Interactions of proteins and phenolics: A case for pulse proteins and onion flavonoids

Abstract

Researchers and the food industry are interested in the development of new plant-based products rich in proteins, considering the environmental, religious, and health concerns of consumers against animal-based proteins. Essentially, plant-based proteins have affordable prices, advantageous qualities, are renewable and biodegradable, and cause comparatively milder allergic reactions (X. Zhang, Wang, et al., 2022). Size, shape, amino acid composition and sequence, net charge and distribution, hydrophobicity/hydrophilicity ratio, secondary, tertiary, and quaternary structures, molecular flexibility/rigidity, and the ability to interact/react with other components are among the physical and chemical characteristics that control protein functionality (Damodaran, 2008). Functional properties of pulse proteins are significant topics for the food industry as alternative protein sources started to be used in different products such as gluten-free products or vegan mayonnaise (Du et al., 2014). Proteins in foods differ in (1) protein amount, (2) amino acid composition, (3) digestibility, (4) rate of protein digestion, and (5) capacity to transfer amino acids for protein synthesis. Animal proteins are efficiently utilized for protein synthesis, contain all of the essential amino acids, and are often well digested. Therefore, animal proteins are regarded as “high quality” (Gilbert et al., 2011). On the other hand, plant-based proteins cannot be widely used in foods as of animal proteins. The use of plant proteins including soy and other legume and oilseed proteins, is limited in conventional foods. These proteins do not function as effectively as animal proteins in most food products, even though they are similar combinations of proteins. There is a lack of knowledge on the precise molecular characteristics of proteins that give rise to several beneficial functions in foods (Damodaran, 2008), and still many research studies are being conducted to overcome these challenges in plant-based proteins and improve their nutritional and techno-functional qualities.

Proteins and phenolic compounds coexist naturally in many foods or food matrices; however, phenolics do not interact directly with proteins since they are found in vacuoles in plants (Rohn, 2014). For phenolics to interact with proteins, plant tissue must be broken down by the effects of food processing and gastrointestinal digestion. Thus, proteins and phenolic compounds in the same environment interact in different ways (Le Bourvellec & Renard, 2012). Protein–phenolic interaction is classified as reversible or irreversible. The protein–phenolic interactions are affected by covalent and non-covalent (hydrogen bonding, hydrophobic interactions, and Van der Waals forces) interactions (Ozdal et al., 2013).

Onion and onion skin contain high levels of antioxidant flavonoids. The most dominant flavonoid in onions/onion skin is flavonols, mainly quercetin/derivatives (a yellow/brown color) (Benítez et al., 2011; Günal-Köroğlu, Turan, & Capanoglu, 2022). It is more difficult for the hydrophobic polyphenols, for instance, quercetin or quercetin-predominant bioactive compounds, to be widely used because of their limited water solubility and bioaccessibility and they quickly degrade when exposed to light, heat, and oxygen (Chen et al., 2018; Lin et al., 2023; X. Zhang, Wang, et al., 2022). Proteins as biopolymer carrier systems for hydrophobic bioactives offer advantages such as surface modification, diverse functional properties, high nutritional value, and eco-friendly products (Lin et al., 2023).

In the literature, studies on protein–phenolic interactions are often conducted by choosing and analyzing food-specific pure proteins and phenolic compounds or phenolic extracts. Eventually, both the dominant protein and phenolic in foods that are generally consumed or present together have been selected and investigated. This paper specifically focuses on the studies dealing with the interaction of pulse proteins with quercetin. The effects of pulse protein–quercetin or onion skin extract complexes in different matrices (emulsions and aqueous solution) as a result of incubation under certain conditions have been investigated (Chen et al., 2018; Fu et al., 2023; L. Han, Lu, et al., 2021; Liao et al., 2022; Lin et al., 2023; Parolia et al., 2022; X. Zhang, Wang, et al., 2022; Y. Zhang, Wang, et al., 2022). Investigations regarding the digestibility of protein–phenolic complexes and bioaccessibility of quercetin have been also covered (Chen et al., 2018; Liao et al., 2022; Lin et al., 2023). The effect of protein–quercetin/quercetin derivative interaction is summarized in Table 1.

The fluorescence quenching method is widely used to examine non-covalent protein–phenolic interactions, primarily regarding the shifts in tryptophan emission spectra (Y. Zhang, Wang, et al., 2022). The maximum wavelength of soy, pea, lentil, or black bean protein (λ_max) was shifted to a lower wavelength (a blue shift) due to the protein–quercetin/onion skin extract interaction since tryptophan in the protein shifted to a hydrophobic environment. Further, a reduction in the intensity of intrinsic fluorescence of pulse proteins was reported with the increase in quercetin or onion skin extract concentration (Fu et al., 2023; Günal-Köroğlu, Yılmaz, et al., 2022; L. Han, Lu, et al., 2021; Parolia et al., 2022; X. Zhang, Wang, et al., 2022; Y. Zhang, Wang, et al., 2022). It is possibly because of the intense hydrophobic interactions of the aromatic amino acid residues on the protein surface (L. Han, Lu, et al., 2021; Parolia et al., 2022).

The fluorescence intensity data were used for the Stern–Volmer equation to pinpoint the protein–phenolic interaction mechanism (Y. Zhang, Wang, et al., 2022). It was also analyzed that the quercetin/yellow onion skin extract-induced fluorescence quenching was in the form of static quenching (Günal-Köroğlu, Yılmaz, et al., 2022; L. Han, Lu, et al., 2021; X. Zhang, Wang, et al., 2022; Y. Zhang, Wang, et al., 2022). The development of a non-fluorescent and ground-state stable compound between the fluorophore and the quencher initiates static quenching (L. Han, Lu, et al., 2021; Liao et al., 2022). It has been emphasized that quercetin has a higher binding affinity to pulse proteins than curcumin/resveratrol (X. Zhang, Wang, et al., 2022), quercitrin/rutin (Fu et al., 2023), and rutin/ellagic acid (Parolia et al., 2022). Possibly, the presence of glycoside and the absence of C-3 active hydroxyl groups increased the steric hindrance of flavonoids (quercitrin and rutin) (Fu et al., 2023; Parolia et al., 2022). Moreover, more hydroxyl groups in the phenolic compound and a catechol moiety of the B ring of quercetin had greater binding to the lysine side chains in proteins (Parolia et al., 2022).

Based on the findings of fluorescence studies, thermodynamic factors such as ΔH, ΔS, and ΔG can be used to infer the non-covalent forces that underlie the interaction between pulse protein and quercetin/onion skin extract (Günal-Köroğlu, Yılmaz, et al., 2022; X. Zhang, Wang, et al., 2022). As indicated in Roos and Subramanian theory (Ross & Subramanian, 1981), pea protein–quercetin/quercetin derivatives interaction was mainly driven by spontaneous hydrogen bonding and Van der Waals forces (ΔH˂0, ΔS˂0, and ΔG˂0) (Fu et al., 2023; X. Zhang, Wang, et al., 2022). Both hydrophilic sides (hydroxyl groups), which can form hydrogen bonds, as well as hydrophobic sides (aromatic rings and aliphatic groups), which give these molecules their lipophilic nature, may contribute to the pea–polyphenol interactions (X. Zhang, Wang, et al., 2022). Nevertheless, it was demonstrated that lentil protein ∼ yellow onion skin phenolic extract or soy/black bean ∼ quercetin complex formation was predominantly driven by hydrophobic forces (ΔH˃0 and ΔS˃0) (Günal-Köroğlu, Yılmaz, et al., 2022; J. Han, Du, et al., 2021; Liao et al., 2022; Y. Zhang, Wang, et al., 2022).

Pulse protein–quercetin interactions cause changes in the secondary structure of proteins. The amide hydrogen molecule's C=O stretching caused a decrease in the number of α-helices in the molecule (L. Han, Lu, et al., 2021). Moreover, the disruption of intramolecular hydrogen bonds caused the protein polypeptide chain to unfold and rearrange (Fu et al., 2023). As a result, the interior hydrophobic groups are partially exposed in the polypeptide chain. Therefore, unfolded proteins support the hydrophobic interactions and hydrogen bonding between pulse proteins and quercetin (Fu et al., 2023; L. Han, Lu, et al., 2021; Parolia et al., 2022).

It was reported that due to protein–phenolic interactions, surface hydrophobicity (H₀) of the protein was decreased (Fu et al., 2023; L. Han, Lu, et al., 2021; Lin et al., 2023; Parolia et al., 2022). The decrease in the surface hydrophobicity actually appears as a result of the decrease in the hydrophobic regions on the protein surface due to the interaction with phenolics (Fu et al., 2023; L. Han, Lu, et al., 2021; Lin et al., 2023). On the other hand, the increase in the negative value of ζ-potential was explained by the substantial amount of ionized carboxyl groups (-COO^-) on the surface of protein molecules above their isoelectric point (Liao et al., 2022). It has been explained that heat treatment had no statistically significant effect on the ζ-potential of the protein–quercetin complex (Liao et al., 2022). However, significant effects of ultrasound application were reported, and the generation of electrically stable SPI dispersions were demonstrated (Lin et al., 2023). On the other hand, ζ-potential may not change since polyphenols are mostly found inside the hydrophobic interior of nanoparticles instead of the surface (X. Zhang, Wang, et al., 2022).

It has been noted that there is an improvement in the emulsion properties of pulse proteins with the interaction of quercetin (Fu et al., 2023; L. Han, Lu, et al., 2021; Y. Zhang, Wang, et al., 2022). Partially unfolded and more flexible proteins may enhance their emulsifying and foaming abilities because they can easily diffuse to the air–water or oil–water interface and lower the surface tension of the air bubbles or oil droplets (Y. Zhang, Wang, et al., 2022). Phenolic compounds were able to improve the protein-derived emulsion properties up to a certain concentration. Since the presence of more phenolic compounds in the environment saturated the interaction (excess of phenolic compounds), the emulsion properties were adversely affected. The emulsions investigated in the literature were mostly oil-in-water emulsions, which have a 5% (J. Han, Du, et al., 2021), 20% (Fu et al., 2023), and 25% (Y. Zhang, Wang, et al., 2022) oil content. However, Günal-Köroğlu, Turan, and Capanoglu (2022) found a distinct influence of yellow onion skin phenolic extract on the emulsion (50% oil) and foaming properties. The impact of higher amounts of phenolic compounds in an emulsion may have been more noticeable with a higher proportion of oil, and it was found that phenolics had a great impact on the emulsification/foaming properties of lentil proteins, depending on the concentration. At low protein concentration or higher phenolic concentration, foaming or emulsion properties were adversely affected due to the increase of free phenolics at the interface (noninteracting), decrease in surface hydrophobicity of proteins, and protein unfolding (Günal-Köroğlu, Turan, & Capanoglu, 2022).

An increase in the solubility of quercetin has been observed with ultrasound or high-temperature applications (Chen et al., 2018; Liao et al., 2022; Lin et al., 2023) thanks to the formation of colloidal complexes in the solution (Lin et al., 2023). A conformational change in proteins provided more hydrophobic groups on their surfaces, and the number of available binding sites was raised for quercetin (Liao et al., 2022). Free and bound quercetin in pulse protein solutions generally describes the total quantity of quercetin. The bound form of quercetin dissolves as protein–quercetin complexes, but the free form is immediately soluble in water (Liao et al., 2022). Further, Chen et al. (2018) and Liao et al. (2022) implied that all proteins (soy, whey, and sodium caseinate) might be utilized to increase quercetin solubility but the efficiency of each protein type varied. The capacity of quercetin to attach to the protein surface and create soluble protein–quercetin complexes is thought to be the cause of this solubility improvement.

Due to its weak water solubility, and chemical transformation under physiological conditions, quercetin has a relatively low bioaccessibility primarily in aqueous-based foods, such as beverages (Chen et al., 2018; Liao et al., 2022). Oil-in-water emulsions are especially well-suited for use as excipient foods (Chen et al., 2018). The poor water solubility of quercetin, which lowers its transport into enterocytes and subsequent absorption, is largely responsible for its limited bioaccessibility. Crystalline forms of free quercetin penetrate the gastrointestinal tract the majority of the time (Lin et al., 2023).

During digestion, quercetin must be partitioned into bile-salt-mixed micelles in the intestinal phase to be available for absorption. The extreme acidity of gastric conditions can cause the breakdown of quercetin's skeletal structure. Quercetin should be shielded by the nanocomplexes in the gastric stage so that it can be released in the intestine (L. Han, Lu, et al., 2021). The portion of quercetin that is liberated from the food matrix to the aqueous micelle fractions and readily absorbed by the intestinal mucosa is known as quercetin bioaccessibility (Lin et al., 2023). Additionally, it is likely that the peptides produced by proteolysis interact to boost micellization and increase the solubility of quercetin (Chen et al., 2018; Günal-Köroğlu, Yılmaz, et al., 2022; L. Han, Lu, et al., 2021; Liao et al., 2022; Lin et al., 2023). Quercetin in emulsions had higher solubility and bioaccessibility. Since the solubility of quercetin within the hydrophobic interiors of the mixed micelles was increased by protein interaction, proteolysis may have also led to micellization, which encourages the solubilization of quercetin (Chen et al., 2018). Günal-Köroğlu, Turan, and Capanoglu (2022) reported that the predominant phenolic compounds in yellow onion skin phenolic extracts are quercetin 7, 4-diglucoside and quercetin. Nevertheless, quercetin in yellow onion skin phenolic extract could not be detected in all solution systems (phenolic solution, lentil protein–phenolic solution, and lentil protein–phenolic emulsion) after the intestinal phase, and the amount of quercetin 7, 4-diglucoside decreased significantly.

Although there are many studies on animal protein–phenolic interactions in the literature, studies on pulse proteins are scarce and mostly limited to soybeans. Some phenolics have a higher binding affinity to proteins, and the binding affinity of phenolics may also vary with different proteins. Along with the changes in protein characteristics, the mechanism of pulse protein–quercetin/quercetin derivative interactions occurs in various ways. For this reason, it is not possible to derive concrete results and thus, the existing data in the literature are conflicting. Therefore, it is not possible to precisely forecast how plant-based proteins and quercetin/quercetin derivatives will behave in foods; however, research on model systems can provide broad insights into these interactions.

The emulsion or foam characteristics were adversely influenced by the presence of non-interacting (free) phenolics at the interface over a certain limit of phenolic concentration, as the interacting sites of the proteins were decreased significantly and the free phenols in the medium were increased. Therefore, it can be concluded that up to a specific concentration limit, phenolic compounds may support the emulsion/foam qualities, while phenolics over a certain concentration have a negative impact. Meanwhile, the solubility and bioaccessibility of quercetin differed in different matrices, for example, emulsion or aqueous solution. In practical terms, phenols are mostly broken down during digestion (unstable), and the complexation of certain phenolics with proteins may increase their bioavailability, according to the literature on model systems. The presence of proteins in different model systems significantly affects the bioaccessibility of each phenolic compound, thus greatly changing the total phenolic content and antioxidant activity after the gastric and intestinal phases. Since the separation of phenolics from the protein–phenolic complex takes time under the digestion conditions, they are released after a set amount of time and, eventually, have a higher bioavailability.

Phenolic additives are added to traditional foods to obtain functional foods. It seems that the form of phenolic additives (extract or pure), and the food matrix are very important. Although these model studies provide an insight about the effects of pulse protein–quercetin/phenolic extract interactions on the food matrix, studies on real food matrixes are limited in the literature. The ratio of phenolics to proteins as well as their structural properties should be carefully taken into account when targeting functional attributes. Since proteins and phenolics interact leading to changes in their functional qualities and bioactivities, it is important to consider these interactions when designing functional foods.

Deniz Günal-Köroğlu: Conceptualization; formal analysis; investigation; writing – original draft. Esra Capanoglu: Conceptualization; project administration; resources; supervision; writing – review & editing

There are no conflicts to declare.

None declared.