• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.1082-1091
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• 2020

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

• Food Science of Animal Resources
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• 제40권6호
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• pp.1033-1043
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• 2020

This study investigated the impacts of gelatin hydrolysate addition on the technological properties and lipid oxidation stability of cooked sausage. Gelatin hydrolysate was prepared from pork and duck skin gelatin, through stepwise hydrolysis using collagenase and pepsin. The cooked sausages were formulated without gelatin (control) or with 1% pork skin gelatin, 1% duck skin gelatin, 1% pork skin gelatin hydrolysate, and 1% duck skin gelatin hydrolysate. The pH, color characteristics, protein solubility, cooking loss, and textural properties of cooked sausages were evaluated, and the 2-thiobarbituric acid reactive substances (TBARS) value was measured weekly to determine lipid oxidation stability during 4 wk of refrigerated storage. Enzymatic hydrolysis of gelatin decreased protein content and CIE L* but increased redness and yellowness (p<0.05). When 1% gelatin or gelatin hydrolysate was incorporated in cooked sausage, however, little to no impacts on pH value, moisture content, protein content, color characteristics, protein solubility, and cooking loss were found (p>0.05). The addition of 1% duck skin gelatin hydrolysate increased the cohesiveness and chewiness of cooked sausages. The inclusion of 1% duck skin gelatin accelerated lipid oxidation of cooked sausages during refrigerated storage (p<0.05), whereas duck skin gelatin hydrolysate caused a lower TBARS value in cooked sausage compared to duck skin gelatin. The results show comparable effects of gelatin and gelatin hydrolysate addition on the technological properties of cooked sausages; however, the oxidative stability of raw materials for gelatin extraction should be evaluated clearly in further studies.

• BMB Reports
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• 제34권3호
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• pp.219-224
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• 2001

To identify the antioxidative peptides in the gelatin hydrolysate of bovine skin, the gelatin was hydrolyzed with serial digestions in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. The second enzymatic hydrolysate (hydrolyzed with pronase E) was composed of peptides ranging from 1.5 to 4.5 kDa, and showed the highest antioxidative activity, as determined by the thiobarbituric acid method. Three different peptides were purified from the second hydrolysate using consecutive chromatographic methods. This included gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an octadecylsilane chloride column. The isolated peptides were composed of 9 or 10 amino acid residues. They are: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp (PI), Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly (PII), and Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp (PIII), as characterized by Edman degradation and fast-atom bombardment mass spectrometry. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability with a methylthiazol tetrazolium assay The results showed that PII had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by the addition of the peptide. These results suggest that the purified peptide, PII, from the gelatin hydrolysate of bovine skin is a natural antioxidant, which has potent antioxidative activity.

• Korean Journal of Fisheries and Aquatic Sciences
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• 제44권5호
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• pp.456-463
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• 2011

We attempted to isolate a collagenase-1 inhibitory peptide from skate Dipturus chilensis skin protein. The protein from skate skin was digested by various enzymes (alcalase, ${\alpha}$-chymotrypsin, neutrase, papain, pepsin, and trypsin) to produce a collagenase-1 inhibitory peptide. The collagenase-1 inhibitory activity of the peptides obtained was measured by gelatin digestion assay. Among the six hydrolysates, pepsin hydrolysate exhibited the highest collagenase-1 inhibitory activity. The peptide showing strong collagenase-1 inhibitory activity was purified by Sephadex G-25 gel chromatography and HPLC using an octadecylsilyls (ODS) column. The amino acid sequence of purified collagenase-1 inhibitory peptide was identified to be Asn-Leu-Asp-Val -Leu-Glu-Val-Phe (961 Da) by quadrupole time of flight (Q-TOF) and electrospray ionization mass spectrometry (ESI-MS) mass spectroscopy. The $IC_{50}$ value of purified peptide was 87.0 ${\mu}M$. Moreover, the peptide did not exhibit cytotoxic effects on human dermal fibroblast cell lines.

• Korean Journal of Fisheries and Aquatic Sciences
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• 제29권2호
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• pp.246-255
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• 1996

To develop a natural antioxidative peptide, the gelatin was extracted from fish (Yellowfin sole) skin by hot $water(50^{\circ}C)$ extraction method and hydrolyzed with Alcalase, pronase and collagenase through a continuous 3-step membrane reactor. Each step enzymatic hydrolysates were determined the antioxidative activity and their synergistic effects, compared with $\alpha-tocopherol$ and butylated hydroxytoluene (BHT). Also, we tried to investigate the antioxidative disposition of peptide which was successfully separated by gel filtration, ion-exchange chromatography, and HPIC in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Second step enzymatic hydrolysate (SSEH) among all hydrolysates and $\alpha-tocoperol$ was showed the strongest antioxidative activity. The optimum concentration of antioxidative activity for SSEH was $1\%(w/w)$ in linoleic acid. The synergistic effects were increased in using the hydrolysate with tocopherol and BHT. In the presence of the peptide isolated from SSEH, supplemented hepatocytes exposed to TBHP showed that delayed cell killing and decreased significantly the lipid peroxidation, compared with hepatocytes not cultured with isolated peptide.

• BMB Reports
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• 제35권2호
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• pp.239-243
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• 2002

Angiotensin I that converts the enzyme (ACE) inhibitory peptide, Gly-Pro-Leu, previously purified and identified from the Alaskan pollack skin gelatin hydrolysate, were synthesized. In addition, the peptides Gly-Leu-Pro, Leu-Gly-Pro, Leu-Pro-Gly, Pro-Gly-Leu, Pro-Leu-Gly, Gly-Pro, and Pro-Leu, which consisted of glycine, proline, and leucine, were synthesized by the solid-phase method. The $IC_{50}$ values of each tripeptide - namely Leu-Gly-Pro, Gly-Leu-Pro, Gly-Pro-Leu, Pro-Leu-Gly, Leu-Pro-Gly, and Pro-Gly-Leu - were 0.72, 1.62, 2.65, 4.74, 5.73, and $13.93{\mu}M$, respectively. The ACE inhibitory activity of these tripeptides was higher than that of dipeptides, such as Gly-Pro and Pro-Leu with $IC_{50}$ values of 252.6 and $337.3\;{\mu}M$, respectively. Among the tripeptides, Leu-Gly-Pro and Gly-Leu-Pro had higher inhibitory activity than Gly-Pro-Leu that was isolated from the Alaskan pollack skin gelatin hydrolysate. Among the different types of tripeptides that were examined, the highest ACE inhibitory activity was observed for Leu-Gly-Pro. It had the leucine residue at the N-terminal and proline residue at the C-terminal.

• Korean Journal of Fisheries and Aquatic Sciences
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• 제42권5호
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• pp.417-425
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• 2009

The potential utility of fish scales to the functional food industry has been investigated due to its antioxidant and antihypertensive characteristics. In this study, we report on the reactive oxygen species (ROS) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities of gelatin hydrolysates processed from Branchiostegus japonicus scales, which are also high in protein content (about 46.1%). We prepared the enzymatic gelatin hydrolysates with four proteases (${\alpha}$-chymotrypsin, Alcalase, Neutrase and trypsin) from B. japonicus scale gelatin, which was prepared according to different reaction times, substrate/enzyme ratios and substrate concentrations. The enzymatic hydrolytic degrees of the gelatin increased time-dependently up to 6 hrs, while the Alcalase gelatin hydrolysates showed the highest hydrolysis degrees compared to the others. Furthermore, gelatin hydrolysates of Neutrase and ${\alpha}$-chymotrypsin showed the highest DPPH radical and $H_2O_2$ scavenging activities ($IC_{50}$ value; 9.18 mg/mL and 9.74 mg/mL), respectively. However, the activities were not significant (P<0.05). We also observed that the four gelatin hydrolysates significantly increased ACE inhibitory activities from approximately 20% to 60% (P<0.05), Among them, the Alcalase gelatin hydrolysates showed the higher ACE inhibitory activity ($IC_{50}$ value; 0.73 mg/mL) compared to the others. These results suggest that the enzymatic gelatin hydrolysates prepared from B. japonicus scales may possess a potentially useful function as an ACE inhibitory agent. As such, the utility of B. japonicus scales should be given due consideration for application in the functional food industry.

• Fisheries and Aquatic Sciences
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• 제12권4호
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• pp.249-257
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• 2009

This study is conducted to partially purify an antioxidative peptide in a two-step gelatin hydrolysate from Alaska pollock surimi refiner discharge, which was obtained by sequential treatment with Pronase E and Flavourzyme. The two-step gelatin hydrolysate was fractionated using chromatographic methods. Based on the same protein concentration of each fraction, the antioxidative activities (85.1-95.4%) of positive fractions fractionated by ion-exchange chromatography were higher than those (27.2-87.8%) from gel filtration. Then, further purification of the positive fractions was performed. Among them, the partially purified A1C1L2G1 and A1C1L2G2 fractions showed 96.2% and 85.1% inhibition, respectively, of linoleic acid peroxidation. The A1C1L2G1 fraction was composed of 15 kinds of amino acids and the predominant amino acids were proline, glycine and alanine. The results obtained in this study suggested that the fraction partially purified through chromatographic methods from the two-step gelatin hydrolysate of Alaska pollock surimi refiner discharge could be useful as a supplementary source for improving health functionality.

• Food Science of Animal Resources
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• 제33권4호
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• pp.493-500
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• 2013

Gelatin is a collagen-containing thermohydrolytic substance commonly incorporated in cosmetic and pharmaceutical products. This study investigated the antioxidant activity of gelatin by using different reagents, such as 2,2-azinobis-(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS), 2,2-di (4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity-fluorescein (ORAC-FL) in a porcine gelatin hydrolysate obtained using gastrointestinal enzymes. Electrophoretic analysis of the gelatin hydrolysis products showed extensive degradation by pepsin and pancreatin, resulting in an increase in the peptide concentration (12.1 mg/mL). Antioxidant activity, as measured by ABTS, exhibited the highest values after 48-h incubation with pancreatin treatment after pepsin digestion. Similar effects were observed at 48 h incubation, that is, 61.5% for the DPPH assay and 69.3% for the ABTS assay. However, the gallic acid equivalent (GE) at 48 h was $87.8{\mu}M$, whereas $14.5{\mu}M$ GE was obtained using the ABTS and DPPH assays, indicating about sixfold increase. In the ORACFL assay, antioxidant activity corresponding to $45.7{\mu}M$ of trolox equivalent was found in the gelatin hydrolysate after 24 h hydrolysis with pancreatin treatment after pepsin digestion, whereas this activity decreased at 48 h. These antioxidant assay results showed that digestion of gelatin by gastrointestinal enzymes prevents oxidative damage.

• Journal of the Korea Academia-Industrial cooperation Society
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• 제17권3호
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• pp.611-621
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• 2016

This study examined the effects of collagen peptide intake extracted from the tilapia fish (TFCP) on UVB-induced skin in hairless mice. To cause photoaging, the hairless mice were exposed to UVB irradiation three times a week and TFCP was administered with 545 and 1090 mg/kg once a day for 12 weeks. The level of wrinkle formation, thickness of the skin, acute erythema, skin hydration, amount of hydroxyproline, expression of MMPs and filaggrin were measured. Wrinkle formation, thickness of the skin, and acute erythema were significantly lower and skin hydration was higher in the TFCP intake group compared to the UVB-control mice group. In addition, the amount of the collagen and hydroxyproline of the collagen peptide intake group were much higher than the UVB-control group. Oral administration of TFCP inhibited the expression of the MMP-3 and MMP-13 mRNA level as well as activated MMP-2 and MMP-9 induced by UV irradiation, and increased the protein expression of filaggrin in hairless mice skin. In summary, the oral administration of TFCP can inhibit wrinkle formation and protect the skin from UV-damage. Therefore, this study suggests that TFCP is a potential candidate material for skin functional foods.