**Acknowledgements**

of up to 200 MPa and at temperatures of up to 40°C [27]. On the other hand, PepY and PepC were more sensitive to pressure and temperature, resulting in inactivation at pressures above 100 and 200 MPa, respectively [27]. For the glycolytic enzymes, a slight increase in activity was observed at pressures of 100 and 200 MPa, but no further changes were observed in the activity of these enzymes above 300 MPa [27]. The HIP process on commercial milkclotting enzymes (recombinant Camel chymosin, calf rennet, bovine rennet, porcine pepsin, protease from *R. miehei*) was able to increase up to 25% the proteolytic activity (up to 300 MPa) or completely inactivate at high pressures (above 550 MPa) [26]. In other study, the leucine aminopeptidase obtained from *Aspergillus* (*A.*) *oryzae* used in the reduction of soy

The results obtained for HPH showed that this process was able to change the optimum temperature of neutral protease from 55 to 20°C after HPH at 200 MPa [28], improve glucose oxidase activity at 75°C after HPH at 150 MPa and increase between 100% and 400% its stability under storage [9]. In addition, it was observed an increase of amyloglucosidase activity at 80°C after HPH at 100 MPa [8] and an increase in the milk-clotting activity of coagulant

The studies of applying HIP and especially HPH, are recent. Therefore, there are some gaps of knowledge that must be fulfilled, allowing complete understanding about the effect and potential of both technologies. Firstly, from the data available in the literature, it is not possible to differentiate the effects of HIP and HPH since few enzymes were evaluated in both processes and, when evaluated, normally is not using the same diluting media, concentration, or method for activity measurement. Therefore, new studies need to be performed in a comparative way (varying just process conditions) to better establish the differences and equivalences of both processes. Ideally, a refined evaluation of molecular structure of enzymes must be determined to quantify the intensity of observed alterations and to describe the sequence of alterations caused specifically by HIP and HPH, helping to explain the enzymes transformation induced and consequently try to establish a mechanistic explanation of the alteration level that induces activation and inactivation of enzymes. From these explanations, it might be possible to predict the type of alteration expected for different enzymes, making these physical methods more interesting for industrial applications. Obtaining these results is mandatory to stimulate application of HIP and HPH, considering the industrial purpose of enzymes activation/modification/stabilization (mainly for enzyme manufactures) or the

requirement of deleterious enzymes inactivation (especially for food processors).

High isostatic pressure and high pressure homogenization can be considered interesting unitary operations to be applied for inducing enzyme changes. Both processes are able to

immunoreactivity was activated at 100–200 MPa/15 min/50°C [89].

enzymes, especially those that have chymosin in its constituents.

**4. Future challenges**

64 Enzyme Inhibitors and Activators

**5. Conclusions**

The authors like to thank the São Paulo Research Foundation (FAPESP) for the financial support.
