Kazeinomakropeptidin jelleşmesinde pH ve transglutaminaz enziminin etkisi
Effects of pH and transglutaminase enzyme on gelation of caseinomacropeptide
Yazar: ESRA KOCAMAN
Danışman: PROF. DR. MERAL KILIÇ AKYILMAZ
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2016
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Kazeinomakropeptid peyniraltı suyundan elde edilen biyolojik aktiviteye sahip bir polipeptiddir. Bu çalışmada, kazeinomakropeptidin jelleşme özelliklerine pH ve transglutaminaz enziminin etkileri incelenmiştir. Kazeinomakropeptid çözeltilerinin pH değeri 3,0-6,7 aralığındaki değerlere ayarlanarak jelleşme sıcaklığı, jel sertliği ve kırılma gerilimi değerleri sıcaklık ve stres taramaları yapılarak belirlenmiştir. Kazeinomakropeptidin tüm pH değerlerinde jel oluşturabildiği, en sert jelin pH 4,5'te, en zayıf jelin ise pH 6,7'de elde edildiği belirlenmiştir. pH değeri düştükçe jelleşmenin daha düşük sıcaklıklarda başladığı saptanmıştır. Kazeinomakropeptidin hidrofobisitesi pH değeri düştükçe artmıştır. Transglutaminaz enziminin kazeinomakropeptidi çapraz bağlayarak polimerler oluşturabildiği ters faz-HPLC ile serbest KMP'deki azalış ve SDS-PAGE ile moleküler kütlenin artması ile belirlenmiştir. Kazeinomakropeptid çözeltisine transglutaminaz enzimi 1-20 U/g aktivitede uygulandığında jelleşmesinin etkilendiği tespit edilmiştir. Transglutaminaz enziminin  kazeinomakropeptidin jelleşmesini enzim aktivitesine, jelleşme pH ve sıcaklığına bağlı olarak farklı şekilde etkilediği bulunmuştur. Jelleşme pH'sı 4,5 olduğunda transglutaminaz enziminin kazeinomakropeptid jelini zayıflattığı tespit edilmiştir. Jelleşme sıcaklığı 90°C olduğunda, 1 U/g transglutaminazın  pH 3'te elde edilen jeli kuvvetlendirdiği bulunmuştur. Jelleşme sıcaklığı 70°C olduğunda ise 20 U/g transglutaminazın pH 3'te elde edilen kazeinomakropeptid jelini kuvvetlendirdiği bulunmuştur. Elde edilen sonuçlar, kazeinomakropeptidin içeriğinde düşük oranda bulunan diğer peyniraltı suyu proteinlerinin de pH ve sıcaklığa bağlı olarak jelleşmeye etki ettiklerini göstermiştir.

Abstract:
Caseinomacropeptide is a polypeptide which has biological and functional properties. In this research, effects of pH and transglutaminase enzyme on gelation properties of caseinamacropeptide were investigated. Commercial caseinomacropeptide (CMP) was used as is. CMP was found to contain 80.4% protein, 4.9% moisture and 5.3% ash. According to the manufacturer, CMP powder had CMP at a level of 63%. CMP solutions were prepared with deionized distilled water by holding overnight at 4°C. Commercial transglutaminase enzyme, Activa MP,  with an enzyme activity of 100U/g was used. Enzyme was used at activities in the range of 1-60 U/g CMP powder.  
Hydrophobicity of CMP was measured by using fluorescent ANS-probe method at pH values of 2.5, 3.5 and 6.7. Excitation wavelength was adjusted to 390 nm and emission wavelength  to 480 nm. Hydrophobicity of CMP increased as the pH was reduced. This was explained by inaccesibility of hydrophobic regions to the probe as a result of aggregation. 
Formation of polymers via cross-linking by transglutaminase was observed by reversed phase HPLC and SDS-PAGE analysis. In the HPLC analysis, amount of free CMP was decreased after enzyme treatment. In SDS-PAGE analysis, formation of large polymers with molecular mass above 66 kDa was seen after enzyme treatment.These results showed that enzyme was able to cross-link CMP. 
Gelation properties of CMP were determined by rheometer. pH values of the CMP solutions were adjusted to pH values in the range of 3,0-6,7. Gelation temperature was  determined by temperature sweep in the range of 25-90oC. After temperature sweep, temperature was kept at 90oC for 30  minutes, then decreased to 25oC in five minutes and then held at 25oC for 10 minutes  to follow gelation.. Gelation temperature was determined when elastic modulus (G') crossed viscous modulus (G''). Separate samples were prepared and held at 90°C for 30 minutes in a water bath for stress sweep measurements. Gel hardness was taken as the maximum G' value obtained in stress sweep measurement.  Break stress was determined as the shear stress value at which G' crossed G'' value.  Frequency sweep was also performed at 25 oC in these samples. CMP was found to form a gel at a concentration of 20%. Among all pH values, the hardest gel structure was obtained at a pH value of 4.5.The weakest gel was obtained at a pH value of 6,7. Gelation of CMP started at a lower temperature as the pH value was decreased. At pH values 3 and 3.5, G' value was found to decrease at temperatures above 70°C during temperature sweep. In temperature sweep of the sample at pH 4,5 there were two clear step increases in G' showing gelation of two different components of CMP powder. Frequency sweep measurements of the samples showed slight differences. 
Effect of transglutaminase enzyme on gelation of CMP was studied at pH 3 and 4.5, enzyme activities of 1 and 20 U/g KMP powder. Enzyme incubation was performed at 40oC for 1 hour. After incubation, solutions were cooled in ice-water bath. Temperature and stress sweep measurements were carried out as done for CMP solutions without enzyme. As it was observed that G' value of the CMP sample at pH 3 decreased after 70°C, additional samples were prepared and allowed to gel at 70°C in a water bath. Treatment with transglutaminase enzyme affected CMP gels differently depending on enzyme activity, gelation pH and temperature. Gelation temperature of CMP was changed after enzyme treatment. Transglutaminase treatment weakened the CMP gels obtained at pH 4.5 regardless of enzyme activity at gelation temperature of 90°C. Gel hardness of CMP treated with 1 U/g transglutaminase increased compared to that of the sample without the enzyme. When CMP gels were prepared at pH 3 and 70°C, transglutaminase at an activity of 20 U/g was found to increase gel hardness and break stress.  
Transglutaminase was found to affect gel properties of CMP depending on enzyme activity, gelation pH and temperature. The results showed that not only CMP but also other minor whey proteins present in the CMP powder contributed to gelation especially at pH 4,5 and temperature of 90°C. Further studies could be conducted to determine optimum transglutaminase activity required for a strong and stable CMP gel.