Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment

Abstract

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

Keywords: anti-inflammatory activity; anti-tyrosinase activity; antioxidants; bioactive peptides; bitter taste; rice by-product.

FIGURE 1

(A,B) Protein and (C,D) antioxidant activity quantifications in (A,C) Alcalase and (B,D) Protamex processes at in-creasing incubation time (up to 120 min) and in 1 and 2 L bioreactor working volume. Conditions 1–6 are reported in Table 1. Protein results are expressed as g of bovine serum albumin equivalent (g BSA eq/L) and antioxidant activity as g of ascorbic acid per liter (g AA eq/L) ± SD (n = 3). ND, not digested initial by-product. Different letters indicate a statistically significant difference (one-way ANOVA test followed by post hoc corrected two-tailed Tukey test, p < 0.05) between the same type of data (proteins, antioxidant activity), from the lowest (a) to the highest (e).

FIGURE 2

(A,B) Protein content, (C,D) antioxidant, (E,F) anti-tyrosinase, and (G,H) anti-inflammatory activities in (A,C,E,G) Alcalase and (B,D,F,H) Protamex processes at 55°C, 120 min, 0.5 U enz/g proteins, 2 L reaction volume (condition 5). Data of liquid samples and of related lyophilized powders are reported; lyophilization was not performed on ND and TD. Fractions were named as follows: ND, not digested initial by-product; TD, total digestate condition 5; retentates of 0.2 μm (R 0.2 μm), 8 kDa (R8), 5 kDa (R5), and 1 kDa (R1), and 1 kDa permeate (P1). Spectrophotometric results are expressed as g of standard compound (BSA, bovine serum albumin; AA, ascorbic acid; KA, kojic acid) equivalent per liter. Cell-based assay results are expressed as fold of NFκB induction in presence of TNFα; results are corrected according to cell viability. Data represent the mean ± SD (n = 3). Different letters indicate a statistically significant difference (one-way ANOVA test followed by post hoc corrected two-tailed Tukey test, p < 0.05) between the same type of data (proteins, antioxidant, anti-tyrosinase, and anti-inflammatory activities), from the lowest (a) to the highest (f).

FIGURE 3

Elaboration of free amino acid contents in total digestate (TD condition 5) and peptide fraction samples from (A,B,E,F) Alcalase to (C,D,G,H) Protamex treatments, in liquid and lyophilized forms. (A–D) Predicted nutritional/structural characteristics and (E–H) taste property contributions were quantified in μmol/L, by elaboration of data reported in Supplementary Table 1. TOT, total amino acid content; EAA, total essential amino acid; BCAA, total branched-chain amino acid; HAA, total hydrophobic amino acid. Data represent the mean ± SD (n = 3). Different letters indicate a statistically significant difference (one-way ANOVA test followed by post hoc corrected two-tailed Tukey test, p < 0.05) between the same type of data (A–D: TOT, EAA, BCAA, HAA; E–H: total levels), from the lowest (a) to the highest (e).

FIGURE 4

Sub-fractionation of samples (A) Alcalase R1, (B) Alcalase P1, (C) Protamex R1, and (D) Protamex P1, by FPLC size exclusion chromatography. Sub-fractions are indicated by numbers. Continuous line: absorbance at 280 nm; dashed line: absorbance at 254 nm.