Milk-Gelling Properties of Proteases Extracted from the Fruits of Solanum Elaeagnifolium Cavanilles

Abstract

There is little information on the milk coagulation process by plant proteases combined with chymosins. This work is aimed at studying the capability of protease enclosed in the ripe fruits of Solanum elaeagnifolium (commonly named trompillo) to form milk gels by itself and in combination with chymosin. For this purpose, proteases were partially purified from trompillo fruits. These proteases had a molecular weight of ~60 kDa, and results suggest cucumisin-like serine proteases, though further studies are needed to confirm this observation. Unlike chymosins, trompillo proteases had high proteolytic activity (PA = 50.23 U_Tyr mg protein^-1) and low milk-clotting activity (MCA = 3658.86 SU mL^-1). Consequently, the ratio of MCA/PA was lower in trompillo proteases (6.83) than in chymosins (187 to 223). Our result also showed that milk gels formed with trompillo proteases were softer (7.03 mPa s) and had a higher release of whey (31.08%) than the milk gels clotted with chymosin (~10 mPa s and ~4% of syneresis). However, the combination of trompillo proteases with chymosin sped up the gelling process (21 min), improved the firmness of milk gels (12 mPa s), and decreased the whey release from milk curds (3.41%). Therefore, trompillo proteases could be combined with chymosin to improve the cheese yield and change certain cheese features.

Figure 1

Schematic diagram of the methodology used in this study.

Figure 2

Sigmoidal four-parameters equation used to analyze the rheological behavior of milk clotted with bovine chymosin, recombinant camel chymosin, or trompillo proteases. η_s=viscosity at gel point, η₀=initial viscosity, η_s/2=half of the maximum viscosity, t_c=time to reach half maximum viscosity, k=rate of caseins aggregation, t₀=time to start the caseins aggregation, and t_s=time to form the milk gel.

Figure 3

SDS-PAGE analysis of the crude extract from Solanum elaeagnifolium fruits (a), and partially purified proteases from the crude extract (b), with its proteolytic zymogram (c). Lane 1=molecular weight marker, lane 2=crude extract, lane 3=molecular weight marker, lane 4=fraction recover sulfate ammonium precipitation (50%) and gel filtration on Sephadex G-100.

Figure 4

Kinetic of caseins hydrolysis by trompillo proteases at 30°C and pH 7. Values represent the average of triplicates (n = 3).

Figure 5

Rheological analysis of milk gelation induced by different proteases at 30°C. η_s=viscosity at gel point, k=rate of caseins aggregation, t₀=time to start the caseins aggregation, and t_s=time to form the milk gel.

Figure 6

Hydrolysis of milk proteins during gel formation at 30°C under the action of different proteases. (a) Milk clotted with trompillo proteases. (b) Milk clotted with recombinant camel chymosin (lane 1) and chymosin from calf stomach (lane 2); samples were taken after 80 minutes of incubation. MWM=molecular weight marker, a=transferrin, b=serum albumin, c=β-caseins, d=α_s1-caseins, e=κ-caseins, f=γ-caseins, g=β-lactoglobulin, h=hydrophobic peptides~14 kDa.

Figure 7

Proteins observed in the whey released by milk gels clotted with Trompillo proteases (lane 1), recombinant camel chymosin (lane 2), bovine chymosin (lane 3), and the mixture (1 : 1 ratio) of recombinant camel chymosin with Trompillo proteases (lane 4). MWM=molecular weight marker, a=immunoglobulins, b=lactoferrin, c=serum albumin, d=β-caseins, e=αs₁-caseins, f=κ-caseins, g=γ-caseins, h=hydrophilic peptides~18 kDa, i=β-lactoglobulin.