86.5% of total protein, respectively. Contents of total glycoalkaloids and potassium in both protein concentrates were significantly lower (P < 0.05) as compared with contents in PFJ dry matter. Both protein concentrates exhibited high nutritional value; values of essential amino acid index (EAAI) were 81.7% and 82.7%, respectively. Fraction of patatin proteins (39-43 kDa) represented with EAAI value of 86.1% the nutritionally improving protein component. Lipid acyl hydrolase activity of patatin family was not negatively affected by cooled ethanol precipitation. It can be thus suggested that biological and enzymatic activities of this protein family are utilizable after this type of precipitation.