Youn Young Shim

New Method Development for Nanoparticle Extraction of Water-Soluble β-(1→3)-d-Glucan from Edible Mushrooms, Sparassis crispa and Phellinus linteus

Sparassis crispa and Phellinus linteus are edible/medicinal mushrooms that have remarkably high contents of beta-(1-->3)-D-glucan, which acts as a biological response modifier, but difficulty in cultivating the fruiting bodies and extraction of beta-D-glucan have restricted detailed studies. Therefore, a novel process for nanoparticle extraction of Sparan, the beta-D-glucan from Sparassis crispa, and Phellin, the beta-D-glucan from Phellinus linteus, has been investigated using insoluble tungsten carbide as a model for nanoknife technology. This is the first report showing that the nanoknife method results in high yields of Sparan (70.2%) and Phellin (65.2%) with an average particle size of 150 and 390 nm, respectively. The extracted Sparan with beta-(1-->3) linkages showed a remarkably high water solubility of 90% even after 10 min of incubation at room temperature. Therefore, it is likely that this nanoknife method could be used to produce beta-D-glucan for food, cosmetic, and pharmaceutical industries.

Identification and Quantification of Cyclolinopeptides in Five Flaxseed Cultivars

Cyclolinopeptides are a group of naturally occurring hydrophobic cyclic peptides found in flaxseed and flax oil that have immunosuppressive activity. This study describes the measurement of flaxseed cyclolinopeptide concentrations using an internal standard HPLC method. In addition, the concentration of cyclolinopeptides in the seed of Canadian flax cultivars grown at two locations over two years is reported. The data are consistent with the formation of flaxseed cyclolinopeptides from two ribosome-derived precursors. Each precursor protein includes the sequences corresponding to three cyclolinopeptides from which those cyclolinopeptides are presumably derived by precursor processing. The concentrations of cyclolinopeptides C and E, which are encoded by the same gene sequence, are highly correlated, and the concentrations of cyclolinopeptides D, F, and G, which are encoded by a second gene sequence, are also highly correlated. The strong correlation between the cyclolinopeptides arising from the same gene may prove to be important in understanding how peptide concentration is controlled. Additional research may lead to approaches to improve flax either as a platform for peptide production or as a source of oil with improved drying properties and flavor.

Effect of Lactobacillus acidophilus KFRI342 on the development of chemically induced precancerous growths in the rat colon.

Lactobacillus acidophilus KFRI342, isolated from the Korean traditional food kimchi, was investigated for its suitability as a dietary probiotic. The effects of L. acidophilus KFRI342 on the development of chemically induced (1,2-dimethylhydrazine; DMH) precancerous cytological changes of the colon were investigated in rats. Forty-five male F344 rats were randomly divided into three dietary groups. The control group received a high-fat diet (HF), a second group received a high-fat diet containing the carcinogen (HFC), and a final group received a high-fat diet containing the carcinogen and L. acidophilus KFRI342 (HFCL). L. acidophilus KFRI342 was administered orally three times per week at 2×10(9) c.f.u. ml(-1). L. acidophilus KFRI342 treatments decreased the number of Escherichia coli in faecal samples, the enzyme activities of β-glucuronidase and β-glucosidase, and plasma triglyceride concentration compared to the HF and HFC treatments (P<0.05). L. acidophilus KFRI342 consumption also decreased the ratio of aberrant crypts to aberrant crypt foci incidence and the number of aberrant crypts in HFCL rats. Therefore, L. acidophilus showed potential probiotic activity as an inhibitor of DMH-induced symptoms in live rats. Our in vivo studies indicate that L. acidophilus from kimchi may be suitable as a probiotic for human use.

Rapid NMR method for the quantification of organic compounds in thin stillage.

Thin stillage contains organic and inorganic compounds, some of which may be valuable fermentation coproducts. This study describes a thorough analysis of the major solutes present in thin stillage as revealed by NMR and HPLC. The concentration of charged and neutral organic compounds in thin stillage was determined by excitation sculpting NMR methods (double pulse field gradient spin echo). Compounds identified by NMR included isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol, and 2-phenylethanol. The concentrations of lactic and acetic acid determined with NMR were comparable to those determined using HPLC. HPLC and NMR were complementary, as more compounds were identified using both methods. NMR analysis revealed that stillage contained the nitrogenous organic compounds betaine and glycerophosphorylcholine, which contributed as much as 24% of the nitrogen present in the stillage. These compounds were not observed by HPLC analysis.

Proposed Systematic Nomenclature for Orbitides

Orbitides are short (5-11 amino acid residue), ribosomally synthesized homodetic plant cyclic peptides characterized by N-to-C amide bonds rather than disulfide bonds. Orbitides can be discovered using mass spectrometry of plant extracts or by identifying DNA sequences coding for the precursor protein. The number of orbitides that have been characterized to date, by a number of different research groups, is modest. The nomenclatural system currently used for the Type VI cyclic peptides has been developed in an ad hoc fashion and is somewhat arbitrary. We propose a systematic naming system specifically for the Type VI cyclic peptides that reflects the taxonomic name of the species producing the orbitides and a numbering system that enables systematic representation of amino acid residues and modifications. The proposed naming system emulates the IUPAC Nomenclature for Natural Products and UniProt, both of which use abbreviations of taxonomic names for the compounds in question. Nomenclature for post-translational modifications also follows the IUPAC precedent, as well as the cyclic peptide literature. Furthermore, the proposed system aims to maintain agreement with the precedents set by the pre-existing literature. An example of the proposed nomenclature is provided using the methionine-containing homodetic peptides of Linum usitatissimum (flaxseed).

Effect of Cyclolinopeptides on the Oxidative Stability of Flaxseed Oil

Polar compounds present in flaxseed oil increase its oxidative stability. Flaxseed oil becomes less stable to oxidation when filtered with silica. This observation may be linked to antioxidant compounds present in flaxseed oil. Flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. The polar fractions from flaxseed oil were then added back to silica-treated flaxseed oil to determine the impact of fractions containing polar compounds on oxidative stability (induction time) at 100 °C. A polar fraction containing mainly cyclolinopeptide A (CLA, 1), but also containing β/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.36 ± 0.28 to 3.20 ± 0.41 h. When oxidative stability was determined immediately after addition of the polar fractions other flaxseed fractions and solvent controls did not affect oil stability. However, when the oxidative stability index (OSI) test was delayed for three days and oil samples were held at room temperature after the addition of the polar fractions to the flaxseed oil, it was observed that the control oil treated with silica had become highly sensitive to oxidation. A polar fraction containing a mixture of CLs (1, 5, 7, 9, 11), improved the oxidative stability of peptide-free oil with respect to the control when the OSI measurement was made three days after adding the fraction. In addition, effects of 1 on the oxidative stability of peptide-free oil containing divalent metal cations was investigated.

Distribution of Cyclolinopeptides in Flaxseed Fractions and Products

Hydrophobic cyclic peptides, termed cyclolinopeptides, found in flaxseed are known for their immunosuppressive activity. This study is the first report of the occurrence of cyclolinopeptides in flaxseed fractions and products produced by aqueous processing and cold pressing. The distribution of cyclolinopeptides in flaxseed was determined after processing of flaxseed by various industrial and laboratory processes. Extracts of the water-soluble mucilage did not contain cyclolinopeptides. The cotyledon had the highest concentration of cyclolinopeptides, whereas seed coat had lower levels. An oil body fraction separated from seed after homogenization in water, followed by centrifugation, had the highest concentration of cyclolinopeptides of the fractions produced by this method. Further washing of the oil body fraction led to a loss of cyclolinopeptides. When oilseed was extruded using an expeller press, cyclolinopeptides were found in greater concentrations in crude oil and the solid sediment present in the oil fraction than in meal or the unprocessed seed. The concentration of cyclolinopeptides in crude flaxseed oil immediately after pressing was much higher than that observed in flaxseed oils purchased from a retail outlet. The effect of oil refining treatments on the removal of cyclolinopeptides was also tested. Acid degumming using aqueous H(3)PO(4) removed cyclolinopeptides from crude flaxseed oil. Alkali refining was less effective as this treatment failed to remove all peptides equally. This work illustrates ways that cyclolinopeptides may be extracted from flaxseed oil that could be developed for large-scale industrial extraction. The ability to extract cyclolinopeptides on a larger scale would allow faster exploitation of commercial applications of these molecules and provide the flaxseed industry with value-added coproducts.

Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

Kinetic Interactions between Cyclolinopeptides and Immobilized Human Serum Albumin by Surface Plasmon Resonance

Cyclolinopeptides (CLs) are octa-, nona-, and decapeptides present in flaxseed (Linum usitatissimum L.) that may have immunosuppressive and antitumor activities, but little is known of their pharmacokinetics. Human serum albumin (HSA), the most abundant blood protein, is an important mediator of organic solute flux, and hence when compounds bind this protein, it potentially affects both their availability and efficacy. Quantitative thermodynamic analysis of the interaction of compounds with HSA is important in the development of biomedical applications. A surface plasmon resonance (SPR) biosensor was utilized to reliably determine binding constants for several CLs with HSA. The maximum binding response of [1-9-NαC]-CLA/HSA was almost 20-fold higher than that of [1-8-NαC],[1-MetO]-CLE/HSA. Through analysis of an array of peptides, it was possible to correlate the impact of structural changes on CL binding. The oxidation of sulfur in methionine (Met) residues formed methionine S-oxide (MetO) and reduced binding significantly. Most strikingly, the further oxidation of MetO to S,S-dioxide (MetO2) produced CLs with stronger binding. The large impact on binding by relatively small modifications of methionine containing CLs suggested that small changes in methionine oxidation can disrupt hydrophobic interaction, the predominant intermolecular force stabilizing the complex between CLs and HSA. SPR binding studies may aid in understanding the fate of CLs after consumption of flaxseed or flaxseed products or the development of CLs as drugs or drug carriers.

Protein Concentrate Production from Thin Stillage

Two-stage fermentation (TSF) of saccharified wheat with a consortium of endemic lactobacilli produced CO2 and induced colloid separation of fermented solution to produce a protein concentrate (PC). Protein-rich slurry (50%, db) was obtained by decanting solution or skimming floating material during or after TSF. Washing and drying processes were explored to improve protein content, extend storage life of slurry, and yield converted stillage for compound recovery. Centrifuging and washing slurry afforded a PC and clarified solution. PC protein content increased to 60% (w/w, db). The PC was dried in a spray dryer or drum dryer or tray dryer. Dried PC water activity ranged 0.23-0.30. The dried PC lysine content was low, but lysine availability (95%) was excellent. Liquid from TSF and washing was readily microfiltered. Mass recovery of protein, glycerol, 1,3-propanediol, lactic acid, acetic acid, and glycerylphosphorylcholine from combined TSF, washing, and filtration were 66, 76, 72, 77, 74, and 84%, respectively.