Alkaline proteases from giant catfish viscera : Recovery by phase partitioning and their food applications

Name: Sunantha Katnawa

Organization : Mae Fah Luang University

keyword: Catfish

LCSH: Fisheries -- By-products

Classification :.LCCS: SH335.8

LCSH: Fishery products

LCSH: Catfishes

Abstract: Fish viscera (5-10% of body weight) generate from fish processing could be a potential and economical source for recovering enzymes such as proteases that may have potential for industrial applications, e.g. in the food, feed, pharmaceutical, leather, silk and detergent industries. The way to extract proteases out from its source should be economical, simple, rapid, scalable and environmental friendly. Hence, the alternative methods like liquid-liquid phase partitioning including aqueous two phase system (ATPS), thermoseparating aqueous two phase system (T-ATPS) and three phase partitioning (TPP) could be used as alternative methods for proteases recovery. These primary downstream processings provide not only the high recovery but also acceptable purity at a time. Therefore, the objectives of this study were to extract and enhance alkaline proteases recovery from farmed Mekhong Giant catfish viscera by ATPS, T-ATPS and TPP. Factors affecting phase separation efficiency (partitioning coefficient, volume ratio, specific activity, recovery and purity) including types and concentration of polymers, type of salts, types of solvent, the ionic strength, pH, NaCl concentration, incubation temperature and number of cycle in partitioning were investigated. The phase partitioning systems providing the highest enzyme recovery was selected to isolate alkaline proteases followed by biochemical characterization of isolated alkaline proteases in terms of pH profile and stability, temperature profile, thermal stability, effect of NaCl, inhibitors and metal ions on alkaline proteases activity, molecular weight and kinetic studies. Utilization of isolated alkaline proteases for protein hydrolysates preparation and carotenoprotein extraction was also investigated in comparison to commercial enzymes (commercial trypsins, Flavourzyme®, Izyme AL® and Alcalase®). From the study, ATPS, T-ATPS and TPP showed effectively separated of target enzyme without activity loss. After studying factors that affected partitioning parameters, the optimum condition with the highest recovery and purity from each partitioning method was obtained. To begin with ATPS, ATPS with 15% polyethylene glycol (PEG) 2000-15% sodium citrate containing 70% (w/w) crude enzyme extract at pH system of 8.5 provided the enzyme recovery of 195.90% and purity of 4.31-fold. The second partitioning method, T-ATPS, the system of 40% Poly (ethylene glycol-ran-propylene glycol) monobutyl ether (EOPO) 3900-10% magnesium sulphate (MgSO4) containing 50% (w/w) crude enzyme extract and NaCl (17%, w/w), induced phase separation at 55°C provided the enzyme recovery of 91.62% and purity of 21.50-fold. The last partitioning method, TPP, showed sodium citrate (50%, w/v) with 1.0:0.5 (v/v) (crude enzyme extract to t-butanol), pH 8.0 at incubation temperature of 25°C gave the recovery of 110.58% with 8.14-fold purification. Hence, ATPS provided the maximum enzyme recovery (196%) followed by TPP (92%) and T-ATPS (111%) while T-ATPS provided the highest purity (22- fold). The obtained alkaline proteases showed high efficacy in both animal (skin gelatin, whey and egg white) and plant (perilla seed, red kidney bean and soybean) proteins hydrolysis by hydrolyzing major protein components into small molecules when the unit of enzyme increased. Due to the highest recovery, ATPS partitioning method was selected to isolate alkaline proteases. The alkaline proteases activity was determined by using N-alphabenzoyl- DL-arginine- p-nitroanilide as substrate showing the optimum pH and temperature of 8.0 and 70ºC, respectively. It also showed high activity and stability over a wide alkaline pH range and also a good stability at high temperatures. Besides, it retained more than 50% of activity at the highest salt concentration (30%, w/v). The alkaline proteases was strongly inhibited by serine protease inhibitors (>80% inhibition), while low inhibition with cysteine-, aspartic- and metallo-protease inhibitors (<20% inhibition). The alkaline proteases activity was extremely inhibited by the metal ion Ag+, Cu2+ and Fe2+. Protein pattern, activity and inhibitory staining confirmed that the proteins with molecular weight of 12 and 31 kDa were a mixture of serine proteases presenting in alkaline proteases. Km and Kcat of the alkaline proteases were calculated to be 0.049 mM and 0.016 S-1, respectively. The alkaline proteases represented the success in production of gelatin hydrolysates with bioactivities. The resultant gelatin hydrolysates showed the highest ACE-inhibitory activity, DPPH, ABTS radical scavenging activity and ferric reducing antioxidant power. For stability study, DPPH radical scavenging activity of the hydrolysates was quite stable over the pH range of 1-11 and slightly increased when heating time increased up to 240 min at 100ºC. The ACE-inhibitory activity of the hydrolysates showed the highest stability at pH 7 but very stable at 100ºC over 15-240 min. The gelatin hydrolysates inhibited human low-density lipoprotein (LDL) cholesterol oxidation and Dipeptidyl Peptidase-IV (DPP-IV) activity by 80.95% and 50.74%, respectively. For molecular weight profile, alkaline proteases provided the gelatin hydrolysates with the lowest molecular weight average of 1.75 kDa. Furthermore, the alkaline proteases can hydrolyze shrimp protein leading to the recovery of more than 73% and 97% of protein and astaxanthin, respectively. The resultant shrimp hydrolysates provided adequate functional properties for food applications by demonstrating excellent solubility (>97%) in a wide pH range (3-10), good oil binding capacity (1.83 g oil/g hydrolysates) and discrete inter-facial properties. Besides, shrimp hydrolysates provided DPP-IV inhibition activity of around 23%. For application of shrimp hydrolysates in fish tofu (0-2%, w/w), the higher incorporation of the hydrolysates resulted in more resistible to lipid oxidation with lower thiobarbituric acid-reactive substances values during 14 days of storage (4°C) in according to rancidity scores by panelists. The counts of microbiological indicators including total viable counts, yeast and molds were significantly (P<0.05) affected by the addition of 2% (w/w) shrimp hydrolysates. For physical properties, colour of fish tofu surface were higher lightness (L*) and yellowness (b*), whilst those value of inner-part were lower lightness (L*) and higher whiteness values from storage day 0 to 14. In addition, softer texture in fish tofu with shrimp hydrolysates was evidenced by the instrumental analysis when compare with control sample. From the study, phase partitioning techniques could be used as an alternative method for the separation of enzymes from fish viscera and other sources with acceptable recovery. It is suggested that the isolated alkaline proteases from this fish viscera can further be used in protein hydrolysates and carotenoprotein production with bioactivities resulting from acceptable antioxidative activities, angiotensin Iconverting enzyme (ACE)-inhibitory, Dipeptidyl peptidase-IV (DPP-IV) and Prolyl oligopeptidase (POP) inhibitory activity. Application of resulting hydrolysates in fish tofu showed that the hydrolysates could be applied as natural antioxidant and antimicrobial.

Mae Fah Luang University. The Learning Resources and Education Media Center

Address: Chiang Rai

Email: library@mfu.ac.th

Name: Saroat Rawdkuen

Role: Advisor

Name: Soottawat Benjakul

Role: Co-Advisor

Name: Oscar Martinez-Alvarez

Role: Co-Advisor

Created: 2014

Issued: 2015

Modified: 2020-04-03

Issued: 2015-10-12

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: Thesis SH335.8 S957a 2014

eng

DegreeName: Dotor of Philosophy

Level: Doctoral Degree

Descipline: Food Technology

Grantor: Mae Fah Luang University

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