• KSBB Journal
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• v.13 no.2
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• pp.147-154
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• 1998

Enzymatic hydrolysis using an immobilized enzyme was carried out to produce chitosan oligosaccharides (COSs) from chitosan effectively. Chitosanase was immobilized on eight different carriers by physical adsorption. The enzyme immobilized on chitin had higher activity than those immobilized on the other carriers in spite of its lower adsorption. The activity of chitin-immobilized enzyme was more than 90% of the original activity. Optimal temperature of the immobilized enzyme increased by about $15^{\circ}C$ and its thermostability was excellent in relatively wide range of temperature. But its effects of pH did not improve compared to the free enzyme. The immobilized enzyme produced 153 mg/g chitosan of the reducing sugar for 3hrs of hydrolytic incubation time. The total content of higher oligomers, tetramer to hexamer, among amount of total COSs obtained for 2hrs was more than 90%. In kinetic parameters for both enzymes, immobilized enzyme showed lower affinity for substrate and reaction rate than free enzyme, however, no reduction of the rate for high substrate concentrations. Consequently, chitin-immobilized could effectively hydrolyse chitosan and produce the higher COSs without activity decrease in comparison with the free enzyme.

• Fisheries and Aquatic Sciences
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• v.2 no.1
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• pp.105-111
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• 1999

In order to produce functional chitin oligosaccharides, a chitinolytic bacterium was newly screened from the viscera of Korean bony fishes, and identified as Bacillus sp. LJ-25. For the production of chitinolytic enzymes, $1.0\%$ nutrient broth and $0.3\%$ colloidal chitin were used as nitrogen and carbon source, respectively. The optimal temperature, initial pH and concentration of NaCl for the enzyme production by Bacillus sp. LJ-25 were $30^{\circ}C$ 6.5-7.0 and $1.0\%$, respectively. The enzyme activity of Bacillus sp. LJ-25 increased until the incubation time of 168 hr, followed by a decrease in activity.

• Journal of Nutrition and Health
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• v.36 no.9
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• pp.908-917
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• 2003

It has been proposed that oxidative modification of LDL (oxLDL) plays a significant role in the pathogenicity of atherogenesis. We tested the hypothesis that chitin and chitosan may function as antioxidants with respect to 0.1 mg cholesterol/ml LDL incubated with 5 $\mu$ M Cu$^2$$^{+}$alone or in the P338Dl mouse macrophage system using L-ascorbic acid as a standard classical antioxidant. The degree of oxLDL formation was ascertained by the relative electrophoretic mobility (rEM) in the combination of thiobarbituric acid reactive substances (TBARS) levels, and the cytotoxicity of oxLDL was detected by macrophage viability. The oxLDL uptake and foam cell formation of macrophages were measured by Oil Red O staining. Incubation with Cu$^2$$^{+}$and macrophages increased rEM of LDL and stimulated TBARS formation. Culture of macrophages with LDL in the presence 5 $\mu$ M Cu$^2$$^{+}$induced macrophage death. In cell-free system 200 $\mu$g/ml water-soluble chitosan and chitosan-oligosaccharide blocked oxLDL formation. Water-soluble chitosan and chitosan-oligosaccharide blocked oxLDL formation near-completely relative to L-ascorbic acid, whereas water-soluble chitin and chitin-oligosaccharide had no measurable antioxidant effect. In macrophage system water-soluble chitosan and chitosan-oligosaccharide blocked oxidation of LDL with a significant increase in cell viability, and decreased TBARS in medium. As for the inhibitory effect on macrophage foam cell formation, chitosan and its oligosaccharide, but not watersoluble chitin, revealed the effectiveness. The endothelial expression of lectin-like oxLDL receptor-1 (LOX-1) was tested by Western blot analysis, and chitosan, chitosan-oligosaccharide and chitin-oligosaccharide blocked LOX-1 expression. These results indicate that water-soluble chitosan and its oligosaccharide showed the inhibitory effect on Cu$^2$$^{+}$-induced LDL oxidation of macrophages, and chitosan, chitosan-oligosaccharide and chitin-oligosaccharide had blocking effect on oxLDL receptor expression in the human umbilical vein endothelial system. Thus, water-soluble chitosan and its oligosaccharides possess anti-atherogenic potentials possibly through the inhibition of macrophage LDL oxidation or endothelial oxLDL receptor expression depending on chemical types.l types.

• Journal of Life Science
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• v.12 no.1
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• pp.77-86
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• 2002

Chitin, a $\beta$-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl rhitobiose and N-acetyl-D-glucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnS $O_4$.4$H_2O$, 37$^{\circ}C$, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and $65^{\circ}C$, respectively. Chi-56A was stable up to $65^{\circ}C$ and in alkaline region. Its $K_{m}$ value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.e.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.5
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• pp.706-713
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• 2003

Two experiments were conducted to evaluate the efficacy of dietary FERMKIT, a commercial toxin binder consisting of probiotic-fermented natural product containing chitin, chitosan and chitosan oligosaccharides ($FERMKITO^{(R)}$, EASY-BIO SYSTEM, Inc., Korea), in binding aflatoxin (AF) and zearalenone (ZEN) and ameliorating their mycotoxicity in meat type ducks. FERMKIT was supplemented to AF contaminated diets (at 120 ppb) at either 0.3 or 0.6% in experiment 1 and to ZEN contaminated diets (at 150 ppb) at 0.6% in experiment 2. In experiment 1 body weight gains were reduced by 37% and mortality was increased by 18% in ducks fed diet contaminated with AF at 120 ppb compared to ducks fed control diet (<10 ppb AF) for the 4-wk experimental period. However, dietary FERMKIT supplementation effectively alleviated overall toxicity induced by AF. The significant treatment-related changes in feather growth, web-toe hemorrhage, leg deformity, liver paleness, organ weights, hematological values and serum biochemical values, as compared to the control, were observed. The FERMKIT supplementation significantly diminished the adverse effects of AF and restored all the parameters measured back (<0.05) toward the control values. These findings indicated that FERMKIT, when added at the levels of 0.3 or 0.6% in the 120 ppb AF diets, could modulate the toxicity of AF with percentage sorption capacity of 52.70% at the level 0.3% and 79.85% at the level 0.6% of the diets (experiment 1). In experiment 2, FERMKIT, when added at 0.6% to the 150 ppb ZEN diets for the 4-wk experimental period, diminished the toxicity as shown by body weight gain, weights of testicles, oviducts, Bursa of Fabricius and cloaca eversion score as compared with the controls (<10 ppb ZEN) and 150 ppb ZEN diet with no added FERMKIT. The findings indicated that FERMKIT could be protective against the effects of ZEN in young growing ducks with percentage sorption capacity of 67.11% as evaluated from toxicity index parameter measured when added at 0.6% of the diets containing 150 ppb ZEN.

• Proceedings of the SOHE Conference
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• 1997.12a
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• pp.39-43
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• 1997

Toha-jeod was manufactured by seven methods ; low salt group (L:15% sodium chloride), high salt g group (H:23% sodium chloride), 50% conventional soybean sauce group (S), low salt group containing 2% w wheat bran (W2%-L), high saIt group containing 2% wheat bran (W2%-H),high salt group containing 2% wheat bran (W2%-H), high salt group containing 4% wheat bran (W4%-H). After these seven groups were refrigerated at $4{\pm}1^{\circ}C$, they were sampled at intervals of three months and analyzed functional components. The free amino acid in Toha-jeod which are omitine, glutamic acid, leucine, alanine, lysine and valine increased gradually up to six months of fermentation and decreased by nine months. Conventional soybean sauce group increased continuously during the fermentation process. Hypoxanthine was altered almost among other nueletides. ATP was not detected, IMP and inosine had disapapted after the six months fermentation. Polyene fatty acids and n-3 fatty acids were decreased and s saturated fatty acids were not altered in the group containing wheat bran during fermentation. In the Hunter values, the group containing wheat bran and high salt group showed lower level than the group n not containing wheat bran and low salt group. Redness indicating the value of Toha-jeod increased as Toha-jeod was fermentated. Low salt group and conventional soybean sauce group were superior to other groups in the extent of redness. As the fermentation of Toha-jeod progressed for a long time, molecular weight distribution tended to become less molecular and the formation of chitin oligosaccharides was increased significantly. After nine months of fermentation, 24.75% chitin oligosaccharides [($GlcNAd_4$ ~ ($GlcNAd_8$, M.W. 823~1789] were created in the high salt group containing 2% wheat bran. [($GlcNAd_6$. M.W. 1236J , that is NACOS-6, which was reported as an antitumor activity material, was present in 4.01~4.37% of total Toha chitin content. 66.30% chitin oligosaccharides were created in conventional soybean sauce.

• Archives of Pharmacal Research
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• v.14 no.3
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• pp.255-260
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• 1991

Chitinase was partially purified from Allium fistulosum L (green onion_. Protein fraction precipitated from ammonium sulfate was passed through CM-Sepharose and Sephacryl HR-200. The specific activity of the chitinase was 6.4 units/mg and total recovery was 6.3%. The analysis of the products from the digestion of N-acetychitohexaose indicated that chitinase was endo in action, with oligerms from N-acetylchitobiose to chitotetraose. N-Acetylglucosaminidase from the same species hydrolyzed oligomers obtained from chitinase reaction to lower oligosaccharides. These data demonstrated that chitinolytic system exists in green onion.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.255-262
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• 2008

Methyl jasmonate (MJ) and yeast extract (YE) induce protein expression and benzophenanthridine alkaloid accumulation in Eschscholtzia californica suspension cell cultures. One hundred ${\mu}M$ MJ primarily induced dihydrosanguinarine $(509.0{\pm}7.4mg/l)$ ; 0.2g/l YE induced sanguinarine $(146.8{\pm}3.8mg/l)$ and an unknown compound. These results occur because dihydrobenzophenanthridine oxidase (DHBO) is induced by YE and not by MJ. YE and chitin (CHI) had similar effects on sanguinarine production and DHBO expression. Differential induction of secondary metabolites was shown in E. californica suspension cultures and the expression of proteins confirmed the metabolite results. Furthermore, treatment by various oligosaccharides helped us to understand the elicitation effect of YE in signal transduction pathways.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2000.05a
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• pp.88-89
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• 2000

Chitosan is derived from chitin by deacetylation in the presence of alkali, which is a copolymer consisting of $\beta$-(1longrightarrow4)-2-acetamido-D-glucose and $\beta$-(1longrightarrow4)-2-amino-D-glucose units with the latter usually exceeding 80% (Arvanitoyannis et al., 1998). Chitosan has been developed as new physiological material since it possesses antibacterial activity, hypocholesterolemic activity and antihypertensive action. However, even though chitosan has very strong functional properties in many areas, its high molecular weight and high viscosity may restrict the use in vivo. In addition, there is little doubt that such properties will influence absorption in the human intestine. Recently, studies on chitosan have attracted interest for converted chitosan to oligosaccharide, because the oligosaccharide possesses not only water-soluble property but also versatile functional properties such as antitumor activity, immune-enhancing effects, enhancement of protective effects against infection with some pathogens in mice, antifungal activity, calcium absorption accelerating effect (Jeon et al., 1999) and antimicrobial activity. There is, however, little information on the toxicity of chitosan oligosaccharide. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.556-562
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• 2007

A total of 126 crossbred weanling pigs (average body weight of $6.3{\pm}0.3$ kg) were used to investigate the effect of chito-oligosaccharide (COS) on growth performance, nutrient digestibility, pH of gastro-intestinal tract (GI), intestinal and fecal microflora of young piglets. Pigs were allocated to three dietary treatments based on body weight and gender in a single factorial arrangement. Treatments were control (No COS), T1 (0.2% COS during starter (6-13 kg) and 0.1% COS during grower (13-30 kg) phases, and T2 (0.4% COS during starter (6-13 kg) and 0.3% COS during grower (13-30 kg) phases, respectively. Each treatment had 3 replicates and 14 pigs were raised in each pen. COS is a low molecular weight water-soluble chitosan that can be obtained from chitin of the crab shell after deacetylation with concentrated sodium hydroxide at high temperature and then further decomposition by chitosanase enzyme in the presence of ascorbic acid. For the starter and grower periods, there were no significant differences (p>0.05) in average daily gain (ADG) and feed to gain ratio among treatments. However, during the overall period (6-30 kg), T2 showed better (p<0.05) feed to gain ratio than other treatments. A digestibility study was conducted at the end of grower phase which showed improvement (p<0.05) in DM and crude fat digestibility in T2 over the control. At 25 kg body weight, 6 pigs per treatment (2 per replicate) were sacrificed to determine the effect of diets on pH and microbial count at different sections of the GI tract. The pH of the cecal contents in pigs fed 0.1% COS was higher (p<0.05) than in the other treatments. Total anaerobic bacterial number increased from cecum to rectum in all treatments. The weekly total bacterial counts showed higher (p<0.05) in feces of pigs fed COS than that of untreated pigs at the $8^{th}$ week. The number of fecal E. coli in untreated pigs at $4^{th}$ wk was 7.35 log CFU/g compared to 6.71 and 6.54 log CFU/g in 0.1 and 0.3% COS-treated pigs, respectively. Similarly, at $8^{th}$ wk, fecal clostridium spp. were lower in pigs fed 0.3% COS (5.43 log CFU/g) than in untreated pigs (6.26 log CFU/g). In conclusion, these results indicated that chito-oligosaccharide could improve feed efficiency in young pigs and inhibited the growth of harmful bacteria.