Abstract
New Zealand cold-pressed hemp, flax and canola seed oils were analyzed for their fatty acid compositions, tocopherols, β-carotene, chlorophyll, total phenolics, flavonoids, color, quality, melting and crystallization characteristics. The dominant fatty acid of canola, hemp and flax seed oils was oleic (57.0 ± 0.0%), linoleic (55.7 ± 0.3%) and linolenic acids (58.7 ± 1.2%), respectively (P < 0.05). Hemp seed oil contained the highest tocopherol, flavonoid and phenolic acid contents. There was a significant difference in color of the oils (P < 0.05) due to the chlorophyll content. Melting and crystallization transitions and enthalpy heat change (∆H) values varied for the three oils in the order canola > flax > hemp. All oils had low moisture and volatiles, unsaponifiable matter and free fatty acids content. Peroxide value, p-anisidine, conjugated dienoic acid, acid value, specific extinction of cold-pressed oils at 232 and 270 nm were under the value of limit allowed in general regulations.
Defatted hemp, flax and canola seed cakes were extracted with different solvent systems namely methanol, ethanol, acetone, methanol 80%, acetone 80% and mixed solvent of methanol:acetone:water (MAW, 7:7:6, v/v/v). Each extract was analyzed for antioxidant capacity using ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. MAW exhibited the highest extraction of phenolic and flavonoid contents in the seed cakes, followed by acetone 80% and methanol 80%. The antioxidant capacity was proportional to the polyphenols recovery in the extracts. Canola seed cakes possessed the highest recovery of polyphenols and antioxidant capacity, followed by hemp and flax seed cakes. MAW extract of canola contained total phenolic content of 2104.67 ± 2.52 mg GAE/100 g fresh weight; total flavonoids of 37.79 ± 0.04 mg LUE.100 g-1 fresh weight; percentage inhibition of DPPH˙ of 33.03 ± 0.38%; total antioxidant capacity (TAC, FRAP assay) of 8.78 ± 0.07 μmol Fe (II).g-1 fresh weight. Identification of individual polyphenol compounds were performed HPLC. MAW extract of canola had the highest (P < 0.05) concentration of all individual polyphenols except gallic acid and catechin. Highest concentration of quercetin and luteolin in MAW extract of hemp was obtained among all solvent systems.
The effectiveness of ultrasonic extraction of phenolics and flavonoids from defatted hemp, flax and canola seed cakes was compared to the conventional extraction method. Ultrasonic treatment at room temperature showed increased polyphenol extraction yield and antioxidant capacity by two-fold over the conventional extraction method. Different combinations of ultrasonic treatment parameters consisting of solvent volume (25, 50, 75 and 100 mL), extraction time (20, 30 and 40 min) and temperature (40, 50, 60 and 70ºC) were selected for polyphenol extractions from the seed cakes. The chosen parameters had a significant effect (P < 0.05) on the polyphenol extraction yield and subsequent antioxidant capacity from the seed cakes. Application of heat during ultrasonic extraction yielded higher polyphenol content in extracts compared to the non-heated extraction. From an orthogonal design test, the best combination of parameters was 50 mL of solvent volume, 20 min of extraction time and 70ºC of ultrasonic temperature.
The use of microwave and pulsed electric field (PEF) processing to optimize polyphenol extraction from defatted hemp seed cake in mixed solvent of methanol, acetone and water (MAW, 7:7:6 v/v/v) was investigated using the Box-Behnken response surface method. A variables combination for microwave processing of time, microwave power and liquid to solid (L:S) ratio and for PEF (ethanol concentration, time, frequency and voltage) were used in the investigation. Following microwave or PEF treatments, the polyphenols were extracted from the samples under ultrasound with fixed variables (200 W of ultrasonic power, water bath of 70°C and 20 min of extraction time). The measured responses were total phenolics (TP), total flavonoids (TF), DPPH˙ scavenging activity and ferric reducing/antioxidant power (FRAP). The optimum variables combination for microwave processing (5 min treatment time, L:S ratio of 6, 700 W power and volume 30 mL) resulted in maximum yields in all measured responses for defatted hemp seed cake. Under the optimum condition of microwave processing except the microwave power, the microwave power 644 W and 633.3 W resulted in maximum yields in all measured responses for defatted flax and canola seed cakes respectively. The PEF-assisted extraction (voltage (30 V), frequency (30 Hz), ethanol concentration (10%) and time (10 s) resulted in maximum yields in all measured responses for all defatted seed cakes.
The bioactive compounds from the cold-pressed hemp, flax and canola seed oils, as well as their oilseed cakes were further investigated. For their effect-directed analysis, planar chromatography was combined with several assays, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) scavenging, acetylcholinesterase inhibition, planar yeast estrogen screen (pYES), antimicrobial Bacillus subtilis and Aliivibrio fischeri assays. The streamlined high-performance thin-layer chromatography (HPTLC)-bioassay method allowed the discovery of unknown bioactive compounds present in these oilseed cake extracts. A first characterization of the unknown bioactive compounds was performed by HPTLC-electrospray ionization (ESI)-mass spectrometry (MS) using the elution-head based TLC-MS-Interface. The results showed that the hemp, flax and canola seed oils, as well as their oilseed cakes possessed various bioactive compounds that can be further characterized by mass spectrometry. In contrast to target analysis, the comprehensive information with regard to effects allowed a first bioactivity evaluation on the feed intake side, which can be used to isolate and yield active compounds for functional food or justify functional feed supplements.
The effect of the defatting process, acid and alkali extractions on the physicochemical and functional properties of hemp, flax and canola protein isolates was studied. The defatting process enriched the protein content from around 35% to 52 – 55% in the defatted oilseed cakes. Further treatment with acid and alkali led to the highest enrichment (P < 0.05) in the protein content (> 90%). The defatting process produced the lightest (P < 0.05) colour compared to the parent oilseed cakes and the protein isolates. Alkali extraction produced protein isolates with the highest (P < 0.05) water holding capacity (WHC) while the original oilseed cakes had the highest oil holding capacity (OHC). Acid and alkali extractions produced protein isolates with the highest emulsifying activity (EA) and emulsion stability (ES). The alkaline soluble flax protein isolate and acid hemp protein isolate had the highest (P < 0.05) creaming stability (CS) and largest droplet size (DS) respectively. Amino acid profiles of protein isolates after acid and alkali extraction were improved, leading to a protein suitable for fortification in food products that meet human nutrition requirements.
AL-HPI & AC-HPI were hydrolysed by proteases namely AFP, HT, ProG, actinidin and zingibain. The proteases were evaluated for their caseinolytic activities. The enzymatic hydrolysis of HPIs was evaluated through the degree of hydrolysis and SDS-PAGE profiles. The bioactive properties of the resultant HPHs were accessed through ORAC, DPPH˙ scavenging and ACE-inhibitory activities. The physical properties of the resultant HPHs were evaluated for their particle sizes, zeta potential and surface hydrophobicity. Among all proteases, HT resulted in the highest bioactivities (ORAC, DPPH˙ scavenging and ACE-inhibitory activities) generated in the shortest time (2 h) compared to the other protease preparations. This was due to the highest caseinolytic and proteolytic activities exhibited by HT. The acidic subunit of edestin in AL-HPI was completely hydrolyzed by HT in 1 h, and the complete digestion of the basic edestin subunit was achieved at 2 h. The particle size of the resultant hydrolysates was gradually decreased upon hydrolysis, depending on the types of proteases and substrates. Al-HPI was the best substrate that led to the higher bioactivities compared to AC-HPI. The HT hydrolysate generated from AL-HPI at 2 h hydrolysis time was selected for further fractionated using OFFGEL and RP-HPLC. The result showed that F10, F12 and F14 from the OFFGEL fractionation exhibited higher ORAC and ACE-inhibitory activities among all the OFFGEL fractions. Hence, F10, F12 and F14 were selected for further purification into several peptides fractions by RP-HPLC prior to ORAC and ACE-inhibitory assays. For the RP-HPLC fractionated peptide fractions from OFFGEL F10, the RP-HPLC F10 exhibited the highest ORAC activity (750.35 µM Trolox.g-1 sample) and ACE-inhibitory activities (80.56%). For the RP-HPLC fractionated peptide fractions from OFFGEL F12, the RP-HPLC F17 had the highest ORAC value (756.34 µM Trolox.g-1 sample) and ACE-inhibitory activities (76.57%). For the RP-HPLC fractionated peptide fractions from OFFGEL F14, the RP-HPLC F17, F18, & F19 exhibited the highest ORAC activity (764.57, 758.65, & 752.47 µM Trolox.g-1 sample respectively) among all the RP-HPLC fractions, and the RP-HPLC F19 possessed the highest ACE-inhibitory activity (75.68%).
In summary, cold-pressed hemp, flax and canola seed oils are healthy oils for human consumption with desirable PUFA content and bioactive compounds. The by-products, oilseed cakes have great potential as new product development in food and pharmaceutical industries such as polyphenols, protein isolates and bioactive peptides.