Chondroitin sulphate (ChS) from the scapular cartilage of the shortfin mako shark (Isurus oxyrinchus) was purified by two-stage enzymatic hydrolysis and a fractional precipitation process using isopropyl alcohol. Characteristics of the ChS fraction were investigated using cellulose acetate membrane electrophoresis and FT-IR spectra. A maximum hydrolysis rate of 78.26% was achieved with 1.35% (w ⁄ w) Alcalase and 1.20% (w ⁄ w) Flavourzyme. A minimum nitrogen content of 2.89% was obtained with 1.43% (w ⁄ w) Alcalase and 1.33% (w ⁄ w) Flavourzyme, as determined by response surface methodology. The precipitation of ChS from the enzymatic hydrolysates was optimised at 40% (v ⁄ v) isopropyl alcohol, which contained 2% (w ⁄ v) NaCl to lower the nitrogen content. The precipitate was further purified via membrane filtration (molecular-weight cut-off, 3 kDa) to remove salt and low-molecular-weight materials. The ChS purified by enzymatic hydrolysis, isopropyl alcohol precipitation and membrane filtration was identified as ChS C by electrophoresis and FT-IR spectra.
Based on the superimposition of four-dimensional response surfaces with respect to TPC, EDA, NSA and SDA, the optimum ranges of extraction conditions were 70-150 W MWP, 30-50% ETC and 8-18 min EXT.
Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy.
Skullcap (Scutellaria baicalensis) is well known for its anti-inflammatory and anti-allergic effects. In our previous study, we found that skullcap could inhibit allergen permeation and regulate Th1/2 immune balance. To reveal the key fractions and components of skullcap, we fractionated skullcap extract into five fractions: hexane, chloroform, ethyl acetate, butanol, and water fraction. Among these fractions, the hexane fraction significantly suppressed the production of Th2-mediated cytokines (Interleukin (IL)-4, 5, 10 and 13) and increased Th1-mediated cytokines (Interferon (IFN)-γ and IL-12). Furthermore, the hexane fraction inhibited the permeation of ovalbumin (OVA), used as an allergen, across the intestinal epithelial cell monolayer. To confirm the active compounds in the hexane fraction, fatty acids were analyzed. Linoleic acid (LA, C18:2 (>59.7%)) was identified as the most important fatty acid in the skullcap hexane fraction. LA significantly suppressed IL-4 production and increased IFN-γ secretion, as well as inhibiting OVA permeation. Thus, LA significantly diminished the permeation of allergen by enhancing intestinal barrier function and regulated allergic responses to maintain Th1/Th2 immune balance.
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