Production of antioxidative protein hydrolysates from corn distillers solubles: Process optimization, antioxidant activity evaluation, and peptide analysis

Highlights

• Response models maximized yield, degree of hydrolysis, and antioxidant activities.

• Artificial neural network models forecasted responses with greater accuracy.

• Peptide fraction of < 3 kDa and 3–10 kDa exhibited stronger antioxidant activities.

• Novel antioxidant corn and yeast peptides including denovo were characterized.

• Predicted active fragments and hydrophobicity of peptides revealed antioxidant effects.

Abstract

Bioprospects of corn distillers solubles containing a unique thermally treated blend of corn and yeast from ethanol industries were evaluated. This includes alcalase hydrolysis optimization of protein concentrate, measurement of antioxidant activities, and peptide analysis. Response surface methodology (RSM) computed optimum values of enzyme: substrate ratio and incubation time, 5.2% (w/w) and 9.3 h. Besides, artificial neural network proved to be a good predictor of responses (R2 ~ 0.94–0.97) in agreement with RSM. The optimum RSM values maximized antioxidant yield, degree of hydrolysis, DPPH-, hydroxyl-, and superoxide-radical activities. The optimal PH and its three ultrafiltered fractions revealed higher scavenging activities against radicals of DPPH (76.15–85.66%), hydroxyl (81.26–91.91%), and superoxide (35.21–54.06%) at 5.0, 5.0, and 3.0 mg. mL⁻¹, respectively. The foremost corn (QQPIIGGA, LPPYLSPA, SNIPLSPL, NPILQPY) and yeast (NIIPSPI) peptides were identified. The greater antioxidative characteristics might be due to more hydrophobic amino acids. The results are potent and pave the way forward for alternative valorized product streams in bioethanol industries.

Introduction

Protein hydrolysates (PHs) and peptides from food and grain by-products have recently fascinated appeal for antioxidants due to quick absorption, smaller molecular weight (M_w), and minor aftereffects (Jin et al., 2016). The antioxidants prevent the oxidation of feed, pet food, and food instigated by free radicals (Hu et al., 2020). The plant-based antioxidative PHs and peptides have been used to stabilize these free radicals, chelate Cu^+2, and Fe⁺² ions, and deactivate reactive nitrogen- and oxygen species (Zou et al., 2016). These reactive species stimulate oxidative stress that can cause diabetes, arteriosclerosis, aging, and chronic diseases (Jin et al., 2016). Presently, butylated hydroxyanisole, tert-butylhydroquinone, propyl gallate, and butylated hydroxytoluene are accessible as synthetic antioxidants (Hu et al., 2020), whose applications are strictly confined under control limits owing to their toxic effects. Therefore, the wastes and agrifood leftovers can be promising sources of proteins and antioxidant peptides (Zhu et al., 2019, Guo et al., 2020, Hu et al., 2020).

Corn distillers soluble (CDS) from novel selective milling technology (SMT) is an undervalued and underexplored by-product, which is generated with an annual amount of 16000 MT (Sharma et al., 2021). The CDS comprises corn and yeast proteins with a protein content of 16.0–22.87% (dry basis) (Adom et al., 2014, Liu, 2017). These proteins were temperature abused during their routine production steps. Moreover, in CDS, heat treatments cause strong binding between proteins and neutral detergent fiber, which leads to structural alterations in the proteins, thereby influencing bioavailability (Reis et al., 2017). Currently, most of the studies on CDS focuses on value-addition to its oil, carbohydrates and glycerol constituents (Reis et al., 2017). The unknown potential of CDS proteins could be retrieved and delivered to high-end applications. The major usage of CDS in the feed area necessitates large energy-requiring evaporation stages, posing a financial hurdle for bioethanol producers. In our recent, protein fraction (PF) was extracted with a content of about 55–57% from CDS of SMT (Sharma et al., 2020). More than half of the hydrophobic and aromatic amino acids (AA) in CDS signified a strong potential for antioxidative PHs (Liu, 2017, Zhu et al., 2019, Sharma et al., 2021). Therefore, this unique blend of corn and yeast proteins of heat-treated CDS in ethanol industries needs to be examined for antioxidant prospects.

Enzymatic hydrolysis, due to enzyme properties such as broad specificity, exclusive functions, and cheapness, is the principal method to release the active peptides at mild conditions (Wang and Mejia, 2005). The PHs possess higher with improved bioactivity, preserved quality, and better functional characteristics than raw protein (Luo et al., 2014). Considering intricate nature of CDS proteins, alcalase may generate the best quality antioxidant PH due to its economic, wide availability, and extensive specificity (Liu et al., 2015). With a circular economy in mind, PHs and peptides from CDS that maintain antioxidant activities could be recovered and valorized for use as functional constituents of high-value products in the feed, food, nutraceutical, and cosmetic industries. Few sequences have been recognized as antioxidant peptides from corn by-products (Zhou et al., 2015, Jin et al., 2016, Zhang et al., 2019) that can offer a structural prototype to generate functional peptides for industrial purposes. For instance, LPF, LLPF, and FLPF from corn gluten meal (CGM) with exceptional radical scavenging activities (RSAs), comprising 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide, hydroxyl, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) have been described (Zhuang et al., 2013b). Jin et al. (2016) purified three novel peptides, CSQAPLA, YPKLAPNE, and YPQLLPNE from CGM hydrolyzed by alcalase and flavourzyme sequentially. The results suggested that CSQAPLA had superior RSAs against superoxide (IC₅₀ = 0.39 mg.mL⁻¹) and DPPH (IC₅₀ = 0.12 mg.mL⁻¹) radicals. Recently, Liu et al. (2020) studied antioxidant peptides from CGM hydrolyzed with alcalase 2.4 L and protex 7 L. They identified antioxidant peptides, LAYPQ, AYLQQQ, YGPQ, LNSPAY, AYPGPQ, PPY, AYPQ, LSPY, and TYSGPK with powerful ABTS scavenging ability. Current studies of corn PHs exists on CGM and corn germ meal by-products (Li et al., 2010, Tang et al., 2010, Lin et al., 2011, Zhuang et al., 2013a, Wang et al., 2014, Wang et al., 2015, Jin et al., 2016, Wang et al., 2019, Zhang et al., 2019, Guo et al., 2020, Liu et al., 2020). But antioxidant PHs and peptides of CDS were unexploited. The potential of CDS proteins has been observed in our studies (Sharma et al., 2020, Sharma et al., 2021). Besides, the present study will expand the scope of adopting other similar resources usually generated in various industrial processing facilities.

It is therefore useful to evaluate the potential of deriving antioxidative peptides and PHs from CDS of SMT. These antioxidant compounds can be remarkable to develop an overarching framework for generating new avenues of CDS. Thus, the objectives were framed as: (1) to optimize the enzymolysis of protein concentrate (PC) with alcalase through response surface methodology (RSM) for producing antioxidative PHs, (2) to characterize the PHs and their different molecular weight fractions in terms of antioxidant activities, (3) to identify and analyze the peptides in the most active fraction of PH. The antioxidant compounds from CDS might be useful as additives for safe storage and supply of food products, and as ingredients in animal feed.