การผลิตสารสกัดจากปลาอย่างต่อเนื่องโดยใช้เครื่องปฏิกรณ์ชีวภาพโปรติเอสตรึงรูปแบบฟลูอิไดซ์ / บุศราภา ลีละวัฒน์ = Continuous production of fish extracts using immobilized proteases fludized bioreactor / Bootsrapa Leelawat
The optimum conditions for preparation of immobilized papain (IP) and Neutrase® (IN) on 35-50 mesh river-bed sand by a covalent bonding method were with 3% and 1% by volume of APTS as carrier activator, 7% and 5% by volume of glutaraldehyde as cross-linker respectively, and 5% by volume of papain solution (169,550 u/g) at pH 8.5 and 3% by volume of Neutrase® solution (172,410 u/ml) at pH 6.0. For the casein hydrolysis using IP and IN, it was found that optimum pH values shifted 0.5 pH units toward the acid side compared to the native enzyme and 0.4 pH units toward the alkaline side, respectively, but the optimum temperatures were the same. Km values of IP and IN was 1.06x10(superscript -3)mM and 4.10x10(superscript -3) mM which was 7.29 and 4.88 times lower than that of free papain and free Neutrase®, respectively. The specific activities of the IP and IN were 452.0 and 337.9 unit/mg protein which were 1.07 and 13.6 times smaller than that of free papain Neutrase®, respectively. The optimum pH rang for reaction stability of IP and IN was at pH 7.0-8.5. The IP and IN were more heat stable than soluble enzymes at 70 degree celsius, but the heat stability of all enzymes at 50 degree celsius was the same. The optimum storage conditions of immobilized enzymes were found at 8-10 degree celsius in 0.002 M EDTA, 0.08 M cysteine in tris buffer at pH 8.5 for IP and IN 2x10(superscript -3)) MCaSO(subscript 4) solution for IN. Under the above storage conditions, the activities of both immobilized enzymes for casein hydrolysis was observed for 65 days. It was found that the activities of both enzymes were stable at 16th-65th day. Moreover, after using the same immobilized enzymes 3 times in casein hydrolysis, the IP retained activity of 29% while the IN retained activity 50%. The proteolytic hydrolysis of fish cooking juice (FCJ) using the three connected immobilized proteases bioreactors 1.7x75 cm. was studied. For Skipjack cooking juice hydrolysis, both IP and IN have an optimum temperature of 50 degree celsius and the optimum space velocity of 5.5(min)(superscript -1), 4 hrs. for the former and 7.9 (min)(superscript -1), 3hrs. for the latter. Under such condition, the maximum degree of hydrolysis using IP and IN was found to be 65% DH, respectively. Fish extracts produced under such conditions contained 92.90% moisture, 4.59% protein, 0.20% fat, 1.73% ash and 1.37% NaCl, which was similar ratio to that of fish extracts converted to concentrated and dried forms. Solubility test on dried fish extracts produced using the immobilized enzymes bioreactors showed much better solubility degree than that of the dried FCJ. Furthermore, organoleptic tests on the concentrated fish extracts as fish flavor enhancer in Yaki-Tori (chicken barbecue) and the dried fish extracts as fish flavor in cubed noodle soup mix showed good ratings for colour, odour, taste and total acceptance.
