In recent years, the demand for “all natural” products is increasing because of the increasing limitations on the use of synthetic antioxidants and enhanced public awareness of health issues. Many natural food components like oils and oilseeds, proteins and protein hydrolysates, fruits and vegetables, oat and rice bran, spices, herbs and tea have antioxidant properties. Natural antioxidants from these food components provide oxidative stability to the food product. The antioxidant activity of proteins is mainly due to interactions between their ability to inactivate reactive oxygen species, chelate pro-oxidative transition metals, scavenge free radicals, and reduction of hydroperoxides. The objective of this study was to verify the antioxidant potential of Pea Protein Isolate ‘NUTRALYS® S85F' through different in vitro tests: 

• TOTAL ANTIOXIDANT RADICAL SCAVENGING ACTIVITY (ORAC ASSAY); 
• SUPEROXIDE RADICAL SCAVENGING ACTIVITY (NBT ASSAY); 
• LIPID PEROXIDATION INHIBITION ASSAY (MDA KIT); 
• HYDROXYL RADICAL SCAVENGING ACTIVITY (HRS ASSAY); 
• NITRIC OXIDE SCAVENGING ABILITY 

NUTRALYS® S85F clearly demonstrates intrinsic antioxidant activities as can be observed from its scavenging activity toward the peroxyl radicals (ORAC), scavenger of the superoxide radicals (NBT), hydroxyl radical scavenging (HRS), prevention of lipid oxidation (MDA) and nitric oxide scavenging (NO) capabilities. Based on our results, NUTRALYS® S85F will provide oxidative stability to food products and health benefits to the consumer.

A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. Many radicals are unstable and highly reactive. The most important oxygen-containing free radicals in many disease states are hydroxyl radical, superoxide anion radical, hydrogen peroxide, oxygen singlet, hypochlorite, nitric oxide radical, and peroxynitrite radical. These are highly reactive species, capable in the nucleus, and in the membranes of cells of damaging biologically relevant molecules such as DNA, proteins, carbohydrates, and lipids. A role of oxidative stress has been postulated in many conditions, including anthersclerosis, inflammatory condition, certain cancers, and the process of aging. Oxidative stress is now thought to make a significant contribution to all inflammatory diseases (arthritis, vasculitis, glomerulonephritis, lupus erythematous, adult respiratory diseases syndrome), ischemic diseases (heart diseases, stroke, intestinal ischemia), hemochromatosis, acquired immunodeficiency syndrome, emphysema, organ transplantation, gastric ulcers, hypertension and preeclampsia, neurological disorder (Alzheimer's disease, Parkinson's disease, muscular dystrophy), alcoholism, smoking-related diseases, and many others. The human body is in constant battle to control free radicals steady state

In recent years, the demand for “all natural” products is increasing because of the increasing limitations on the use of synthetic antioxidants and enhanced public awareness of health issues. Many natural food components like oils and oilseeds, proteins and protein hydrolysates, fruits and vegetables, oat and rice bran, spices, herbs, and tea have antioxidant properties. Natural antioxidants from these food components provide oxidative stability to the food product. The antioxidant activity of proteins is mainly due to interactions between their ability to inactivate reactive oxygen species, chelate pro-oxidative transition metals, scavenge free radicals, and reduction of hydroperoxides. 

The objective of this study was to verify the antioxidant potential of Pea Protein Isolate ‘NUTRALYS® S85F’ through different  in vitro  tests.

NUTRALYS® S85F was weighed and reconstituted at a final concentration of 10 mg/ml in 100 % H2O (stock solution) and mixed for 30 minutes at room temperature, this was followed by centrifugation and the clarified supernatant was diluted and used for the subsequent assays:1, 10, 20, 100, 200, 500, 1,000 µg/ml final concentration for all assays. The above-mentioned stock solutions were separated into several aliquots and stored at - 20 °C. Once an aliquot was thawed it was immediately used for a specific assay and never re-frozen.

All dilutions prepared for each experiment were maintained at room temperature until use in the assay.

Catechin, Ascorbic acid, Quercetin and Trolox are used as positive controls. 

Total antioxidant radical scavenging activity (ORAC assay):

Antioxidant activity of NUTRALYS® S85F was determined by the ORAC method, based on BMG Labtech protocol. The method measures the antioxidant scavenging activity against the peroxyl radical, induced by 2, 2’-azobis-(2-amidinopropane) dihydrochloride (AAPH), at 37°C for 30 minutes. 

TE over considered concentration range = Slope regression curve (Trolox®) / Slope regression curve (sample)

NUTRALYS® S85F has significant scavenging activity toward the peroxyl radicals however, the Trolox equivalent is significantly lower than that of any of the reference antioxidants

Lipid peroxidation inhibition assay (MDA kit): 

The lipid peroxidation inhibition assessment of NUTRALYS® S85F was performed using the MDA assay kit (Sigma/MAK085) 

NUTRALYS® S85F has a moderate effect on the prevention of lipid oxidation when compared to antioxidant references

Nitric oxide scavenging activity can be estimated by the use of Griess IlIosvoy reaction (Garrat, 1964) as described by Parul et al. (2013), 

NUTRALYS®S85F does demonstrate scavenging of the NO when compared to antioxidant references 

Superoxide radical scavenging activity (NBT assay): 

The superoxide radical-scavenging activity of NUTRALYS® S85F was determined according to the method of Chang et al. (2001) and Tung et al. (2009), 

The percent inhibition was calculated according to the following equation: 

% inhibition = [(absorbance of control - absorbance of sample) / absorbance of control] x 100.

NUTRALYS®S85F at the highest test concentration (1 mg/ml) displayed 90% inhibition.

Hydroxyl radical scavenging activity (hrs assay): 

NUTRALYS® S85F was tested at 7 concentrations in presence of iron-EDTA solution (mix solution: ferrous ammonium sulfate/EDTA/DMSO/ascorbic acid), as described by Halliwell and Gutteridge (1981)  

NUTRALYS® S85F has an equivalent antioxidant effects toward this particular ROS when compared to antioxidant the references 

NUTRALYS® S85F has an equivalent antioxidant effects toward this particular ROS when compared to antioxidant the references 

• Significant scavenging activity toward the peroxyl radicals (ORAC)
• A scavenger of the superoxide radicals (NBT) , with 90% inhibition at the highest test concentration (1000 µg/ml)
• Significant hydroxyl radical scavenging (HRS) activity however, higher concentrations of test compound was required to produce the same antioxidant effect as the reference compounds.

NUTRALYS® S85F clearly demonstrates intrinsic antioxidant activities as can be observed from its scavenging activity toward the peroxyl radicals (ORAC), scavenger of the superoxide radicals (NBT), hydroxyl radical scavenging (HRS), prevention of lipid oxidation (MDA) and nitric oxide scavenging (NO) capabilities. Based on our results NUTRALYS® S85F will provide oxidative stability to food products and health benefits to the consumer.