###### Abstract  
Among various food sources, milk proteins remain the major vector for functional peptides endowed with several biological activities. Particularly, the proteolytic activity of lactic acid bacteria during milk fermentation has been one of the most followed strategies to produce bioactive peptides. In the present study, the exploration of the activity of several starter cultures, at different fermentation times, was firstly investigated by reversed phase-high performance liquid chromatography. Among the tested strains, _Lactobacillus helveticus_ showed a higher proteolytic activity and it was submitted to further investigations by changing the fermentation substrate (skim milk, brain heart infusion, peptone water) as well as the extraction strategy (trichloroacetic acid vs. glass beads). The chromatographic analyses and the in vitro antioxidant and antihypertensive assays highlighted considerable differences for _L. helveticus_ hydrolysates from different substrates, while a negligible impact by the two extraction protocols emerged. Furthermore, nano-high pressure liquid chromatography coupled with a high resolution mass spectrometry analyzer allowed the preliminary discrimination of fractions from fermented skim milk, likely responsible for the found activity. The obtained results suggest the possibility of varying the fermentation parameters in order to maximize the functional effects of the bioactive peptides. bioactive peptides; _Lactobacillus helveticus_; fermentation substrate; liquid chromatography; antioxidant activity; ACE inhibitory activity +
Footnote †: journal: Medical  
precursors, BPs contribute to the modulation of different body functions exerting, inter alia, antioxidant, digestive, immunomododulatory, hypotensive, antithrombotic, antibacterial, and opiate-like properties. Such potential health benefits justify the emerging employment of BPs and protein hydrolyses as valuable sources for physiological functioning and human well-being promotion [1; 2; 3]. Although potential BPs have been identified from various food proteins (of either animal or plant origin) [1; 4] and their intake could be regularly associated with the current diet, milk proteins remain the major source of functional BPs endowed with a wide range of biological activities. The occurrence of biofunctional peptides with high nutritional value has been also widely reported in fermented milk products such as yogurt, cheese, sour milk, or kefir, and some of them have been shown to contain health beneficial properties [2; 5]. This feature accounts for the growing interest by food scientists in exploiting protein hydrolyses and bioactive peptides from milk and dairy products for applications as functional foods as well as for potential uses in nutraceuticalids [2; 5]. The proteolytic activity of lactic acid bacteria (LAB) during milk fermentation has been, and still is, among the most pursued strategies to produce functional peptides. In this scenario, the research of novel tailored strategies to exploit dairy fermented foods as sources of health peptides is widely increasing. As reported in literature [5; 6; 7; 8], BPs generated during milk fermentation mostly range from 2 to 20 amino acids and some of them are known to display multi-functional properties. BPs have been mainly screened for their capacity to exhibit specific biological properties, with particular attention paid to the antioxidant [9; 10], angiotensin-converting enzyme-inhibitory (ACEi) [11; 12; 13], and antimicrobial [14; 15] actions among others. Although numerous approaches dealing with the production of BPs from a variety of sources are described in literature [16; 17], often misleading and incomplete evidences still drive the rational selection and fermentation conditions of specific strains for the generation of active components. Moreover, the lack of systematic approaches, allowing overcoming of challenging peptide extractions and purifications, represents one of the major limitations in BP production.