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A study of antimicrobial and surfactant properties of N-lauroyl amino acids and development of similar compounds from canola meal proteins



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The present study investigated an alternative approach to utilize canola meal proteins. In commercial canola oil production, two different processes are used to extract oil: pre-pressed solvent extraction (PSE) and expeller-pressing. The former is a more widely used process that produces desolventized-toasted (DT) meal; whereas the latter affords cold-pressed (CP) meal. Both DT and CP meals are primarily used in animal feeds. This study investigated the hypothesis that amino acids (AAs) released from meal protein hydrolysis can be chemically modified to generate compounds with useful functions. Pre-treatment of DT and CP meal with ethanol (99%, v/v) and following protein separation from the pre-treated meal were studied as process condition optimizations to obtain protein containing a minimum amount of non-protein components and maximize meal protein recovery. The optimum conditions for ethanol treatment were achieved at 50°C for 30 min at a meal-to-ethanol ratio of 1:4 (w:w), reducing the oil content of the meal to 1%. The protein recovery using aqueous extraction was found optimum at pH 12 with a meal:water ratio of 1:10 (w:v), resulting in 73% and 33% recovery of protein from ethanol pre-treated CP and DT meals, respectively, in a single extraction. Repeated extraction of ethanol pre-treated meal increased protein recoveries to 79% and 38% from CP and DT meal, respectively. Untreated meals and ethanol pre-treated meals were then hydrolyzed with 6 M HCl (protein:acid ratio of 5 mg:2 mL) for 24 h at 110°C. The untreated CP meal released 279 mg AA/g of dbm (dry biomass), and AA recovery was improved to 373 mg AA/g dbm after ethanol pre-treatment. However, untreated DT meal released 400 mg AA/g dbm and no improvement in AA recovery after ethanol pre-treatment. Hydrolysis of separated protein fractions from ethanol pre-treated CP and DT meals yielded 544 mg AA/g dbm and 382 mg AA/g dbm, respectively. H2SO4 was examined as an alternative acid. More than 80% of the total AAs of CP proteins were released with 3 M H2SO4, while for DT meal proteins, a 5-M concentration was needed to achieve the same level of hydrolysis. H2SO4 hydrolysis released less free AAs and more peptides than did HCl at lower acid concentrations of 0.5-1.5 M. Results for the extent of hydrolysis and AA yield indicated that hydrolysis of DT meal protein was less efficient when compared to CP meal proteins. N-acylated (N-lauroyl) derivatives were prepared from six commercially available reagent grade AAs (glutamic acid, lysine, leucine, proline, valine, glycine) selected based on their abundance in canola protein, using reaction with lauroyl chloride. The structures of the resulting compounds were confirmed by FTIR and NMR spectroscopy. Aqueous solutions (0.1%, w/v) of N-lauroyl AA derivatives reduced the surface tension of pure water. The critical micelle concentration (CMC) was comparatively lower than sodium dodecyl sulphate (SDS) for all of the tested sodium N-lauroyl AAs except for the proline derivative. Among all the acylated derivatives tested and SDS, the sodium N-lauroyl AA mixture imparted the lowest surface tension (29.5 mN/m), the lowest CMC (1,100 ppm), and the highest foam stability (38% after 180 min). Solutions of the sodium N-lauroyl glutaminate had the lowest foaming capacity and stability. The in-vitro growth inhibition of sodium N-acylated AAs was studied against pathogenic and non-pathogenic three Gram-negative (Escherichia coli TOP10F, Pseudomonas fragi, Salmonella enteritidis) and three Gram-positive (Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes) bacteria. The sodium N-lauroyl derivatives of glycine, leucine, proline, and valine at 25 ppm inhibited growth (> 90%) of all organisms tested. The N-lauroyl derivatives of lysine and the AA mixture had lower solubilities than the others and exhibited the lowest growth inhibitions. To confirm the potential of canola meal protein hydrolysate in those functions, N-lauroyl products of the acid hydrolysate of DT meal proteins were prepared and tested. Protein extraction at pH 12 with a meal:water ratio of 1:10 (w:v) followed by ultrafiltration (5 kDa MWCO) gave the highest protein concentration (830 mg/g dbm) and generated the highest free AA yield upon hydrolysis. Prior to acylation, the hydrolysate was separated into three fractions based on charge and polarity, resulting in Fraction 1: containing negatively charged and uncharged polar AAs, Fraction 2: AAs with hydrophobic side chains, and Fraction 3: positively charged AAs. All the separated fractions and the unfractionated hydrolysate were acylated to obtain their N-lauroyl products. The unfractionated protein hydrolysate and the Fraction 1 afforded measurable quantities of N-acylated product that could proceed to further purification and preparation of their sodium salts. Sodium lauroyl products from the unfractionated hydrolysate gave the highest inhibitory activity at 250 ppm exhibiting higher than 87% of growth inhibition against all bacterial strains tested. The sodium salt of acylated Fraction 1 gave complete growth inhibition against L. lactis at 125 ppm and 70% growth inhibition against all of the strains except P. fragi. This study showed that 1) the differences in the oil extraction process conditions was a significant factor to determine the achievable degrees of hydrolysis and free amino acid content due to acid hydrolysis, 2) AAs obtained from DT and CP meals can be modified to obtain useful molecules, 3) N-lauroyl AAs were bi-functional molecules having surface-active and anti-microbial activities. These findings lead to uses of canola meal proteins derived AAs. Canola meal proteins can be converted to AAs to generate useful compounds, showing an alternative to using industrially processed oilseed meals.



Canola meal protein, Pre-treatment, N-acylation, acid hydrolysis, amino acid, Surfactant, Antimicrobial



Doctor of Philosophy (Ph.D.)


Food and Bioproduct Sciences


Food Science


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