Development and validation of an analytical method to ensure quality requirements of hydrolysed proteins intended for agricultural use as biostimulants

Abstract

Plant biostimulants, as defined by EU Reg.1009/2019, represent a promising solution to increase agricultural production, improving the efficiency of fertilizers and thus reducing their environmental impact. At present, protein hydrolysates are considered the main class of biostimulants as they increase yield and product quality of various crops. Several sources of proteins are used to produce protein hydrolysates, but in this context of environmental protection and green economy, the development of biostimulants from industry by-products is of great interest. Industry by-products of animal origin (ABPs) are classified into three categories based on the degree of risk for animal, human and environmental health, according to EU Reg. 1069/2009. In particular, tanneries are known to produce large quantities of Category 3 ABPs, which are associated with no health risk. According to EU Reg. 142/2011, ruminant proteins derived from ABPs Category 3 must be hydrolysed to a molecular weight below 10.000 Daltons (Da). This research aims to set up and validate an analytical method for a reliable determination of the high limit molecular weight of hydrolysed proteins derived from Category 3 ABPs. A gel filtration chromatography method was developed and validated by evaluation of linearity, repeatability, reproducibility (intermediate precision) and measurement uncertainty. The validation process was carried out according to Eurachem and ISO guidelines and performance characteristics were further verified by testing the method in a second and independent laboratory. Acceptance criteria of linearity R2>0.990, repeatability RSD%≤5, intermediate repeatability RSD%≤10 and uncertainty measurement U ≤ 20% were met. Therefore, the method was considered suitable to assess the compliance of the hydrolysed proteins to 10.000 Da limit in biostimulants from animal by-products.

Keywords

• Biostimulant
• Protein hydrolysate
• Animal by-products
• Gel filtration chromatography
• Reliable analytical method
• molecular weight cut-off