**4. Future challenges**

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).
