5. Effects of food protein-derived renin inhibitory peptides in vitro and in vivo

The most widely utilized method for assessing the renin inhibitory potential in vitro is a fluorometric assay utilizing a human recombinant renin (Cayman Chemical, MI, USA). Recent data indicate that some food protein-derived hydrolysates and peptides possess in vitro renin inhibitory activity. Inhibiting activity against human recombinant renin has been reported, for instance, for hemp seed, pea, bovine blood, and chicken skin protein-derived hydrolysates produced by various food grade proteases (Table 1).

Among the protein hydrolysates, the highest renin inhibitory activities have been reported for hemp seed protein hydrolysates with IC50 values of 0.08–0.81 mg/ml [27, 44]. These activities are at the same level with the synthetic renin inhibitor Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Lys- (Boc)-OMe [22]. Alcalase has yielded to very active renin-inhibiting hydrolysates (e.g., Refs. [2, 30]), and also pancreatin and papain have produced high renin inhibitory activity (e.g., Refs. [21, 27]). Papain has also exhibited good prospects in silico in releasing renin inhibitory peptides from, for instance, bovine fibrinogen [37]. Moreover, simulated food protein hydrolysis with gastrointestinal enzymes has also resulted in products with renin inhibitory activities [27, 51]. Taken together, the efficiency of the protease to release renin inhibitory peptides seems to depend on the parent protein matrix. Thus, in silico tools are recommended to be utilized prior to the in vitro experiments to predict the efficacy of proteases with the particular protein matrixes.

Spontaneously hypertensive rats (SHR) are widely used animal model to assess the antihypertensive effects by in vivo experiments. This animal model is applied in short- and long-term manners, for example, to study the antihypertensive effects of milk protein-derived peptides [18, 23]. Recently, food protein-derived renin-inhibiting peptides and protein hydrolysates have induced antihypertensive effects when orally administered to spontaneously hypertensive rats. Decreases in SBP by 19–33 mmHg have been reported for instance, for enzymatic hydrolysates of chicken skin, red seaweed (Palmaria palmata), hemp seed, and pea protein (Table 1). Generally, the purified renin inhibitory peptides and RP-HPLC fractions have exerted the antihypertensive activities at lower dosage (30 mg/kg bw) compared to the crude protein hydrolysates and membrane-filtrated fractions, which has shown similar antihypertensive effects with 100–200 mg/kg bw (Table 1). Hydrolysates and peptides have shown dual inhibition against renin and ACE, or modulation capacity on the RAAS gene expression. Thus, the antihypertensive effects are not solely due to the renin inhibition (e.g., Ref. [41]). For example, egg-derived pentapeptide RVPSL has been recently shown to decrease renin mRNA expression in the kidney of SHRs with a dosage of 50 mg/kg bw administered daily for 4 weeks [79]. Also, weakly active renin-inhibiting peptides have been shown to display physiological antihypertensive activity. A weakly active pea protein hydrolysate (19% renin inhibition at 1 mg/ml) exhibited SBP lowering effects in SHRs and in a kidney disease rat model and was found to downregulate renal expression of renin mRNA in the rat model (Table 1). Also, the pea protein hydrolysate showed antihypertensive effects in hypertensive humans in a 3-week intervention trial (Table 1). This indicates that antihypertensive food protein-derived peptides may be acting at the same time via multiple pathways at the protein level as well as at the gene level modulating the RAAS.

would be important. A consensus for a static process to model the digestion of plant secondary metabolites has been constructed based on in vivo data [3]. Indeed, the future research should focus more on the in vivo bioavailability of the peptides and based on the correlation with

The peptides are exposed to peptidolytic digestion also on the brush border membrane of the intestine. There are number of peptidases with varying specificities bound on the intestinal epithelial cells. It has been suggested that dipeptide and tripeptide tend to resist the gastric and duodenal digestion and also the hydrolytic action of peptidases at the brush border membrane. These small peptides can be absorbed by active transcellular transport or by passive process [63]. To study the absorption in vitro, the monolayer of intestinal cell lines, such as Caco-2 cells, simulating intestinal epithelium, is commonly utilized. Clinical data concerning the bioavailability of bioactive peptides are very restricted; however, ACE-inhibitory lactotripeptides, Ile-Pro-Pro and Val-Pro-Pro, have been detected in human and animal circulatory

5. Effects of food protein-derived renin inhibitory peptides in vitro and

The most widely utilized method for assessing the renin inhibitory potential in vitro is a fluorometric assay utilizing a human recombinant renin (Cayman Chemical, MI, USA). Recent data indicate that some food protein-derived hydrolysates and peptides possess in vitro renin inhibitory activity. Inhibiting activity against human recombinant renin has been reported, for instance, for hemp seed, pea, bovine blood, and chicken skin protein-derived hydrolysates

Among the protein hydrolysates, the highest renin inhibitory activities have been reported for hemp seed protein hydrolysates with IC50 values of 0.08–0.81 mg/ml [27, 44]. These activities are at the same level with the synthetic renin inhibitor Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Lys- (Boc)-OMe [22]. Alcalase has yielded to very active renin-inhibiting hydrolysates (e.g., Refs. [2, 30]), and also pancreatin and papain have produced high renin inhibitory activity (e.g., Refs. [21, 27]). Papain has also exhibited good prospects in silico in releasing renin inhibitory peptides from, for instance, bovine fibrinogen [37]. Moreover, simulated food protein hydrolysis with gastrointestinal enzymes has also resulted in products with renin inhibitory activities [27, 51]. Taken together, the efficiency of the protease to release renin inhibitory peptides seems to depend on the parent protein matrix. Thus, in silico tools are recommended to be utilized prior to the in vitro experiments to predict the efficacy of proteases with the particular

Spontaneously hypertensive rats (SHR) are widely used animal model to assess the antihypertensive effects by in vivo experiments. This animal model is applied in short- and long-term manners, for example, to study the antihypertensive effects of milk protein-derived peptides [18, 23]. Recently, food protein-derived renin-inhibiting peptides and protein hydrolysates have induced antihypertensive effects when orally administered to spontaneously

in vivo data, a harmonized in vitro method could be proposed.

system after oral ingestion [25].

248 Renin-Angiotensin System - Past, Present and Future

produced by various food grade proteases (Table 1).

in vivo

protein matrixes.

### 6. Production of food protein-derived renin inhibitory peptides

A general challenge is how to process the protein hydrolysates further into peptide products with high yield and biological efficacy. Careful choice of suitable enzymes and conditions such as temperature, hydrolysis time, degree of hydrolysis, and enzyme-substrate ratio are crucial for production of peptides with targeted bioactivities and functional properties. Hydrolysis process is recommended to be performed as a continuous process rather than traditional batch process to reduce the enzyme consumption and increase the efficacy [45, 76]. One advantage of enzymatic hydrolysis process is the feasibility in pilot and industrial scale production [6, 7, 28].

To enhance the bioactivity, the active peptides should be concentrated after protein hydrolysis. Size, net charge, and hydrophobicity of the peptides have an important role to select the most suitable techniques to enrich the active peptides. The commonly used techniques include ultrafiltration membranes and chromatographic techniques to obtain an uniform product with the desired range of molecular mass (e.g. [15]). For example, ultrafiltration with 1 kDa membrane has been utilized to concentrate renin inhibitory peptides from rapeseed protein hydrolysate into permeate [36, 38, 48]. In addition to separation based on molecular size, ultrafiltration can be applied to separate peptides according to the net charge. This electrodialysis-ultrafiltration can be utilized to separate anionic, cationic, and neutral peptides of corresponding size range [5, 16, 17]. Large-scale chromatographic methods, used in sugar recovery and wastewater treatments, have been used to enrich peptides from hydrolysates and to separate off ineffective peptides or further undesirable components of the hydrolysates, such as colors, abnormal flavors, and/or salts [10]. Large-scale food-grade processing protocols for designed peptides fractions are needed for further development. Understanding the structural characteristics of peptides with targeted bioactivity and exploitation of these characteristics is a crucial requirement for this approach.
