Extraction, Partial Purification and Characterisation of a Protease Extracted from Kesinai (Streblus Asper Lour.) Leaves, and its Potential Application in the Production of Cheddar Cheese

Calf-rennet is a conventional milk-clotting enzyme, which is most widely used coagulant in cheese-making all over the world. The world wide reduced supply of calf rennet and increase of cheese production have led to a systematic investigation of calf-rennet substitutes. Extracts of ‘kesinai’ (Streblus asper) leaves have been investigated as a source of enzymes to be used in cheese-making as an alternative source of calf rennet. The overall aim of this study was to evaluate the properties and application of Streblus asper leaf protease as a rennet substitute in cheese-making. A milk-clotting protease was extracted from kesinai leaves using 100 mM Tris-HCl buffer (pH 7.4) and several other buffers. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatography. Results obtained showed that 100 mM Tris-HCl buffer (pH 7.4) was found to be the most effective extraction buffer. A higher yield of the kesinai protease was obtained using acetone precipitation as compared to ammonium sulphate precipitation. The ratio of cold acetone to crude extract of 1.25:1 was found to be the most suitable ratio for the partial purification of the protease. After the final purification step, the enzyme was partially purified 3.3 fold with a 42.3% of recovery. The partially purified protease was characterised and it showed an optimum activity at 60 oC and pH 7.4. The enzyme was stable up to 70 oC for one hour, and residual activities at pH 6.0 to 10.0 were 72.4% and 70.2% of the optimum activity, respectively. The enzyme was found to have a higher temperature stability at -10 oC and 4 oC. It retained more than 98% of its activity 7 days after storage at both -10 and 40C. The enzyme was inhibited by PMSF and trypsin inhibitor by 98% and 95.87% relative to the initial activity, respectively, suggesting the presence of serine residue at the active site. Ca2+ had a slight stimulating effect while, Hg2+, Zn2+ and Pb2+ had strong inhibitory effects on the enzyme activity. The partially purified enzyme appeared as a single band on SDS PAGE with an apparent molecular weight of 75.8 kDa. The maximum coagulation activity was observed at pH 6.0 and at temperature 70 oC. The presence of CaCl2 up to 10 mM increased the milk-clotting activity while addition of NaCl decreased the milk-clotting activity. The milk-coagulation was also strengthened by increase enzyme concentration Cheddar cheese was prepared using the acetone-precipitated enzyme and commercial enzyme (chymosin), and the physico-chemical, biochemical and sensory propertieswere determined throughout two months of ripening. The yield of cheese produced using the commercial enzyme was 6% higher (p<0.05) than the yield obtained with kesinai enzymes. Cheeses made from kesinai enzyme and commercial enzyme were proximate compositionally alike except for moisture, protein contents and pH. The moisture content (42.67 ± 0.47%) of cheese made with commercial enzyme was lower than the moisture (43.88 ± 0.99%) of cheese made with kesinai enzyme, while the protein content (21.02 ± 0.41%) and pH (4.79 ± 0.03) were (p<0.05) higher in cheese made with commercial enzyme than the protein content (18.87 ± 0.32%) and pH (4.72 ± 0.01) obtained from cheese made from kesinai enzyme. In both cheeses,the moisture contents declined (p<0.05) with ripening, while the pH increased with ripening time. Non protein nitrogen (NPN) (0.04 ± 0.00%) and non-casein nitrogen (NCN) (0.09 ± 0.00%), were higher (p<0.05) in the whey obtained with kesinai enzyme compared to those (NPN and NCN were 0.03 ± 0.00% and 0.06 ± 0.00%,respectively), obtained with the commercial enzyme. Protein nitrogen (PN) (2.75± 0.06%) and casein nitrogen (CN) (2.56% ± 0.05) contents of cheese made with kesinai enzyme were (p<0.05) lower than those (CN and TN were 2.93 ± 0.11% and characteristics such as hardness (811.8 ±36.6 g for cheese made from kesinai enzyme and 984.5 ± 24.2 g for cheese made from commercial enzyme), gumminess (622.8 ± 39.2 g for cheese made from kesinai enzyme and 820.9 ± 20.9 g for cheese made from commercial enzyme), and chewiness (534.7 ± 17.1 g mm for cheese made from kesinai enzyme and 735.3 ± 14.6 g mm for cheese made from commercial enzyme),increased in both types of cheese during the early stage of ripening and decrease gradually at end of ripening whereas springiness and cohesiveness showed similar changes in both cheeses during the ripened period. It is concluded that kesinai protease could be used for the production of Cheddar cheese or it can be used together with another commercial enzyme in cheese production

Data-Driven Control of Linear Time-Varying Systems

An identification-free control design strategy for discrete-time linear time-varying systems with unknown dynamics is introduced. The closed-loop system (under state feedback) is parametrised with data-dependent matrices obtained from an ensemble of input-state trajectories collected offline. This data-driven system representation is used to classify control laws yielding trajectories which satisfy a certain bound and to solve the linear quadratic regulator problem - both using data-dependent linear matrix inequalities only. The results are illustrated by means of a numerical example

A Nash Game Approach to Mixed H2/H∞ Control for Input-Affine Nonlinear Systems

With the aim of designing controllers to simultaneously ensure robustness and optimality properties, the mixed H2/H∞ control problem is considered. A class of input-affine nonlinear systems is considered and the problem is formulated as a nonzero-sum differential game, similar to what has been done in the 1990s by Limebeer et al. for linear systems. A heuristic algorithm for obtaining solutions for the coupled algebraic Riccati equations which are characteristic of the linear quadratic problem is provided together with a systematic method for constructing approximate solutions for the general, nonlinear problem. A few numerical examples are provided

Approximate infinite-horizon optimal control for stochastic systems

The policy of an optimal control problem for nonlinear stochastic systems can be characterized by a second- order partial differential equation for which solutions are not readily available. In this paper we provide a systematic method for obtaining approximate solutions for the infinite-horizon optimal control problem in the stochastic framework. The method is demonstrated on an illustrative numerical example in which the control effort is not weighted, showing that the technique is able to deal with one of the most striking features of stochastic optimal control

Finite-dimensional characterisation of optimal control laws over an infinite horizon for nonlinear systems

Infinite-horizon optimal control problems for nonlinear systems are considered. Due to the nonlinear and intrinsically infinite-dimensional nature of the task, solving such optimal control problems is challenging. In this paper an exact finite-dimensional characterisation of the optimal solution over the entire horizon is proposed. This is obtained via the (static) minimisation of a suitably defined function of (projected) trajectories of the underlying Hamiltonian dynamics on a hypersphere of fixed radius. The result is achieved in the spirit of the so-called shooting methods by introducing, via simultaneous forward/backward propagation, an intermediate shooting point much closer to the origin, regardless of the actual initial state. A modified strategy allows one to determine an arbitrarily accurate approximate solution by means of standard gradient-descent algorithms over compact domains. Finally, to further increase robustness of the control law, a receding-horizon architecture is envisioned by designing a sequence of shrinking hyperspheres. These aspects are illustrated by means of a benchmark numerical simulation

Molecular simulations of the large conductance mechanosensitive (MscL) channel under mechanical loading

AbstractThe MscL channel is a mechanosensitive channel which is gated by membrane stress or tension. Here, we describe a series of simulations which apply simulated mechanical stress to a molecular model of the MscL channel using two methods – direct force application to the transmembrane segments, and anisotropic pressure coupling. In the latter simulations, pressures less than that equivalent to a bilayer tension of 12 dyn/cm did not cause the channel to open, while pressures in excess of this value resulted in the channel opening. These results are in approximate agreement with experimental findings