The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

The nutritional value of flaxseed meal (FSM), a by-product of the flax crushing industry, has not been evaluated properly for use within swine rations. A series of experiments were conducted to determine the nutritional profile of this novel feed ingredient for pigs.<p>The analysis of FSM revealed that it contains, on a dry matter (DM) basis, 133 g/kg ether extract (EE), 345 g/kg crude protein (CP), 60 g/kg ash, 164 g/kg ADF, 250 g/kg NDF, 102 g/kg crude fibre, 14 g/kg starch and 9 g/kg phosphorus. The gross energy (GE) content of the meal was 5.2 Mcal/kg DM. The ether extract fraction was characterized by, as a percent of total fat, 46.6% á-linolenic acid, an omega-3 fatty acid. Palmitic, stearic, oleic and linoleic acids accounted for 9.5, 4.8, 20.7 and 18.4% of the total fat content respectively. The crude protein content was well balanced for all amino acids with the exception of lysine (4.1% of CP), the level of which falls below that of the requirements for growing pigs (5.3% of CP for pigs 20-50 kg). The apparent digestibility of DM, nitrogen, ash, EE and GE as well as determination of the DE and NE content of FSM was determined for both growing pigs (32 pigs, initial weight 70 ± 3 kg) and gestating sows (26 pigs, parities 2 4). Animals were fed wheat/barley based diets containing 0, 10, 20 or 30% FSM. Faecal grab samples were collected for 3 days after a dietary adaptation period. The apparent digestibility of nutrients in FSM was determined both by regression and by difference calculations. As calculated by difference, the apparent digestibility coefficients for DM, nitrogen, ash, and GE were 63.0, 60.8, 22.3 and 60.5% respectively for growing pigs. The values obtained for sows were 64.1, 58.8, 20.8, 94.9 and 65.4% for DM, nitrogen, ash, EE and GE respectively. The DE content was 3.37 Mcal/kg for growing pigs and 3.52 Mcal/kg for sows. Net energy was then estimated by use of a prediction equation to be 2.34 and 2.44 Mcal/kg for growing pigs and sows. <p>An experiment was conducted to evaluate the growth performances and carcass fatty acid profiles of pigs fed with graded levels of FSM. A total of 200 pigs (100 barrows, 100 gilts; initial weight 32 ± 4 kg) were blocked by gender and housed in groups of 5 pigs per pen. The experiment was divided into three phases for pigs 32-60 kg, 60-85 kg and 85-115 kg. Each group was assigned to one of four dietary treatments containing 0, 5, 10 or 15% FSM at the expense of wheat and soybean meal. At the time of market, 6 pigs per treatment group were randomly selected for carcass fatty acid analysis, and backfat and rib-end loin samples were collected. The average daily gains, average daily feed intakes and gain to feed ratios were not affected by dietary treatment (P > 0.05). Inclusion of 15% dietary FSM increased the ALA content from 11 to 47 (± 0.8) mg/g of backfat (P < 0.001) and from 5 to 10 (± 0.4) mg/g of loin tissue (P < 0.001). Increasing dietary FSM decreased the saturated fatty acid content of backfat (P < 0.01). <p> The final experiment was designed to determine the availability of phosphorus in semi-synthetic diets containing FSM, and to determine the effects of microbial phytase inclusion of this availability. Five treatment groups, 8 barrows (45 ± 4 kg initial weight) each, were fed a diet containing 30% FSM with increasing levels of phytase (0, 575, 1185, 2400 and 2570 FTU/kg). Apparent P digestibility increased from 20.6 to 61.3% with the inclusion of up to 2570 FTU/kg microbial phytase (P < 0.001), and followed a quadratic response pattern with an R2 value of 0.96. A broken-line analysis estimated the optimal phytase inclusion level to be 1415 FTU/kg of diet. Inclusion of just 575 FTU/kg accounted for half of the response, improving the apparent P digestibility by 20% and reducing P excretion by 850 mg/kg dry matter intake.

Two studies on potential approaches for processing low-salt, low-fat (LSLF) bologna were completed. In study 1, the effects of three factors, namely salt type (sea salt vs. regular NaCl), NaCl concentration (0.75%, 1.00%, 1.25% and 2.00%) and holding of stuffed batter before cooking (cooked immediately (CI) vs. delayed cooking (DC)), on the quality of LSLF bologna were investigated. There was no difference between salt type for most of the parameters measured. The holding factor significantly improved the water holding capacity (WHC) and texture of bologna samples containing 0.75% NaCl, as shown by lower (p<0.05) expressible moisture. However, holding factor did not affect WHC and instrumental texture of samples with 1.00%, 1.25% or 2.00% NaCl. A NaCl level by hold effect (p<0.05) was observed for texture profile analysis (TPA) in which there was significant improvement in the texture of samples containing 0.75% NaCl that were subjected to DC, but no effect at other NaCl levels. Panelists were able to detect the positive effect (p<0.05) of DC on the texture of samples with 0.75% or 1.00% NaCl. This study showed that DC is effective in improving the texture of bologna samples with extremely low NaCl (0.75%) content. The biggest challenge in this first study was the difficult sample handling experienced during slicing. Since bologna is commonly sold as thin slices, the bologna must be firm enough for ease of slicing. The second study focused on improving bologna firmness by the addition of microbial transglutaminase (MTG), known for its functionality as a protein cross-linker, and of flaxseed meal (FSM), known for its excellent water holding capacity. The physico-chemical and sensory characteristics of 12 treatment combinations (0, 0.15% and 0.30% MTG; 0, 0.5%, 1.0% and 1.5% FSM) were determined. In general, results showed that MTG significantly improved the textural quality of bologna, but resulted in a higher purge loss during storage of vacuum packaged slices. On the other hand, FSM significantly reduced the expressible moisture content and purge loss of the product. In terms of product colour, MTG had no effect but FSM when added to the formulation at level as low as 0.5%, affected the colour as determined by both instrumental and sensory evaluation. The overall results of the project indicated that texture in LSLF bologna is not a major issue, since processing conditions and combinations of ingredients can be manipulated to improve texture. The biggest challenge, however, is in the area of flavour – improving the flavour of low-salt processed meats warrants further research.

The nutritional value of flaxseed meal (FSM), a by-product of the flax crushing industry, has not been evaluated properly for use within swine rations. A series of experiments were conducted to determine the nutritional profile of this novel feed ingredient for pigs.<p>The analysis of FSM revealed that it contains, on a dry matter (DM) basis, 133 g/kg ether extract (EE), 345 g/kg crude protein (CP), 60 g/kg ash, 164 g/kg ADF, 250 g/kg NDF, 102 g/kg crude fibre, 14 g/kg starch and 9 g/kg phosphorus. The gross energy (GE) content of the meal was 5.2 Mcal/kg DM. The ether extract fraction was characterized by, as a percent of total fat, 46.6% á-linolenic acid, an omega-3 fatty acid. Palmitic, stearic, oleic and linoleic acids accounted for 9.5, 4.8, 20.7 and 18.4% of the total fat content respectively. The crude protein content was well balanced for all amino acids with the exception of lysine (4.1% of CP), the level of which falls below that of the requirements for growing pigs (5.3% of CP for pigs 20-50 kg). The apparent digestibility of DM, nitrogen, ash, EE and GE as well as determination of the DE and NE content of FSM was determined for both growing pigs (32 pigs, initial weight 70 ± 3 kg) and gestating sows (26 pigs, parities 2 4). Animals were fed wheat/barley based diets containing 0, 10, 20 or 30% FSM. Faecal grab samples were collected for 3 days after a dietary adaptation period. The apparent digestibility of nutrients in FSM was determined both by regression and by difference calculations. As calculated by difference, the apparent digestibility coefficients for DM, nitrogen, ash, and GE were 63.0, 60.8, 22.3 and 60.5% respectively for growing pigs. The values obtained for sows were 64.1, 58.8, 20.8, 94.9 and 65.4% for DM, nitrogen, ash, EE and GE respectively. The DE content was 3.37 Mcal/kg for growing pigs and 3.52 Mcal/kg for sows. Net energy was then estimated by use of a prediction equation to be 2.34 and 2.44 Mcal/kg for growing pigs and sows. <p>An experiment was conducted to evaluate the growth performances and carcass fatty acid profiles of pigs fed with graded levels of FSM. A total of 200 pigs (100 barrows, 100 gilts; initial weight 32 ± 4 kg) were blocked by gender and housed in groups of 5 pigs per pen. The experiment was divided into three phases for pigs 32-60 kg, 60-85 kg and 85-115 kg. Each group was assigned to one of four dietary treatments containing 0, 5, 10 or 15% FSM at the expense of wheat and soybean meal. At the time of market, 6 pigs per treatment group were randomly selected for carcass fatty acid analysis, and backfat and rib-end loin samples were collected. The average daily gains, average daily feed intakes and gain to feed ratios were not affected by dietary treatment (P > 0.05). Inclusion of 15% dietary FSM increased the ALA content from 11 to 47 (± 0.8) mg/g of backfat (P < 0.001) and from 5 to 10 (± 0.4) mg/g of loin tissue (P < 0.001). Increasing dietary FSM decreased the saturated fatty acid content of backfat (P < 0.01). <p> The final experiment was designed to determine the availability of phosphorus in semi-synthetic diets containing FSM, and to determine the effects of microbial phytase inclusion of this availability. Five treatment groups, 8 barrows (45 ± 4 kg initial weight) each, were fed a diet containing 30% FSM with increasing levels of phytase (0, 575, 1185, 2400 and 2570 FTU/kg). Apparent P digestibility increased from 20.6 to 61.3% with the inclusion of up to 2570 FTU/kg microbial phytase (P < 0.001), and followed a quadratic response pattern with an R2 value of 0.96. A broken-line analysis estimated the optimal phytase inclusion level to be 1415 FTU/kg of diet. Inclusion of just 575 FTU/kg accounted for half of the response, improving the apparent P digestibility by 20% and reducing P excretion by 850 mg/kg dry matter intake.

The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

Vitamin B6, or more specifically the active form pyridoxal 5ʹ-phosphate (PLP), plays a crucial role as a cofactor for numerous enzymes linked to carbohydrate, fatty acid, and amino acid metabolism. There is a high prevalence of moderate vitamin B6 deficiency in the population that may be further exacerbated through the ingestion of vitamin B6 antagonists present in the food supply. For example, flaxseed contains the anti-pyridoxine factor 1-amino D-proline (1ADP) in the form of a dipeptide called linatine. In order to address these issues, the current study was designed to: 1) characterize and quantify the total amount of anti-pyridoxine factors present in flaxseed through the use of UPLC/ESI-MS analysis, 2) investigate the in vivo effects of synthetic and flaxseed-derived 1ADP on amino acid metabolism using a rat model of moderate B6 deficiency, and 3) identify novel biomarkers of vitamin B6 inadequacy using a LC-Qtof-MS based non-targeted metabolomics approach. The total anti-pyridoxine content, measured as 1ADP equivalents, in the flaxseed extract was found to be 177-437 μg/g of whole flaxseed, depending on the variety tested. Plasma biochemical analyses revealed that B6 vitamers, particularly PLP concentrations were reduced (P≤0.001), due to 1ADP ingestion (10 mg/kg diet) irrespective of the sources. Oral ingestion of flaxseed-derived 1ADP in moderately vitamin B6-deficient rats increased plasma cystathionine (P≤0.001), and decreased plasma α-aminobutyric acid (P≤0.001) and glutamic acid (P=0.017) concentrations compared to the controls. However, the ingestion of synthetic 1ADP elicited greater perturbations in amino acid profile compared to the flaxseed-derived 1ADP, which was predominantly in the form of the dipeptide linatine. Additionally, oral ingestion of the synthetic as well as the flaxseed-derived 1ADP significantly (P≤0.05) inhibited the activities of hepatic PLP-dependent enzymes involved in transsulphuration reactions of methionine metabolism. The use of a non-targeted metabolomics approach identified ten potential lipophilic markers of vitamin B6-insufficiency: glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone, orthothymotinic acid, cystamine and 3-methyleneoxindole. These data serve to highlight potential deleterious effects of anti-pyridoxine factors linked to flaxseed in a population at risk for moderate vitamin B6 deficiency. / October 2015

Breast cancer molecular subtypes, based on expression of estrogen, progesterone and human epidermal growth factor 2 receptors, alter prognosis and treatment options. &alpha;-linolenic acid (ALA) is a complementary therapy, however its effectiveness across breast cancer types and estrogen environments is unclear. This research determined the effect of ALA on growth, apoptosis, fatty acid profile, and gene changes in four breast cancer cell lines with varying receptor expression with or without (&plusmn;) estradiol (E2). ALA (50-200uM) &plusmn; E2 reduced growth in all cell lines. 75&mu;M ALA +E2 increased phospholipid % ALA in all cell lines and induced apoptosis in cell lines lacking the three receptors. Cellular % ALA was positively associated with apoptosis and inversely associated with cell growth. ALA altered expression of cell cycle, apoptosis and signal transduction genes. In conclusion, ALA incorporates into breast cancer cells, reduces growth and induces apoptosis regardless of receptor status or E2 level.

Experiments were conducted to investigate the effects of dietary fat, vitamin E(Vit. E) and selenium(Se) supplementation on the performance, fatty acid(FA) composition, lipid oxidation on blood and the oxidative stability of muscle tissues (breast and thigh) during refrigerated storage of broiler chickens fed high levels of flaxseed(FXS). Four hundred and eighty d-old broiler chicks were fed experimental diets for a 3 wk period, which consisted of the combination of two dietary fat sources: fullfat soybean(FFSB) and FXS; 3 Vit. E levels: 10, 40 and 80 IU/kg and two Se levels: 0.15 and 0.30ppm (Exp. I). Four hundred and forty d-old chicks were fed on FFSB diets supplemented with 10, 80, and 160 IU of Vit. E/kg, FXS diets supplemented with 10, 40, 80, 120, 160 and 200 IU of Vit. E/kg, corn-soybean based diet (control), and on a commercial diet for 21 days (Exp. II). For Exp. III, 1680 (840 of each sex) d-old broiler chicks were raised up to 41d. Three basal diets containing 10% FXS and 3 levels of Vit. E: 60-, 90- and 160 IU/kg were formulated for 3 age periods: starter (0--3wk), grower (3--5 wk) and finisher (5--6wk). Three groups of birds were fed these basal diets, while 3 other groups were fed the diet containing 160 IU of Vit. E either during the starter, grower or finisher phase (diets supplemented with 60 IU/kg were given in the two remaining phases). (Abstract shortened by UMI.)

Incorporation of full-fat flaxseed, and to a lesser extent, canola seed in diets to produce n-3-enriched products has attracted interest in the poultry industry. However, high amounts of nonstarch polysaccharides (NSP) in oilseeds compromise their nutritive value. The objectives of the current research were to develop enzyme supplements effective in cell wall depolymerization and viscosity reduction, particularly in flaxseed; to evaluate the effects of enzyme addition and feed processing on oil utilization and egg n-3 fatty acid deposition in broiler chickens and laying hens fed oilseed-containing diets; to characterize the NSP hydrolysis products and to investigate the effects of diet type and enzyme addition on growth performance and the incidence of necrotic enteritis (NE) in broiler chickens challenged with Clostridium perfringens. Results showed that diets containing high levels of flaxseed reduced egg production and shell quality in laying hens, and impaired final body weight and feed conversion ratio (FCR) in broiler chickens. Reducing flaxseed particle size via grinding did not improve the growth performance of broiler chickens, whereas diet pelleting showed more pronounced and beneficial effects in improving the nutritive value of flaxseed, particularly when intact seeds were used. Multicarbohydrase supplementation resulted in a significant depolymerization of cell wall polysaccharides in soybean, canola and flaxseed meals, which was followed by the production of water-soluble NSP hydrolysis products, and the reduction of flax mucilage viscosity in vitro was also evident. Enzyme addition to flaxseed-containing diets improved FCR of broiler chickens and egg production performance of laying hens, and facilitated egg n-3 fatty acid deposition. The C. perfringens challenge caused intestinal NE lesions and increased the mortality of broiler chickens with the highest NE mortality and intestinal C. perfringens counts observed in those fed flaxseed-containing diets. Enzyme supplementation to diets containing high levels of water-soluble NSP (wheat/barley- or wheat/barley/flaxseed-based) facilitated post-disease compensatory growth in pathogen challenged birds. This was accompanied by a numerical reduction of intestinal C. perfringens by 1.4 log10 cfu/g in birds fed the flaxseed-containing diets. Such findings indicated that enzyme addition may be used as a nutritional strategy to reduce the risk of NE development in broiler chickens.

Food protein-derived peptides have increasingly become important sources of ingredients for the formulation of therapeutic products. The main aim of this work was to study the in vitro and in vivo bioactive properties of structurally diverse group of peptides produced through enzymatic hydrolysis of flaxseed proteins (FP). Hydrolysis of FP with seven proteases followed by fractionation into low-molecular-weight (LMW) cationic fractions yielded multifunctional peptides that inhibited angiotensin converting enzyme (ACE) and renin activities, which are molecular targets for antihypertensive agents. The LMW peptides also exhibited antioxidant properties by scavenging free radicals and inhibiting amine oxidase activity. The peptide fractions showed inhibition of calmodulin-dependent phosphodiesterase, an enzyme that has been implicated in the pathogenesis of several chronic diseases. Moreover, FP hydrolysis with thermolysin and pronase followed by mixing with activated carbon yielded branched-chain amino acids (BCAA)-enriched multifunctional peptide mixture (Fischer ratio of 23.65) with antioxidant properties and in vitro ACE inhibition; Fischer ratio of 20.0 is considered minimum for therapeutic purposes. The BCAA-enriched peptide product can be used in clinical nutrition to treat muscle wasting symptoms associated with hepatic diseases. Furthermore, an arginine-rich peptide mixture (31% arginine versus 11% in the original flaxseed protein) was produced by hydrolysis of FP with trypsin and pronase followed by separation using electrodialysis-ultrafiltration. Arginine plays important physiological roles especially as precursor to vasodilator, nitric oxide. The arginine-rich peptide mixture exhibited in vitro ACE and renin inhibition and led to decreased systolic blood pressure (–17.9 and –11.7 mmHg, respectively at 2 and 4 h) after oral administration to spontaneously hypertensive rats. For the first time in the literature, we showed that arginine peptides have superior physiological effects when compared to the amino acid form of arginine. Lastly, quantitative structure-activity relationship studies using partial least squares (PLS) regression yielded two predictive models for renin-inhibiting dipeptides with z-scales amino acid descriptors. The PLS models indicated that hydrophobic and bulky side chain-containing amino acids contribute to renin inhibition if present at the amino- and carboxyl-terminal of dipeptides, respectively. Based on this study, Ile-Trp was discovered as potent renin-inhibiting dipeptide, and may serve as a useful template for the development of potent antihypertensive peptidomimetics.