Extraction de la pepsine et utilisation dans la coagulation du lait en vue d’une valorisation des proventricules de volailles au profit de la filière lait en Algérie

Abstract

This study aimed the extraction of the pepsin from chicken proventriculus and characterization in order to ensure its use as a rennet substitute in the milk coagulation. For this purpose, we approached the study of its coagulant, proteolytic activities and biochemical, rheological and microstructural pepsin gel aspects with comparison to the rennet gel. The chicken pepsin crude extract presented a strength coagulation of 13142. The partial purification by FPLC gave 45 fractions. The 18 fraction presented maximum coagulant activity (AC) and proteolytic activity (PA). The ration AC/AP of the pepsin crude extract increased from 7.15 to 34.25 after its partial purification. This increase is widely appreciated in cheese making. The coagulant activity for chicken pepsin and rennet have been defined for temperature intervals included between 50 ° C and 55 ° C, pH 5.0 and 5.6 and 0.04 M CaCl2 respectively. The coagulation was further estimated by the turbidimetry and the conductimetry. The turbidimetry showed similarity (p>0.05) between the micelles fusion evolution in the formation of the pepsin or rennet gel. However, the conductimetry has shown a difference (p< 0.05) between the minerals conductivity distributed between the aqueous and the micellar phase during gelation. Rheological approach allowed to study the pepsin and rennet gel behavior by application of different shear rates. The apparent viscosity in non-destructive mode showed a similar viscous character evolution after, it tended to its maximum to indicate the formation of the two gels. Flow curves showed a plastic character for the two gels (Bingham liquid). On the other hand, the rennet gel seems to be viscoelastic ( = 0.55) relatively notable than gel pepsin ( = 0.60). The rennet and pepsin gels breaking points are reached respectively to 0.34 N and 0.38 N. Syneresis of the two gels showed at the beginning of coagulation rapid expulsion of whey. This expulsion tends to increase for gel pepsin but for rennet gel a stabilisation is shown for the same time of coagulation. Proteolysis study was evaluated by the estimation of NPN/TN and NCN/TN fractions. NPN fractions have not shown a significant difference (p> 0.05) ; 11.4% versus 11% for pepsin and rennet respectively. Nevertheless, the NCN fractions have presented differences (p<0.05) of 58.4 % for the pepsin gel and 49.8 % for the rennet gel. In urea-PAGE, pepsin degradation products αs1I-CN and β I-CN of αs1-CN and et β-CN caseins respectively were observed in the early stages of incubation. Those of rennet appeared late from 6 hours of incubation. In SDS-PAGE, only para κ-CN was obtained as a κ-CN degradation product after pepsin or rennet hydrolysis. The interactions study revealed the bonds type involved in the formation of two gels. The rate of their proteins dissociation showed an abundance in hydrophobic interactions, followed by hydrogen and calcium bonds. The difference between the rate of dissociated proteins of two gels is not significant (p>0.05). The microscopic study of gels showed the evolution of the micelles fusion and the sol - gel system transition. Similarity of pepsin and rennet gels protein structure networks is visualized. This resemblance is characterized by compactness and the appearance of many cavities of heterogeneous size and amorphous zones. The spatial structure of two gels seems to be homogeneous. To ensure stability of the chicken pepsin crude extract during a 135-day storage, two modes of drying were studied and compared, freeze-drying (lyophilization) and drying under reduced vacuum. While the partial vacuum drying generated residual activity of 45.8% which decreased to 26.2% after 135 days of storage, freeze-drying ensured better results with 48.5% at the end of drying operation and more than 41% for the same conservation period. The residual activity seems to be better if the pepsinogen is activated after lyophilization. Furthermore, the addition of lactose to the pepsin extract provides more interesting residual coagulant activity. Theses results tend and converge towards the rennet substitution possibility by chicken pepsin in the milk coagulation. Also, it would be possible to use it in other areas for biological interest or industrial technology purposes.