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Linseed, the multipurpose plant Magdalena Zuk a,b,∗ , Dorota Richter c , Jan Matuła c , Jan Szopa a,b,c a

Faculty of Biotechnology, Wroclaw University, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland Linum Foundation, Pl. Grunwaldzki 24a, 50-363 Wroclaw, Poland c University of Natural Sciences, Pl. Grunwaldzki 24a, 50-363 Wroclaw, Poland b

a r t i c l e

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Article history: Received 19 January 2015 Received in revised form 5 May 2015 Accepted 7 May 2015 Available online xxx Keywords: Linseed plant Oil Seedcake Fiber Shives biochemistry Application of linseed products Value-added linseed products

a b s t r a c t The oilseed flax (linseed) (Linum usitatissimum L.) is predominantly the source of valuable oil, in which the most appreciated are omega-3 fatty acids. The extensive biochemical analysis of linseed oil resulted in the identification of its other components with potential application in improvement of human health. The focus is now on them and they are of particular interest for human nutrition, cosmetic and pharmaceutical industry. Linseed plant also provides seedcakes (linseed expeller), fibers and shives as by-products. The recent development of analytical methods allowed to determine several valuable compounds in these materials have made them thereby more appealing to industry. Besides lignocellulose biomass, which is mainly used for components of polymer composites or liquid/gas fuel production, phenylpropanoids and terpenoids are the major constituents that contribute to bioactive properties of the linseed byproducts. Antibacterial, antifungal, anticancer, and anti-inflammatory activities are assigned to these compounds. These findings led to the diversification of linseed plant application. This review outlines oil, seedcake, and fiber and shives biochemistry and describes their potential application. It is expected that the development of value-added products from linseed plant might greatly improve the economic viability of its cultivation. Moreover, the use of linseed plant as a whole to produce innovative industrial products would enhance the sustainability of natural resources. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Flax (Linum usitatissimum L.) is generally regarded as a dualpurpose crop plant due to its main products, the fiber and seed. Since many year ago, the fiber has been converted to yarn, which served as a major source to manufacture textiles for table or bed coverings and clothing, whereas seeds have been pressed to extract edible oil. Marginally, shives and straw, mainly from linseed flax, were also used to seal and thermally insulate homes. Probably originating from Middle East, flax plant was subsequently introduced to several other world regions including Europe. Disruptive selection through thousands of years of flax domestication has resulted in its diversification into oilseed and fibrous plant types. Both types differ substantially in phenotype and physiology. Linseed flax grows up to 40–60 cm tall with highly branched stem, while fibrous plant grows up to 80–120 cm and is less branched.

∗ Corresponding author at: Faculty of Biotechnology, Wroclaw University, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland. Tel.: +48 71 3756326; fax: +48 71 3252930. E-mail address: [email protected] (M. Zuk).

While linseed cultivars are rather grown in continental climate regions, fiber flax cultivars are preferably grown in cool, moist climate condition. Cultivation region of the world, water stress, high temperature and disease occurrence affect growth parameters. For example, flax seedlings can withstand a temperature of −4 ◦ C, but very high temperatures (exceeding 32 ◦ C) could shorten stem length and flowering period, thereby affecting fiber or seed yield. The flax popularity lasted until the middle of the last century, when flax fiber was gradually forced out of the market by synthetic fibers and cotton. However, the recently growing demand for flax as a source of new raw materials has triggered the renewal of its cultivation in the world. According to the last available issue of FAO statistics (http:// faostat3.fao.org/faostat-gateway), in 2013 the global world linseed production (area harvested) was 2,252,104 ha The significant linseed flax cultivation areas were located in Canada (412,000 ha), – the “traditional” leader of linseed cultivation, followed by Russian Federation (410,000 ha) and Kazakhstan (384,300 ha) – the two countries indicating the highest stable increase in linseed production during last five years, India (338,000 ha), China (312,890 ha). Far lower area was reported for United States of

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America (56,960 ha), Ukraine (55,000 ha), Argentina (14,600 ha), Egypt (5000 ha) and of the EU countries the highest area of linseed cultivation was located in United Kingdom (34,000 ha), France (8510 ha), and Belgium (7900 ha), Spain (7000 ha) while the other countries like Sweden (4400 ha), Germany (3700 ha), Romania (3042 ha), Italy (3000 ha), Netherlands (1881 ha), Czech Republic (1513 ha) and Poland (1470 ha) cultivated the linseed rather for local niche market and experimental purposes than for global industry. The top producers of linseed oil for global market were China (120,765 t), Belgium (104,916 t) and USA (102,965 t), followed by India (42,000 t) and Germany (41,100 t), supplying all together 75 % of the world linseed oil production. According to the FAO statistics, there has been a decline in linseed production over last decade almost everywhere in the world. Depending on the country, there was a 10–60% drop in production, which was perhaps associated with linseed offering lower returns to farmers than most other grains (e.g. wheat and corn) or oilseeds (e.g. rapeseed). The decrease of linseed area harvested can be observed in case of Western Europe countries in the last decade (after 2000 year). The possible reason of such situation can be removal of fiber flax and linseed subsidies that growers had been receiving to grow this crop. Thankfully at the same time in Eastern Europe especially in Russian Federation, Ukraine, Belarus the significant increase (more than 2.5 fold during last five years) of linseed production was noticed. There are also other signs that linseed has once again taken a prominent position in studies of crop plants. One of the most significant is the fact that recently the sequence of flax nuclear genome was assembled and published. (Wang et al., 2012). It boosted the genomics research on flax evolution, selecting improved phenotypes, breeding lines and designing new varieties. The goal of the total utilization flax GENomics (TUFGEN) project launched by Genome Prairie was maximizing the utility of flax by developing genomic-based tools to help crop breeding, improve field performance and enhance, seed and fiber properties in period of July 2009–December 2014. Now and in the future, flax is very likely to be included in more projects involving analysis of the genomic information aiming to improve its traits and yield to make it more economically feasible. Basically, linseed products might be used in different branches of industry. Linseed plant utilization for food, feed, and fiber, as well as processing of flaxseed has been recently deeply reviewed (Singh et al., 2011). This paper presents an extensive review of literature on the biochemical composition and biomedical application of linseed common products, including the fiber and oil, together with newly recognized products, like the extracts from shives and seedcake. Another aim of this review is indicating the possible direct or after processing way of commercial utilization for almost every part of linseed plant. Numerous prospective applications of linseed are pointed out in this review, including its use as a valuable source of compounds important in human anticancer and anti-atherosclerosis prophylaxis, as well as the production of preparations that might be used as an alternative antibiotic against pathogenic microbes and the moisture-absorbing bandages rich in antioxidants. Thus, considering such promising and wide spectrum of linseed applications, there is a hope that linseed will again become popular and highly prized plant in the nearest future. One of the concerns of “Plants for the Future”, a strategic research plan for 20 years of development of European agriculture run by the European Technology Platform (ETP) (European Technology Platform, 2007) was improvement of plant fibers and innovations in fiber applications. In their view, for fiber various applications it is crucial to gain control of cell wall composition and the interaction of cell wall components to determine fiber characPlease cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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teristics. Understanding of the complex genetic and biochemical background of fiber formation is of high importance to industry and would enable us to deliver valuable fibers corresponding to the high demands of industry. Another problem addressed by this document is the progressive decline of minor crops cultivation. The increase of crop biodiversity would benefit human diet by diversification of food sources. The lipid composition of linseed seeds was pointed out as well-suited source of essential fatty acids for human and animal nutrition. Linseed was recognized as one of the oil crops good for production of advanced biofuels by European biofuels technology platform (EBTP). The flax is now also covered by the USA – United Stage States Department of Agriculture (USDA program, 2015) and the EU. Implementation of direct payments by the EU aims to increase productivity, stabilize farmers’ income, and secure crop diversification and availability of goods for the market. According to the Regulation (EU) No. 1307/2013 of the European Parliament and of Council (European Commission, 2013), the coupled support may be granted by Member States to production and sectors of particular importance for economic, social or environmental reasons, as well as those undergoing difficulties, and flax was included in them.

2. Structure and biochemistry of linseed products 2.1. Seeds and oil Mature seeds are a source of several important products for industrial purposes. For example, leguminous species are a source of valuable proteins, cereals provides carbohydrate and oily seeds serve as a source of lipids. Total fat content in the linseed plant was 35–46%, total protein accounted for 18–25%, and carbohydrate constituted 23–30%. Linoleic acid concentration was in the range from 16 to 75% and alpha-linolenic acid varied from 1.7 to 59%. The ash content of linseed varieties was between 3 and 5%. Genotype and environmental conditions significantly affect linseed properties (Singh et al., 2013; Soto-Cerda et al., 2014; Thambugala et al., 2013). The seed comprises the seed coat, embryo, endosperm and storage material in the form of carbohydrates, proteins, fats, etc. The outer seed coat layer, the testa, is thick, wavy and shiny, while the inner layer, called the tegmen, is thin (Fig. 1). Below is a single layer of epidermal cells, which covers the next 1–5 layers of parenchyma cells. They are called ring cells and might contain tannin and chlorophyll, which contribute to the seed color. The mostly colorless sclerenchyma is a unicellular layer located below the ring cells. At least the next two cell layers are formed by transversal cells with irregular orientation. Another layer of cells is the endosperm, containing oil and protein. It surrounds the embryo, which has two cotyledons. Usually, the cotyledons are white or yellowish and also contain oil. The embryo consists of an embryonic axis, at which both ends are growing points. The upper growing point forms the shoot system while the lower forms the root system. The seed is attached to the ovary wall by the funiculus, a short stalk. The average yield of linseed oil pressed from the seeds was 35–50% of the seed weight. The most frequently found fatty acids in linseed oil are: palmitic acid (6%), stearic acid (2.5%), oleic acid (19%), linoleic acid (13%) and alpha-linolenic acid (55%). The oil content and fatty acid composition often change due to crop adaptation to regional growing seasons as well as environmental effects (Cloutier et al., 2011). For example, in Canada the oil content varied up to 15% in individual farm samples (Duguid, 2009). Also the percentage of alpha-linolenic acid might increase (ca. 5%) in a cool environment (Fofana et al., 2006). Linseed,

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Fig. 1. Seed morphology. Longitudinal section (A) and cross-section (B) of linseed (Linum usitatissimum L.): embryo (em); seed coat (sc) also called the testa; cotyledons (seed leaves), (sl); endosperm (en); epicotyl (epc); hypocotyl (hy); radicle (ra); mucilage cell (muc); epidermis (ep); parenchymatous layer (pl); sclerite layer (sl); membraniform layer (ml); brown layer (bl); endosperm (en).

The high percentage of alpha-linolenic acid causes the oxidative instability of linseed oil, and therefore it is mainly used in the chemical industry as a component of paints, inks and varnishes. Improved oxidative stability has been obtained by altering the fatty acid profile, for example by lowering alpha-linolenic and increasing linoleic acid content. Ethyl methane sulfonate (EMS)-mediated mutagenesis caused a point mutation in fatty acid desaturase 3 (FAD3), resulting in non-functional enzymatic activity and thus reducing the level of alpha-linolenic acid and accumulation of linoleic acid (Vrinten et al., 2005). Genetic engineering has also been used for altering the fatty acid profile in the linseed plant. Examples are expression of an exogenous gene from potato (Solanum sogarandinum L.) of glucosyl transferase modifying flavonoid compounds (GT plant type) or simultaneous expression of three key genes of the flavonoid pathway coding chalcone synthase, chalcone isomerase and dihydroflavonol reductase, all derived from petunia (Petunia hybrida L.) Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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(W92 plant type) and silencing of the endogenous chalcone synthase gene (W86 plant type), resulting in variations of fatty acid profiles. W86 oil showed a 10-fold increase in the omega-3 fatty acid level. In contrast, a 10% increase in the level of omega-6 was detected in W92 type (Table 1). Besides favorable changes in fatty acids profile, these results showed for the first time the regulatory connection between separate metabolic pathways (i.e. phenylpropanoid and fatty acid). The variations in polyunsaturated fatty acid (PUFA) content affect oil stability. The thiobarbituric acid reactive substances (TBARS) measurements show a decrease in the level of oxidation products by 10.21, 50.93 and 86.53% for W86, GT and W92 oil, respectively, in comparison to control plants (data not published yet). Since the levels of omega-3 fatty acid in GT and W92 types were almost the same, while oxidative status substantially changed, the intriguing question arises: what are the other compounds involved in fatty acid protection against oxidation? To identify the compounds that might participate in oil stability, content of water- and lipid-soluble antioxidants in the oil was recently determined (Table 2). Of water-soluble components (0.05 ␮g/g FW) vanillin was identified as the most abundant (45%) phenolic compound in linseed oil. The other identified components are non-hydrolysable (proanthocyanidins) and hydrolysable tannins (5.4 and 3.0%, respectively), p-coumaric acid, ferulic acid, caffeic acid, coniferyl and syringic aldehyde (10, 16, 3, 9 and 7%, respectively) and small amounts of flavonoids, probably luteolin and kaempferol derivatives. Lipid-soluble secondary metabolites are highly (0.9 mg/g FW) represented in oil. Among these components, ␥-tocopherol (50%), plastochromanol-8 (44.5%), lutein (3%) and ␤-carotene (1.5%) have been found in linseed oil. The highest positive correlation coefficient was obtained for total phenolic content and oil stability. Thus, it suggests that accumulation of compounds from the phenylpropanoid pathway is required for linseed oil stability improvement. High oxidative stability of linseed oil enhances its application not only in the chemical industry but also for biomedical application. Several clinical research studies in humans have aimed at assessing the efficacy of linseed bioactivity in health and disease. For example, many research reports suggest the beneficial impact of a diet high in alpha-linolenic acid from linseed oil on reducing markers of oxidative stress and inflammation associated with risk of many common chronic diseases (e.g. atherosclerosis) (Goyal et al., 2014). From a biochemical point of view, polyunsaturated fatty acids intake modulates membrane lipid composition. Consumption of linseed oil protects against the negative consequences of unbalanced human diet, and prevents or delays the onset of chronic diseases (for example atherosclerosis) through reducing the burden of oxidative stress and generation of anti-inflammatory mediators. In the light of recent experiments, it is evident that a wellbalanced diet might contribute to cardiovascular disease and breast cancer treatment in a positive manner. In a very recent study, the effect of flax seed daily ingestion on blood pressure of peripheral artery disease patients was examined (Rodriguez-Leyva et al., 2013). Those patients that entered the trial with blood pressure (BP) ≥ 140 mm Hg and ingested daily 30 g of milled flaxseed with the food for 6 months showed a significant reduction (ca. 15 mm Hg) in BP. The reduction in BP correlated with circulating alpha-linolenic acid and lignan levels. Thus, it was concluded that linseed included in the daily diet induced an anti-hypersensitive effect. Linseed intake might be associated with decreased risk of breast cancer. Its daily ingestion of 25 g increased the tumor apoptotic index and reduced cell proliferation among breast cancer patients (Flower et al., 2013). Linseed,

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4 Table 1 Fatty acid composition in GM and control flax oil.a,b

Control (mg/gFW) 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 24:0 Saturated fatty acids Polyunsaturated fatty acids Total fatty acids

12.62 0.16 0.12 0.10 7.52 41.75 147.00 4.43 0.26 0.19 0.12 0.10 0.03 20.55 151.65 214.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.44 0.00 0.64 0.01 0.34 4.64 3.34 0.64 0.00 0.03 0.02 0.00 0.00 2.44 3.64 5.87

GT (mg/gFW)

W92 (mg/gFW)

W86 (mg/gFW)

10.09 ± 0.35 0.20 ± 0.05 0.14 ± 0.01 0.13 ± 0.04 8.50 ± 0.05 41.49 ± 3.87 181.72 ± 3.24 5.51 ± 7.98 0.23 ± 0.00 0.33 ± 0.04 0.14 ± 0.00 0.19 ± 0.04 0.04 ± 0.00 18.55 ± 2.34 247.54 ± 4.85 304.61 ± 5.64

13.89 ± 1.02 0.27 ± 0.01 0.18 ± 0.00 0.15 ± 0.02 7.03 ± 0.53 32.11 ± 3.47 197.84 ± 4.44 6.06 ± 0.88 0.16 ± 0.00 0.30 ± 0.02 0.15 ± 0.00 0.20 ± 0.00 0.05 ± 0.00 21.28 ± 2.15 204.23 ± 5.24 258.39 ± 4.98

12.53 ± 0.94 0.21 ± 0.01 0.16 ± 0.00 0.12 ± 0.01 7.38 ± 0.98 54.22 ± 4.08 102.88 ± 2.94 132.83 ± 6.91 0.24 ± 0.01 0.27 ± 0.00 0.15 ± 0.00 0.18 ± 0.00 0.05 ± 0.00 20.35 ± 1.99 235.99 ± 5.04 311.22 ± 6.02

a GT – plants with overexpression of glucosyl transferase, W92-plants with simultaneous expression of three key genes of the flavonoid pathway coding chalcone synthase, chalcone isomerase and dihydroflavonol reductase, W86-plant with silencing of the endogenous chalcone synthase gene b Fatty acid methyl esters (FAMEs) were extracted from the oil using 0.5 M KOH in methanol. The methyl esters were quantified by gas chromatography (Agilent Technology 6890N with FID detector) using pentadecanoic acid as an internal standard (Zuk et al., 2011a,b, 2012; Lorenc-Kukula et al., 2009).

Table 2 Biochemical composition of flax seeds, seedcake and oil.a Compounds

Seeds (mg/g FW)

Ferulic acid Ferulic acid glucoside Coumaric acid Coumaric acid glucoside Caffeic acid Caffeic acid glucoside 3,4-dihydroxybenzoic acid Secoisolariciresinol diglucoside (SDG) Vanillin Syringic aldehyde Coniferyl aldehyde Vitexin Proanthocyanidin Hydrolysable tannins ␥-Tocopherol Plastochromanol-8 Lutein ␤-carotene

6.03 15.28 3.66 10.34 0.66 7.16 0.04 133.1 0.01 0.02 0.02 0.28 0.25 0.48 8.337 1.148 0.046 0.014

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.022 0.026 0.018 0.041 0.01 0.009 0.001 0.387 0.00 0.001 0.003 0.005 0.002 0.001 0.61 0.07 0.01 0.01

Seedcake (mg/g FW)

Oil (␮g/g)

5.73 ± 0.029 14.88 ± 0.041 3.41 ± 0.019 1.06 ± 0.035 0.61 ± 0.01 6.36 ± 0.009 0.04 ± 0.001 120.1 ± 0.387 0.01 ± 0.00 n/d 0.02 ± 0.001 0.13 ± 0.001 0.21 ± 0.002 0.43 ± 0.001 2.467 ± 0.55 0.256 ± 0.042 0.013 ± 0.002 0.004 ± 0.001

0.008 ± 0.001 n/d 0.005 ± 0.001 n/d 0.0015 ± 0.005 n/d 0.0001 ± 0.000 n/d 0.0227 ± 0.001 0.0037 ± 0.001 0.0047 ± 0.001 n/d 0.0015 ± 0.001 0.0027 ± 0.001 459.1 ± 4.95 403.54 ± 2.06 29.97 ± 0.36 13.98 ± 0.431

n/d –not determined. a Alkali hydrolyzed materials were methanol extracted – for phenylpropanoid compounds analysis or chloroform extracted – for hydrophobic compounds analysis, obtained extracts were resolved on UPLC–MS and quantify using commercial standards (Zuk et al., 2011a,b).

Supplementation of the diet with omega-3 fatty acids, lignans and a large group of antioxidants (phenylpropanoids, terpenoids) from linseed should be recommended for prevention of human civilization diseases. 2.2. Seedcake (linseed expeller) Biochemical analysis of methanol extract from linseed expeller revealed the presence of several compounds from the phenylpropanoid pathway. A higher number of compounds was identified after seedcake alkali hydrolysis. Based on retention time and UV spectra of respective standards, the UPLC analysis of seedcake extract obtained by alkali hydrolysis revealed the presence of phenolic acids and their glucoside derivatives (21%), lignan (78%) and low quantities of other phenolics. All these compounds are regarded as strong antioxidants, and thus it is suggested that they might be involved in protection of fatty acids against oxidation during seed storage and industrial oil extraction. Indeed, engineered linseed plants accumulating phenylpropanoids (mainly hydrolysable tannins) produces oil with a significantly increased quantity (5-fold) of omega-3 fatty acids in comparison to control plants (Zuk et al., 2012). Thus, the obtained results strongly suggest Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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that fatty acid composition and yield depend on the antioxidative status of seeds. The antioxidant capacity of aqueous extracts of seedcakes was shown to be used to prevent lipid oxidation in pork meatballs. The aqueous extract from the seedcakes protect the lipids against oxidation to a higher extent (Waszkowiak and Rudzinska, 2014). In another report, the effect of ethanol linseed extracts on lipid stability and changes in nutritive value of frozen-stored meat products was determined. During 150-day storage of meat products the lipid oxidation was monitored, and the data showed that the ethanol extract significantly limited lipid oxidation in stored meatballs and burgers (Waszkowiak et al., 2014). It should also be pointed out that linseed seedcakes are a rich source of secoisolariciresinol diglucoside (SDG). It is important to note the beneficial role of this compound for human health. There are several reports confirming the role of SDG in protection against different kinds of cancer (Flower et al., 2013; Goyal et al., 2014). 2.3. Fibers Flax is mainly a source of fibers that are produced in the outer region of its stem. The straws from fibrous flax have been studied Linseed,

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over a long period of time. The anatomical structure of straw from fibrous flax is well described in numerous publications; for review see (Akin, 2003). The stem cross-section reveals the cuticle layer on the surface of the epidermis, parenchyma cells within the epidermis surrounding fiber bundles, which are formed by bast fibers arranged in bundles, cambium cells and woody core cells. The lignified woody core cells constitute the fraction called shives produced during fiber extraction from straw. The linseed stem anatomy shows the same arrangement. However, the linseed phenotype is exceptional. The linseed plant grows up to 40–60 cm tall. Its leaves are grayish green, 20–40 mm long and 3 mm broad. In over 60% of cases, the flowers are blue in color, 15–20 mm in diameter and have five petals. The seeds are medium brown (ca. 95% cultivars) or yellow (ca. 4% cultivars), 4–7 mm long and become sticky when wet. The color of the seeds depends on the variety. The linseed life cycle consists of 116–130 days of vegetative period, 45–60 days of flowering period and a maturation period of ˛ et al., 2005). 60–90 days (Zajac Fiber yields vary according to variety, environment or agronomic practices, but total fiber yields of ca. 15% of straw dry weight are possible. In the case of fibrous flax fiber, yields are almost 2 times higher (25–30% of straw dry weight), and the yield of seed production is 2–2.5 times higher (Heller et al., 2015) in the case of linseed plants. For greater seed production, linseed varieties are sown at low densities (ca. 750 plants/m2 ). Plants grown in these conditions are intensively branching and when they achieve full seed maturity have thick stems and produce fiber of low quality. In summary, the linseed plant phenotype differs from its fibrous counterpart due to its shorter and highly branched stem, producing a higher quantity of seeds but a smaller amount of low-grade fibers. The plant cell is surrounded by the cell wall, which is a relatively rigid structure composed mainly of saccharide and phenolic polymers. After plant maturation and harvesting, all that remains of the elementary fiber is the cell wall. Flax fibers are classified as stem fibers. They are cellulose-rich bast fibers that contain little lignin and can be found in the outer non-woody stem tissues. It should be noted that lignin, which is relatively low in quantity, has an extremely important impact on both the mechanical and chemical properties of plant fibers, and therefore represents a major target for engineering. Similarly to fibrous plant, linseed fiber exists in bundles of individual fiber strands (Fig. 2). The outer secondary cell wall (densely packed) G-layer of the secondary cell wall is rich in cellulose microfibrils and crystalline cellulose and poor in galactan (hemicelluloses), while the Gn-layer of the secondary cell wall is rich in galactan and poor in cellulose microfibrils (loosely packed). The elementary fibers are bound together end to end by pectin to form bundles. Each bundle consists of 10–40 individual fibers, that are about 20–30 mm long and 0.015–0.020 mm in diameter. The fiber lengths vary depending on the position in the stem. The bundles that are oval in shape display high quality while those irregularly shaped show poor quality (Van Sumere, 1992). Linseed straw offers low-grade fibers, which cannot be used for fine linen textile. Therefore for years, linseed straw was treated as a by-product, which was burned or chopped to spread on fields. However, recently linseed fiber has managed to meet the demands of the composite and specialty paper industry by providing value-added products and in this way might improve farming economy. Cellulose, hemicellulose and pectin are structural carbohydrates and are the main chemical constituents of nearly all plant cells and tissues. These biopolymers are the greatest sources of natural and renewable compounds for putative application in industry, in particular the power branch, and in medicine. Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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Table 3 Biochemical composition of flax fibers and shives.a Compounds

Fibers (ug/g DW)

Ferulic acid Ferulic acid glucoside Coumaric acid Coumaric acid glucoside Caffeic acid Caffeic acid glucoside p-Hydroxybenzoic acid 3,4-Dihydroxybenzoic acid Vanilic acid Vanillin Acetovanillone Acetosiringon Syringic aldehyde Syringic acid Coniferyl aldehyde Vitexin Proanthocyanidin Hydrolysable tannins Canabidiol Phitosterols Lutein Sqalene

82.5 ± 0.5 291.6 ± 0.14 0.54 ± 0.04 0.24 ± 0.05 0.05 ± 0.01 0.03 ± 0.01 2.91 ± 0.01 26.82 ± 0.25 6.56 ± 0.1 193.44 ± 0.1 73.32 ± 0.31 110.52 ± 0.45 18.13 ± 0.01 0.75 ± 0.01 2.15 ± 0.05 1.65 ± 0.01 0.83 ± 0.01 1.09 ± 0.01 0.004 ± 0.00 0.123 ± 0.01 0.001 ± 0.00 0.003 ± 0.00

Shives (ug/g DW) 34.85 ± 4.7 169.03 ± 2.5 72.56 ± 7.8 60.78± 5.7 6.37 ± 0.82 10.02 ± 8.7 n/d 32.77 ± 4.91 41.90 ± 0.1 319.9 ± 3.4 34.18 ± 4.08 56.46 ± 8.95 39.04 ± 1.34 12.27 ± 0.27 86.63 ± 5.76 1.78 ± 0.46 1.23 ± 0.31 2.65 ± 0.56 0.007 ± 0.00 0.228 ± 0.01 0.002 ± 0.00 0.007 ± 0.00

n/d –not determined. a Alkali hydrolyzed materials were methanol extracted – for phenylpropanoid compounds analysis or chloroform extracted – for hydrophobic compounds analysis, obtained extracts were resolved on UPLC–MS and quantify using commercial standards(Zuk et al., 2011a,b).

Cellulose is the basic structural component (60–65%) of flax fibers. The other component important for fiber structure is lignin (2–5%), which provides rigidity to the plant. Incrustation of the cell wall by lignin hardens it and reduces its water content, which results in a lower elasticity. Pectins and hemicelluloses, the other fiber constituents (5–7 and 15%, respectively), are mainly responsible for binding single fibers into bundles, and are predominantly released during retting. However, they are also present in the primary wall of individual fibers, and thus are constituents of the fibers themselves (Preisner et al., 2014). Apart from lignocellulose polymers, the fiber contains a number of secondary metabolites from the phenylpropanoid and isoprenoid pathways. A recently developed fiber extraction method based on alkali hydrolysis and organic phase extraction followed by UPLC–MS analysis has allowed those compounds to be isolated and identified (Preisner et al., 2014; Zuk et al., 2011a,b). The fiber contains mainly ferulic acid. Its glycosidic derivative and simple phenylpropanoids from the benzoic route in the total quantity of 0.5 mg/g DW, vanillin, acetovanillone and hydroxybenzoic acid are included in this route. Flavonoids in a low concentration were represented by vitexin (Table 3). Apart from the crucial function in plants, such as growth regulation, protection from environmental stress and defense signaling, many phenylpropanoids possess beneficial properties for human health. For example, they exhibit antioxidative properties (flavonoids, phenolic acids, lignins), bacterio- and mycostatic properties (vanillin and its derivatives, benzenoid derivatives) or anti-neoplastic and anti-inflammatory properties (naringenin, kaempferol, lignans). Moreover, benzenoid derivatives are crucial structural elements of many important compounds of plant metabolism, such as glucosinolate esters (Arabidopsis thaliana L.), phenolic glycosides and xanthones (Hypericum androsaemum L.), cocaine (Erythroxylum coca Lam.) and taxol (Taxus cuspidate L.). Recently it has been shown that cinnamic acid, vanillin and coumarin enhance transformation of green algae by Agrobacterium better than commonly used acetosyringone (Cha et al., 2011). Very important for potential biomedical application of linseed fibers is presence of canabidiol–the potentially anti-inflammatory comLinseed,

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Fig. 2. Stem morphology. Cross-section of linseed phloem fibers (Linum usitatissimum L.): (A) epidermis (ep), hypodermis (hd), bundle of phloem fibers (bph), endodermis (en), primary phloem (pph), secondary phloem (sph), cambium (ca), secondary xylem (sxy); (B) a distinct border between G-layer and Gn-layer is visible in phloem fibers (arrows); (C) elementary phloem fibers (eph), middle lamellae (ml), plasmalemma (pm), lumen (lu), secondary cell wall (scw), primary cell wall (pcw); (D) elementary phloem fibers, gn – newly deposited gelatinous layer of secondary cell wall, g – mature gelatinous layer of secondary cell wall.

pound (Styrczewska et al., 2012). In the fiber the small content of fatty acids, fitosterols (predominantly ␤-sitosterol) and lutein were noticed (Styrczewska unpublished data). All this compound which are identified in flax fiber will be released to body fluids when we use linen dressing or positively influence our condition when we wear or use linen textile. Recently, FT-IR spectroscopy has been found to be very suitable to detect the major chemical components of flax fiber (Zuk et al., 2011a,b). The broad absorption band at 3400 cm−1 corresponds to the stretching (OH) mode of the free hydroxyl groups and those involved in the intra- and inter-molecular hydrogen bonds

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O H· · ·O of the glucopyranose system. The changes in the intensity of the 3400 cm−1 band components resulted from different cellulose polymer conformation (Fig. 3). It has commonly been accepted that the most diagnostic region characterizing the pectin and lignin constituents of fibers is the 1400–1800 cm−1 region of IR spectra (Wrobel-Kwiatkowska et al., 2009). The characteristic band of pectin is that at 1733 cm−1 , which corresponds to the vibration of the free carboxyl group. The component at about 1610 cm−1 corresponds to the symmetric stretching vibrations of ionized COO− groups of pectins. It should be noted that the degree of fiber retting (pectin release) follows the inten-

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Flax shive components show significant biological properties. For example, they possess antioxidant activity beneficial for their application as “alternative to antibiotics”. This is mainly due to phenolic compounds from the phenylpropanoid pathway that are esterified with pectin and lignin constituents of shives. Examples of compounds identified in flax shives are presented in Table 3. Shives contain mainly vanillin and its derivatives and ferulic acid and its glycosidic form in the total quantity of 0.41 mg/g DW and 0.20 mg/g DW, respectively. Flavonoids are represented by vitexin (0.002 mg/g DW) in a low concentration. The presence of vanillin and its derivatives strongly suggest that shives can have antimicrobial properties. Indeed, the growth of several species of human pathogenic bacteria (hospital strains) was inhibited by a shives extract (data unpublished). Linseed shives will be used as a very productive medium for mushroom cultivation causing increase in their yield and health in relation to cultivation on cereal straw. That is important the previous sterilization of medium based on linseed shives was not necessary. (Sobieralski et al., 2011) Fig. 3. Example of IR vibrational spectra of linseed fiber.

3. Multipurpose application

sity of the pectin bands. Therefore, it was suggested that the data for the pectin content measured by FT-IR is more precise than those from the biochemical method. For lignin, the characteristic bands are observed at 1661 and 1510 cm−1 . The band at 1661 cm−1 has been considered as originating from water associated with lignin and that at 1510 cm−1 as corresponding to the stretching of the aromatic skeleton of the lignans. The integral intensity of this band measured for lignin in flax fiber confirms the data from biochemical analysis. 2.4. Shives Shives are the waste material after fiber extraction from flax straw. They are comprised of lignocellulose polymers and contain a number of heterogeneous compounds from phenylpropanoid and isoprenoid pathways. The quantitative analysis of linseed shives is for the first time reported in this review. Lignocellulose is a complex assembly of cellulose, hemicelluloses and pectin and the phenolic polymer of lignin. The amounts of these polymers, their proportions and monomer composition are different than in fiber. Cellulose accounts for about 56% of shives weight, whereas hemicelluloses constitute approximately 15% of it. The last two components, pectin and lignin, are present in shives in different amounts, compromising together approximately 23% of shives weight. The remaining 6% are phenolics (0.13%) and ash consisting of waxes and inorganic compounds. As in fibers, cellulose fibrils in shives are embedded in lamella composed of pectin and hemicelluloses. The structure is reinforced by lignin, which is covalently bonded to hemicelluloses. Lignin is a highly complex, heterogeneous biopolymer with no defined primary structure. The knowledge on shives constituents and their quantity is mainly derived from chemical methods. The sequential release of shives components and measurements of their levels are commonly performed. Recently infrared spectroscopy has been applied to characterize the vibrational properties of the major components of flax shives at the molecular level. As in other cases, a good diagnostic probe for lignocellulose complexes from shives is the bands at 3200–3400 cm−1 corresponding to the (OH· · ·O) vibrations of the intra- and intermolecular hydrogen bonds (HB) of cellulose and the 1500–1800 cm−1 region corresponding to four types of carboxyl groups present in pectin and lignin constituents. The data from IR spectra confirmed those derived from chemical measurements (Wróbel-Kwiatkowska et al., 2009; Zuk et al., 2011a,b). Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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For centuries, flax has been used by human society for various applications. Flax is generally regarded as a dual-purpose plant providing two main products, fiber and seeds. The fiber derived from the flax stem is characterized by high strength and durability. The seeds provide oil rich in omega-3 fatty acids, digestible proteins, and lignans. In addition, linseed is a good source of high quality protein and soluble fiber and has considerable potential as a source of phenolic compounds. Thus, linseed appeared as one of the most important oilseed crops for industrial purposes, as well as in terms of being a source of food and feed, and fiber. This aspect of linseed utilization was comprehensively reviewed recently (Singh et al., 2011). However, difficulties associated with flax cultivation and processing, unpredictable quality together with the appearance of cheaper and more resilient cotton fibers on the market, in combination with flax oil oxidative instability and the introduction of rapeseed oil onto the market, resulted in falling values of flax in the world market. However, renewed interest in flax products has been noted recently. This is due to the research data suggesting that the flax raw material provides a variety of industrial and health benefits. 3.1. Seeds and oil For years, flax seeds were recommended in the human diet, because of their high content of components beneficial for human health. Besides polyunsaturated fatty acids, they contain relatively high quantities of secoisolariciresinol diglucoside (SDG), phenolic acids and flavonoids. Flax oil contains high quantities of the essential polyunsaturated fatty acids alpha-linolenic acid (ALA) and linoleic acid (LA). It has been widely proven that a high level of ALA in the diet can reduce the risk of cancer and cardiovascular diseases and limit the production of arachidonic acids and other pro-inflammatory eicosanoids. The linseed fatty acids have been reported to affect cytokine gene expression. For example, calves supplemented with flax oil (but not fish oil) tended to show a decrease in the expression of IL-4 and IL-8 genes (Karcher et al., 2014). The simultaneous consumption of omega-3 and omega-6 acids in a proper ratio is essential in cancer prevention and inflammation reduction. It is fairly well established that lowering of the risk of diseases by omega-3 fatty acids is related to cholesterol oxidation. Currently, the typical Western diet contains an excessive amount of omega-6 acids; thus, its supplementation with a high level of omega-3 fatty acids from flax oil could be beneficial for consumer health. The most recent reports Linseed,

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strongly support this point of view. For example, two groups of patients (155 subjects) with idiopathic mild to moderate carpal tunnel syndrome (the most common entrapment neuropathy in human) were treated for 4 weeks with placebo or linseed oil. It was found that linseed oil was effective in the management of mild and moderate carpal tunnel syndrome, especially in improving the severity of symptoms and functional status (Hashempur et al., 2014). Linseeds used for poultry feeding resulted in more nutritional eggs known as “omega eggs”. Linseed oil affects bone metabolism in broilers. Birds fed on linseed oil alone or in combination with palm oil showed enhanced digestibility of calcium, reduced serum calcium and increased tibia calcium concentrations (Zhong et al., 2014). Also interesting are recent reports on the form of oil that is applied. For example, rats fed on linseed oil in the form of microemulsions showed higher levels of docosahexaenoic acid (DHA) in the brain synaptic membrane in comparison to rats that were given oil without emulsion formation. This finding is of great importance because alpha-linolenic acid needs to be converted to DHA through the action of desaturase and elongase enzymes, and this conversion is at a minimal level (<2%) in mammals. A study indicated that application of micro-emulsions can enhance the synaptic membrane DHA levels and influence the functions associated with the brain in a beneficial manner (Sugasini and Lokesh, 2014). In another report, it was suggested that a significant increase in permeability parameters of skin might be obtained using linseed oil together with Span 80, Transcutol P and distilled water in a nanoemulsion formulation. The optimized nano-emulsion formulation used as a trans-dermal carrier of therapeutic agents had a small average globule diameter of 117 nm with a polydispersity index of 0.285 (Kumar et al., 2014). However, the high content of omega-3 fatty acids makes flax oil readily oxidized, and thus it has a minor role in the human diet. Therefore, only a limited number of cultivars with low alphalinolenic acid contents (e.g. Linola) are suitable for the commercial preparation of edible oil (Przybylski, 2005), and even this oil has a very short shelf life. In flax grains, lipids can be protected against oxidation by various mechanisms, for example, by the activity of antioxidants such as phenylpropanoids (flavonoids, phenolic acids), carotenoids or tocochromanols (Amalesh et al., 2011; Smirnoff, 2010). The antioxidant capacity of flavonoids is related to the presence of OH groups, which may directly bind free radicals and chelate metals (Mira et al., 2002). By contrast, carotenoids are supposed to act as free radical scavengers by electron transfer to their double-bond structure. However, even after cold extraction, most of these mechanisms are no longer operative. Perhaps lipid-soluble antioxidants (carotenoids) are not effective enough and phenolics are not effectively extracted with oil. To avoid the rapid onset of rancidity, flax oil is often supplemented with lipid-soluble vitamins A and E and stored in dark glass jars. As none of these protection methods are fully satisfactory, genetic engineering has been applied. Three approaches have been successfully used to manipulate the content of antioxidants from the phenylpropanoid pathway in transgenic flax. The overproduction of the flavonoid aglycones, the accumulation of glycosylated phenylpropanoids or the increased biosynthesis of tannins resulted in a significant increase in the content of these compounds in flax seeds and oil. In all cases, the increase in phenylpropanoid content resulted in the expected enhancement of the antioxidant properties of the oil extract and increased oil stability. In comparison to control, the highest increase in stability, measured as malonyldialdehyde (MDA) content, was detected for the oil from plants overproducing flavonoids (86%). A medium increase (50%) was observed for oil from plants over-accumulating glycosylated derivatives. The lowest (10%) was Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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obtained in the case of plants overproducing tannins, but it was still significant for this oil. These data fairly well concurred with those derived from other studies describing analyses of oil oxidation in relation to their PUFA content. As seen from studies of the peroxidation of lipid standards, an increasing number of unsaturated C C bonds enhances the susceptibility to oxidation (Przybylski, 2005). Thus, it was observed that higher PUFA levels resulted in easier oil oxidation. However, it is interesting to note that among the investigated oils, the one from the W86 plant seeds, despite being richest in omega-3 fatty acids, was the most stable when exposed to high temperature. This suggests that the lowered stability caused by a high PUFA content may be overcome when high enough levels of effective antioxidants are present. It is obvious that mainly the antioxidant content in the oil determines its susceptibility to oxidation at high temperature. The data from the measurements of phenolics correlate fairly well with oil stability. This is in contrast to the terpenoid compounds content, in which case no such correlation has been found, but there is no doubt that these compounds affect the total antioxidant capacity of the oil as well. Experiments performed on both groups of compounds (using standard substances) and our results obtained on transgenic plants with overproduction or reduction of phenylopropanoid or terpenoid compound allow us to claim that this first group has higher influence on oil stability. The general conclusion from this study is that water-soluble antioxidants are more suitable for fatty acid protection against oxidation rather than lipid-soluble ones, and the most effective of them are phenolic acids. Furthermore, the engineering of the phenylpropanoid pathway in flax is beneficial for flax seed oil stability, and the extent of lipid protection depends on the antioxidant concentrations. In addition to preventing rancidity, both types of antioxidants (phenylpropanoids and terpenoids) could increase the commercial value of food products based on flax oil and might be beneficial for human health. When consumed together with PUFA, they can reduce the risk of various diseases (Heber, 2007). Recently, the positive effects of feeding with supplements rich in omega-3 fatty acids on beef cows reproduction were determined in one of the studies. In another study the dietary treatments had no effect on pregnancy rates, but the levels of plasma prostaglandin F and serum progesterone were affected (Richardson et al., 2013; Scholljegerdes et al., 2014). The prostaglandins are a group of hormone-like lipid compounds that are derived enzymatically from fatty acids and have a variety of strong physiological effects, such as regulating the contraction and relaxation of smooth muscle tissue. Progesterone, also known as the hormone of pregnancy, is a steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis in humans and other species.

3.2. Seedcake The market potential of flax might be further strengthened by the use of seedcakes, a material which up to now has been used only marginally for animal feeding. Recently, flax seedcake extract, rather than intact seedcakes, has started to be considered as a feasible source of bioactive compounds. So far, the extract has been studied as a potential antitumor and anti-bacteria agent. For example, the lignan-rich extract from linseed hulls was investigated as a potent agent reducing the risk of some chronic hormonal conditions, such as benign prostatic hyperplasia (BPH). Rats with BPH induced using the testosterone propionate were fed with a diet containing different quantities of extract. It was found that the lignan-rich extract significantly inhibited testosterone propionate-induced prostate size, and this effect was dose dependent (Bisson et al., 2014). Linseed,

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Another new aspect of seedcake extract utilization is its potent antimicrobial activity. The worldwide increase in multidrug resistance of pathogenic bacteria has led to an increasing need for topical antimicrobial products that can be applied for therapy of infections. Many of the products are admittedly highly cytotoxic toward microbial cells, but unfortunately at the same time they have side effects on human tissue. Thus, searching for products that effectively kill bacteria but do not cause side effects has become an issue of great importance in modern bio-medical sciences. It was found that seedcakes from flax plants are a rich source of phenolic compounds potentially active against bacteria. Indeed, the results of the recent studies on flax plants, including genetically modified plants, resulted in a potential new, fully natural pharmaceutical product containing biologically active compounds that exhibit antibacterial properties. For example, in one study a seedcake extract was tested against sensitive and multidrug-resistant clinical bacterial strains (Zuk et al., 2014). The alkali hydrolyzed seedcake extract from genetically modified flax was prepared and its biochemical composition was carefully analyzed. It was found that seedcake extract is a rich source of strong antioxidant metabolites such as coumaric acid, ferulic acid, caffeic acid, and lignan. Antimicrobial activity of the extract was examined using a minimal inhibitory concentration (MIC) test on four bacterial species, normally used as model organisms for analysis of antibiotic resistance, and also clinical strains that acquired resistance to more than three groups of antibiotics. The extract exhibited strong germicidal activity against all these species. The antimicrobial activity of standard substances was also analyzed, and the obtained results suggest that phenolic acids are responsible for antimicrobial activity of flax seedcake extract. The data lead to the suggestion that flax seedcake extract may be a suitable candidate for unselective antimicrobial treatment and that flax-derived natural products are a promising substitute or even alternative to antibiotic therapy. A potential mechanism of action based on DNA disintegration and topoisomerase II (gyrase) inhibition was proposed (Zuk et al., 2014). 3.3. Fiber Linseed is cultivated mainly as a source of oil for human consumption and manufacturing of environmentally friendly paints or other industrial products. However, there is a growing demand for linseed as a source of valuable fiber in the world market, which also contributed to an expansion of linseed cultivation recently. The probable reason of this situation will be still growing attention put on using of linseed fiber – in this situation this plant will be more valuable for farmers. The big problem, to solve, is the different dates of maturity of the seeds and maturity of fibers contained in the stems of linseeds (however, this problem exists mainly in the cultivation of fibrous flax). This hassle can be omitted in a special good weather condition flowed by special agricultural treatment (for example fertilization, planting density) the process of maturation of seeds can be shortened and if the seeds were collected as soon as possible the straw can be also used to get fiber. The linseed fibers are being increasingly used in the automobile and construction industries as an eco-friendly composite material. Tested biomechanical features of generated composites that contain flax fiber showed improved tensile strength (Li et al., 2013). Moreover, recently due to legislation requirements the automotive industry was forced to use innovative and eco-friendly components in cars. In recent studies, the influence of fabric made from linseed fibers on proliferation of cultured fibroblasts was tested. Since cytotoxicity and allergy tests on human cultured cells (keratinocytes, fibroblasts) and laboratory animals were proven to be negative, the fabrics were used for preparing and testing a wound dressing and tissue scaffold (Qualley et al., 2012). Active and healed ulcers (scars Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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after ulcers) occur in approximately 1% of the population, with occurrence of the pathology stated to be more often for women. Successful treatment that stimulates healing is an essential step toward eliminating this morbidity, improving quality of patients’ life, and reducing healthcare costs. Subjects (22 persons) suffering from chronic ulceration of venous origin for at least two years were treated with a wound dressing. Four-week application of flax bandage resulted in faster healing, specifically reduced wound exudates and decreased wound size in 55% of patients. Interestingly and importantly, patients reported that the bandage diminished the pain accompanying chronic venous ulceration (SkórkowskaTelichowska et al., 2010). A composite containing flax fiber embedded in polylactide was recently used for tissue repair in a rat model. A composite plate implanted into muscle was very well adopted as a scaffold and tolerated by surrounding tissue (Gredes et al., 2010). Linseed fibers are considered as favorable substrates for energy production. Different methods can be used for utilization of linseed lignocelluloses, including direct combustion, gasification, pyrolysis, and enzymatic conversion to ethanol and other organic compounds. Same research has shown that retted and decorticated linseed stalks can be processed into a fiber of surprising fineness and with little loss in strength per fiber. (Anthony, 2002) The short fibers from linseed straw can be used to generate cottonized flax. When cotton took over the spinning market, almost all spinning and weaving equipment was designed to use fibers with the approximate length and diameter of cotton fibers. Hence, a mechanical process was developed to break down flax fiber that could be spun on the cotton equipment. The only problem is that fibers suitable for cottonization should have a linear density of less than 1 tex (like fibrous flax), while linseed fibers are much thicker. The cottonization of linseed fiber is more difficult and complicated than fibrous flax. This fact makes the cottonization and blending with cotton much more difficult. However is possible if you use good quality storm with specially prepared retting system to obtain good quality fiber. The problem is not with length of fiber because cottonizing of flax fiber involves reducing the length of the fibers to that suitable for cotton machinery. This is normally done by cutting and can be done also on short fiber from linseed. The only one problem is thickness of oil flax fiber – but it can be improved by using good retting system and enzymatic treatment. Of course the problem is in the costs of this process but this is possible in same conditions. During the last decade, a new method of cottonising technical bast fibers (including linseed and hemp fiber) has been developed at Institute of Natural Fibers, Poznan, Poland and implemented to Polish industry. The method is based on the use of enzymatic preparation (Pektopol PT) to facilate de-gumming of the fiber bundles and their easier separation into smaller bundles (elementary fibers) which can be used to cottonization or to textile production. (Sedelnik et al., 2006; Cierpucha et al., 2004) Such flax is generally referred to as cottonized flax, and it is used for clothing production with the proportion 50–80% with cotton fibers. The potential of flax fiber as a geotextile to produce mesh that protects soil from erosion or to reduce the level of dust and erosion along roads, railroads and building sites should also be pointed out. The linen non-woven can be applied with under sown grasses or seeds of other plants to reinforce slopes and embankments and after taking over this role by rooting plants, mat decomposes and does not pollute the environment. Moreover, linseed fiber might also be used for reinforcement of recycled paper. Paper recycling requires re-pulping, which causes a loss of paper strength in the next generation. It is believed that the addition of flax fibers to the pulp might improve the paper strength. Linseed fiber finds application in production of insulation batts that might replace glass fiber batts. The mentioned examples prove linLinseed,

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seed fiber to be a prospective component of valuable industrial products. 3.4. Shives The market potential of linseed might be additionally supported by the use of shives, application of which used to be limited to sealing and thermally insulating homes. The prospective approach might be the conversion of shives’ lignocellulose polymers into bio-products. Lignocellulose biomass derived from shives is a promising renewable resource for the production of bio-based materials, including transportation fuels. The majority of energy stored in biomass is contained within the polymers of the cell wall. The cell wall is also the major component of dried biomass by weight. It constitutes approximately 70–90% of seasoning or drying straw biomass (Allison et al., 2010; Hodgson et al., 2010). Currently bio-energy production from agricultural residues is based on hydrolysis of lignocellulose as the initial step, then enzymatic depolymerization by specific bacteria and finally saccharide fermentation to energy products (Yee et al., 2012). The concentration and composition of the cell wall affect its recalcitrance to enzymatic deconstruction (saccharification) (Ding et al., 2012). For example, utilization of the fermentable sugars stored in the carbohydrate polymers of the cell wall is limited by the presence of lignin. Thus, the reduction of lignin content is frequently the central approach for biomass improvement (Chen and Dixon, 2007; Li et al., 2008). Indeed, down-regulation of CAD, the gene controlling the last step in monolignol biosynthesis, resulted in generation of a far better substrate for biogas production. Low lignin poplar trees showed as much as a 15% increase in the efficiency of bioconversion and almost complete hydrolysis of the cellulosic polymer upon alkaline pre-treatment (Mansfield et al., 2012). In agreement with this are our own as yet unpublished results. The efficacy of biogas production was improved when shives from cinnamyl alcohol dehydrogenase (CAD) engineered flax were used. Lignin reduction (by about 30%) in these shives with the use of RNAi technology resulted in a 25% increase in the efficiency of biogas production. On the other hand, decreasing lignin content might enhance plant susceptibility to pathogen infection. For example, in flax down-regulation of the CAD gene by RNAi suppression increased susceptibility to the pathogenic fungus Fusarium oxysporum. It has been shown that the percentage of infected seedlings was two-fold higher in CAD RNAi lines in relation to control plants (WróbelKwiatkowska et al., 2007). In A. thaliana L., the CAD double (cad-c and cad-d) mutants showed increased susceptibility to both an avirulent and a virulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato relative to control plants. In contrast, in a hybrid poplar (Populus tremula L. × Populus alba L.), it has been reported that no increase in disease incidence was observed in antisense COMT or CAD lines in relation to controls (Halpin et al., 2007). The brown midrib (bmr/bm) mutants of sorghum and maize, which have reduced lignin content, showed increased resistance to specific fungal pathogens. These and other studies from a variety of plants indicate that reducing lignin content and altering its composition do not necessarily increase the susceptibility of bioenergy feedstocks to pathogens (Porter et al., 1978; Sattler and Funnell-Harris, 2013). The reason for this is that perhaps pathogen infection leads not only to synthesis of “defense” lignin but also to the production of numerous other phenylpropanoid compounds including phenolic phytoalexins, stilbenes, coumarins, and flavonoids. These compounds are also implicated in plant defense. In flax, the up-regulation of flavonoids and phenolic acids derived from the phenylpropanoid pathway induced flax resistance against pathogen infection (Lorenc-Kukuła et al., 2009). Please cite this article in press as: Zuk, http://dx.doi.org/10.1016/j.indcrop.2015.05.005

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The GM linseed with increased level of phenylpropanoid compounds indicated also higher tolerance for cold condition and can be sown two weeks before control. Winter resistance of flax can lead to more and better yield of linseed. For research in this direction could be likewise used perennial flax varieties that yield for many years and can cope well with winter survival. Flax shives contain a heterogeneous mixture of compounds known to have antimicrobial activity. Recently, an engineered flax plant has been used as a source of antimicrobial compounds. The antibacterial activity of shives extract with a unique composition of different phenylpropanoid compounds (vaniline, ferulic acid, pcoumaric acid and their glycosides) was detected. Growth of several bacterial strains of clinical relevance (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa) was strongly inhibited when treated with this extract (Czemplik et al., 2011). Preliminary data suggest that the mechanism of the bactericidal effect is based on bacteria gyrase inhibition and bacteria genome disintegration by phenylpropanoid compounds. This finding indicates that crop plants might be promising sources of diverse compounds with antimicrobial activity against quickly mutating bacteria resistant to available antibiotics. The worldwide increase in multidrug resistance of pathogenic bacteria has led to an urgent need to identify an alternative strategy to counter bacterial infection. The preparation based on flax shive extract will be a good alternative to conventional treatment. The rest material after extraction of antimicrobial compounds is still valuable material for biomedical application – can be used as a natural drug carrier as a substance which are added to pharmaceuticals to improve it delivery and effectiveness. After alkali hydrolysis, performed to rely of well bounded compounds, the shives cellulose, the basic structural component of flax shives and fiber, with free functional groups/bounds will be obtained. Such active groups can bond pharmaceuticals. After process of 20 h of ball-milling treatment the decreased particle size will be obtained. This method results in structural changes in biopolymers (cellulose, hemicellulose, pectin, and lignin) and increasing of functional group content (Dyminska et al., 2012). The pilot study confirms this hypothesis that fibers and shives, initially subjected alkali hydrolysis and after that micronized effectively binds hydrophobic and hydrophilic compounds (data unpublished).

4. Future outlook The research data presented in this review suggest that the flax raw material provides a variety of industrial and health benefits, as summarized in Fig. 4. Previously used only for textile and oil production, flax raw material is now being considered for biodegradable composite, implants, wound dressing, alternative antibiotics, anti-atherosclerotic, anti-tumor and anti-inflammatory preparation production and marketing. However, to accomplish this beneficial goal it is necessary to further develop the technology that might help to increase flax productivity and enhance the quality of raw materials. Genetically modified organism (GMO) technology is the most exploited method for generation of transgenic organisms for both scientific and commercial purposes in modern plant biotechnology. However, according to Eurobarometer over 60% (61–90% depending on the country) of members of society do not accept GMO, which locks the door for the commercialization of GM plants and products. Thus, the challenge for new biotechnology is to develop a method of modulation rather than modification of the genome, which would still produce effects similar to those of agrotransformation. The method for the non-invasive modulation of the genome involves the induction of changes in methylation–demethylation of the endogenous gene by treatment of plants with short length Linseed,

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Fig. 4. Scheme of multipurpose application of linseed raw products.

oligodeoxynucleotides (oligos), which are sense/antisense to the coding region. Since the method does not include genomic DNA breaking, no heterologous DNA incorporation into the genome takes place in this case, and the process does not need to be mediated by Agrobacterium infection. It can therefore be regarded as natural due to not involving genetic modification. Successful application of epigenetic tools for plant improvement has recently been reported. For example, antisense oligos were used to reduce the expression of the nucleus-encoded phytoene desaturase in tobacco, the chlorophyll a/b-binding genes in wheat, the chloroplast-encoded psbA gene in A. thaliana L. (Dinc et al., 2011), the SUSIBA2 transcription factor, in sugar-treated barley (Sun et al., 2005) and the GNOM LIKE 1 gene in tobacco (Liao et al., 2013). An experiment with Taraxacum officinale F.H. Weeg showed that 74–92% of the variation in the level of methylation induced by jasmonic acid and salicylic acid was preserved in the next generation (Verhoeven et al., 2010). In the most advanced study, the gene coding for ␤-glucanase was up-regulated by treatment of flax plants with oligos, and this feature accompanied by gene demethylation was stably inherited up to the third plant generation (Wojtasik et al., 2014). In the last three years pilot experiments, using oligos, on linseed have also been performed – the modulation of chalcone synthase and lycopene ␤-cyclase genes’ activity was achieved. The induced changes were stable for at least two generations (data unpublished). Thus, the new method based on epigenetic modulation of the genome appears to be a future tool for investigation of gene function and improvement of crop plants as well. The other scientific and perhaps industrial challenge with flax as a subject is the synchronization of fiber and seed maturation. It is well established that stem maturation and seed maturation are independent processes for both the fibrous and linseed plant varieties. Fibrous flax is harvested when stems are almost entirely defoliated and the development of the secondary phloem fiber cells is complete. At this time flax combines good fiber yield and quality. However, harvesting the plant at this stage means risking loss of sowing seed quality. On the other hand, later harvesting

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causes increasing lignification and results in poor fiber quality. Thus, more synchronized maturation of stem fibers and seeds will reduce harvest risks. A breeding program aiming to synchronize these maturation processes in flax in order to reduce harvest risks during the production of both high quality fiber and flax sowing seed was initiated (in FLAX: breeding and utilisation: Proceedings of the EEC Flax workshop, Brussels, Belgium 1988). Even after almost three decades no report is available on the progress of the program. However, a few reports concerning the search for clues on the early domestication history of flax have appeared lately. The recent finding that the cultivated flax with spontaneously opening capsules (dehiscent flax) displays close relatedness to its wild progenitor (pale flax) and winter flax (required vernalization), which is closely related to linseed or the fibrous type and distantly related to its progenitor, suggests that flax’s early domestication history might involve multiple events of domestication for oil, fiber, capsular indehiscence and winter hardiness (Fu et al., 2012). Based on the fact that capsular dehiscence and winter hardiness are major characteristics of pale flax and that cultivated flax originating from the warm Middle East was spread to cold world regions including Europe, it was hypothesized that winter hardiness was among the early domesticated traits (Fu et al., 2012). Nowadays, the growing interest in flax attracts more attention to linseed straw and fiber as by-products of its cultivation in North America. The associations between stalk fiber content and quantitative (plant height, number of days from emergence to end of flowering, petal width, seed weight, seed oil content and proportion of linolenic acid to total fatty acids) or qualitative (stalk branching, petal color, petal overlapping, petal margin folding and seed color) plant characteristics were investigated (Diederichsen and Ulrich, 2009). The results showed that the wide variation in fiber content of flax germplasm will be a highly useful tool for determination of germplasm relevant for breeding dual purpose flax. For the same reason, that is to make one flax cultivar of a dual purpose crop, genomic regions controlling both stalk fiber and seed quality traits were investigated (Soto-Cerda et al., 2013). The conclusion was that

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