The Influence of Flavonoid Dihydroquercetin on the Enzymatic Processes of Dough Ripening and the Antioxidant Properties of Bread

Abstract

Bread is an integral part of the diet of the world population. Development of bread enriched with biologically active substances, including antioxidants, could be good nutritional support for human health. Among well-studied antioxidants, we can highlight dihydroquercetin, a flavonoid with outstanding antioxidant properties, such as anti-inflammatory activity, immunostimulatory properties, anti-cancer properties, and others. At the same time, the technology of bread enrichment must consider the possible negative effects of the additive on the technological processes and properties of the final product. The present work was carried out to evaluate the effect of dihydroquercetin on the enzymatic processes occurring during dough maturation and the antioxidant properties of the finished bread. Dihydroquercetin was added in amounts of 0.05 g, 0.07 g, and 0.1 g per 100 g of wheat flour and fermented with commercial baker’s yeast (Saccharomyces cerevisiae). The kinetics of pH and total titratable acidity (TTA) during dough fermentation showed that dihydroquercetin caused slight slowing of enzymatic processes. However, the dosage of dihydroquercetin did not cause statistically significant changes in the yeast concentration, which reached a level of 108 KOU/g after 2 h in all dough samples. Loss of dihydroquercetin during fermentation was established at a level of 20–25%. At the same time, an increase in the total amount of flavonoids in the dough after 2 h of fermentation and an increase in values of antioxidant activity were noted. The antioxidant properties of the bread also increased when it was enriched with dihydroquercetin (about 3.5–4 times) despite the fact that the total quantitative loss of antioxidant in the technological process was considerable (about 40%). A protective effect of the bread matrix on flavonoids during digestion was shown. Dihydroquercetin loss was about 25% regardless of the amount applied. This work clearly showed that addition of dihydroquercetin to a bread formulation represents a promising strategy for increasing the antioxidant properties of bread.

1. Introduction

Bread is the essential component of most people’s diet. Bread consumption depends on habits, social factors, and economic opportunities, but, on average, it is 20–25% of the total food consumed. Bread is also of great psychophysiological importance as a cheap source of energy and some nutrients necessary for normal human life activities [1,2,3]. However, the general analysis of bread nutritional value indicates a chemistry imbalance. Use of natural ingredients as a source of antioxidants and functional additives in bread and other bakery products is a global trend. Numerous studies in the specialized literature highlight an improvement in the nutritional characteristics of wheat flour through incorporation of new functional ingredients and development of healthy nutritional products [4,5,6,7,8,9]. In this sense, bakery products enriched with dietary fiber, amino acids, and bioactive compounds from flours obtained from whole grains or pseudo-cereals, which prevent diseases associated with metabolic syndrome, such as cardiovascular diseases, arteriosclerosis, and colon cancer, are an alternative [4,10]. Bread enrichment with vegetable antioxidants could be one of the possible ways to develop a range of improved types of bread [4,5,10,11].

Antioxidants are a natural way to provide human cells adequate protection against reactive oxygen species. Although oxidation processes in the human body are a natural biological reaction, the resulting free radicals can damage cell membranes and other cell structures [12,13,14,15,16,17]. The damage caused by overloading the body with free radicals over time can become irreversible and lead to certain diseases, including heart disease, liver disease, and certain cancers (such as oral, esophageal, stomach, and intestinal cancers). Therefore, antioxidants are of interest to many scientists. The protective effect of antioxidants is actively studied worldwide. A huge group of antioxidants is plant polyphenols, including flavonoids, which are increasingly being considered as functional food supplements [18,19,20,21].

Lee et al. reported that flavonoids have the most potent antioxidant activity because their chemical structures contain a ο-diphenol group, a 2–3 double bond conjugated to the 4-oxo function, and hydroxyl groups at positions 3 and 5. Flavonoids efficiently absorb hydroxyl and peroxyl radicals, form complexes with metals, and inhibit metal-initiated lipid oxidation [12].

According to various studies, a wide range of pharmacological activities of flavonoids, including antioxidant, antiangiogenic properties, antidiabetic, cardioprotective, neuroprotective, and anti-Alzheimer’s disease, have been identified, mainly due to their hydroxylation degree, structural class, other substitutions and conjugations, polymerization degree, and metal chelate activity. Recent studies show that higher intake of flavonoids in a diet is inversely related to threat of mortality and some types of depression [22,23,24,25,26].

Dihydroquercetin is a flavononol, a well-studied antioxidant extracted from the rump of larch, with an extensive list of pharmacological properties, such as anti-inflammatory activity, immunostimulatory properties, anti-cancer properties, and others. It is a powerful antioxidant with a well-documented effect in prevention of several malignancies in humans. Dihydroquercetin has shown promising inhibitory activity against inflammation, malignancies, microbial infection, oxidative stress, cardiovascular disease, and liver disease. The anti-cancer activity is relatively significant compared to other activities investigated in vitro and in vivo, with little or no side effects to normal healthy cells [27,28,29,30,31]. Dihydroquercetin is associated with antioxidant activity and capillary protecting action. It is shown that dihydroquercetin has antioxidant activity in vivo, which was evaluated in Wistar rats suffering from tetrachloromethane induced hepatitis [27,28,29]. Antioxidant activity of dihydroquercetin is also exhibited through its neuroprotective effects via inhibition of oxidative neuronal injuries in rat cortical cells, which was supported by DPPH radical scavenging activity and inhibition of lipid peroxidation [27]. Furthermore, dihydroquercetin is also associated with inhibition of leukocyte infiltration and COX-2 and iNOS in the brain [27]. Several studies have been reported that dihydroquercetin shows significant hepatoprotective activity in rotenone-induced hepatotoxic rats. The double bond in C2–C3 position in the C ring of dihydroquercetin is essential for hepatoprotective activity [27]. Additionally, hepatoprotective efficacy of dihydroquercetin has also been assessed in mice with acute liver injury. Dihydroquercetin decreases liver lesions, vacuole formation, neutrophil infiltration, necrosis, and increases activity of antioxidant enzymes [27]. Enrichment of bread with dihydroquercetin can be a nutritional support for the human body provided that the bioavailability of the antioxidant is ensured.

As for the beneficial effect of a compound, one of the main concerns is its bioavailability. Generally, the low water solubility of pure dihydroquercetin supplement largely limits its absorption in the body. Food matrices can act as efficient delivery systems for dihydroquercetin and affect its absorption.

Quality of bread as a food matrix for antioxidant enrichment depends on many factors. Biochemical transformations caused by yeast fermentation processes play a major role in baking technology. Baking yeasts Saccharomyces cerevisiae, as biological leavening agents, form the porous structure and define consistency of bread but also generate its flavor and taste. Yeast is not always able to maintain high activity and fill the food system with necessary metabolic products. Active development of new formulations of bakery products, including those enriched with vegetable polyphenols, determines the need to study the impact of additives on the technological properties of yeast [1,3,10,32].

Numerous studies in the specialized literature demonstrate conflicting data on the effect of polyphenols on yeast metabolic processes. Several studies demonstrate dose-dependent inhibitory effects of some polyphenols on fermentation activity, ethanol synthesis by yeast, and yeast biomass growth. Other authors convincingly prove the ability of S.cerevisiae yeast to convert certain phenolic compounds during fermentation, for example, to convert ferulic acid to 4-vinylgvaicol and coniferyl aldehyde to coniferyl alcohol. However, the metabolic features of these processes are still poorly studied [2,32,33].

Developing a new type of bread implies minimization of a possible negative impact of a phenolic antioxidant on the processes of bread production and preservation of the bioactive properties of the antioxidant as part of the food matrix of bread.

Our research is aimed at examining the effect of dihydroquercetin on the technological properties of Saccharomyces cerevisiae during dough fermentation and establishing formation of antioxidant properties of bread when enriched with dihydroquercetin (Figure 1). It has been suggested that the bread matrix can maintain the amount and bioactive properties of dihydroquercetin and ensure its bioavailability.

2. Materials and Methods

2.1. Materials

Taxifolin is the active ingredient of dihydroquercetin, manufactured by Taxifolia LTD, Belgorod. Certificate of state registration No. RU 77.99.003 E.018404.05.11. Purity is 98–99%. Commercially available ingredients, including dried yeast Saccharomyces cerevisiae baking yeast “Saf-momentum”, wheat flour produced by «Soyuzpichsheprom» LLC, Chelyabinsk, were used for bread making.

2.2. Preparation of Bread

Bread was prepared in a steamless way. The dough was kneaded for 10 min from the total amount of raw materials and water to a dough moisture content of 45% according to the recipe.

The calculated recipe included 1000 g of wheat flour, 20 g of yeast, 10 g of salt, and 530 mL of water. In order to study the effect of dihydroquercetin on fermentation processes, a prescription dosage was chosen (0.05 g, 0.07 g, and 0.1 g per 100 g of flour).

The temperature of the dough after kneading was 31 ± 1 °C degrees C. The fermentation process was carried out for 120 min at a temperature of 30 ℃. The resulting dough was divided into an experimental sample and control sample.

Experimental laboratory bread, 300 g, was baked at a temperature of 220 °C [34].

After baking, the bread samples were left to cool down at room temperature and examined 3 h after baking.

2.3. Evaluation of the Dihydroquercetin Effect on Fermentation Processes

Dough fermentation was followed by pH, total titratable acidity (TTA), and microbiological analyses conducted soon after production (0 h) and at the end of fermentation (2 h). Drop in pH was measured electrometrically using the pH meter. TTA was determined by titration with 0.1 N NaOH (expressed in terms of mL of NaOH) on 10 g of dough.

The microbiological counts were performed on 15 g of each sample of dough. Dough samples were suspended in 135 mL of Ringer’s solution, homogenized by means of a stomacher for 2 min, and then serially diluted. Inoculation, cultivation, and incubation of the different microbial groups occurred as follows: total yeasts were spread plated on yeast extract peptone dextrose (YPD) agar, incubated at 28 °C for 48 h. Plate counts were performed in triplicate.

2.4. Extraction of Bioactive Compounds

The freshly prepared dough and the dough at the end of the fermentation process (2 h) were lyophilized (48 h) and milled to pass through a 30 mesh sieve for further analysis.

Bioactive compounds from the freeze-dried dough were extracted in 70% aqueous methanol as reported in Skendi et al. [35]. Freeze-dried dough (1.5 g) was first homogenized in 4 mL of 70% methanol on a vortex for 30 s and then the extraction was performed in an ultrasonic bath for 15 min. Then, the sample was centrifuged at 2500× g for 15 min and the supernatant was collected. Extraction of the bioactive compounds from the bread samples was carried out at least in triplicate.

In the bread samples, the crumb was separated from the crust and lyophilized for 48 h. The extraction of biologically active substances was carried out in the crumb and crust + subcrustal layer separately. This approach was used to compare quantitative losses of biologically active substances in the crumb and crust and to establish the potential thermal stability of dihydroquercetin.

Extracts for determining the content of dihydroquercetin, total flavonoid, and antioxidant activity of bread samples were obtained according to the procedure by Arranz, S.& Calixto, F. S. [36] with a slight modification. Briefly, a 0.8 g sample of breadcrumbs was extracted using 20 mL of acidic aqueous methanol (MeOH:H2O 50:50, v/v, pH 2) followed by 20 mL acetone:water (70:30 v/v). For extraction, 1 h mechanical shaking was used for extraction at room temperature. Acid hydrolysis has been used to enhance the release of insoluble polyphenols bound to cell walls.

2.5. Chemical Composition and Antioxidant Properties of Dough and Breads

In the resulting extract, the mass fraction of dihydroquercetin was determined by HPLC method. The samples obtained after filtration by a Phenex 0.45 μm PTFE syringe filter were analyzed using a reserved-phase C18 Sunfire column (250 mm × 4.6 mm/5 μm; Waters, Wexford, Ireland) at 30 °C on a Shimadzu HPLC system (Shimadzu, Japan) coupled with a diode array detector (DAD). The sample injection volume was 20 μL. The flow rate was 1 mL/min. Two mobile phases (A: 1% acetic acid; B: 100% acetonitrile) were used in the ratio of 6:4. Dihydroquercetin was quantified by external calibrations ranging from 0.25 to 25.0 μg/mL. The detection limit (DL) for dihydroquercetin was 1.07 μg/mL. The quantification limit of (QL) for dihydroquercetin was 3.24 μg/mL.

The total flavonoid content was measured using the procedure described by Shafii et al. [37]. 0.5 mL of sample extract was mixed with 0.1 mL of 10% (w/v) ethanol solution of aluminum chloride, 0.1 mL of 1 M sodium acetate, and 4.3 mL of distilled water. After 30 min in the dark, absorption at 415 nm was measured using a spectrophotometer (SF 56, Russia). Quercetin (0.01–0.5 mg L−1; R2 = 0.997) was used as the standard. The total flavonoid content was expressed in mg EQ/g.

The antioxidant capacity of the freshly prepared dough and after two fermentations was evaluated according to DPPH methods. DPPH analysis was carried out on the basis of the research by Ragaee et al. [11]. Briefly, 200 μL of extract plus 800 μL methanol/water was added to 200 μL freshly prepared 0.1 m Mol/L of DPPH solution and the mixture was kept at room temperature in a dark room for 30 min. The absorbance was measured at 517 nm compared to the control sample (as 100%) and the percentage of scavenging effect was expressed as [1 − (A517 of sample/A517 of control)] × 100].

2.6. Potential Bioavailability of Dihydroquercetin

The potential bioavailability of dihydroquercetin was found in bread samples based on the bioavailability index (Ibav) by the method in [38,39] with some modifications.

In vitro digestion simulation included 3 stages:

Cleavage in the oral cavity. Five grams of bread crumb sample were mixed with 4 mL of α-amylase in SSF buffer (75 U/mL in the final mixture) and 1 mL of 7.5 mM CaCl2 solution. The mixture was vortexed for 15 s and then incubated for 15 min in a thermostatically controlled shaker at 37 °C.

Cleavage in the stomach. Samples obtained after the oral phase were mixed with 8 mL of pepsin in buffer (5000 U/mL in the final mixture), 5 μL of 0.3 M CaCl2 solution, 1.345 mL of water, and 1 M HCl to reach pH 2.5. The mixture was then incubated with stirring in a thermostatically controlled shaker at 37 °C for 2 h.

Cleavage in the intestine. Gastric chyme was mixed with 16 mL of pancreatin in buffer (100 U/mL for trypsin activity in the final mixture), 0.18 g of bile extract (10 mM in the final mixture), 80 μL of lipase (20 U/mL in the final mixture), 40 μL of 0.3 M CaCl2, and 3.48 mL of water. In order to reach pH 7.0, 1 M NaOH was added.

The resulting mixture was centrifuged and filtered through a membrane (0.45 microns).

$${\mathrm{I}}_{\mathrm{bav}}=\frac{{\mathrm{A}}_{\mathrm{conc}}}{{\mathrm{A}}_{\mathrm{orig}}}\times 100$$

(1)

where A_conc—is the amount of dihydroquercetin after the digestion process; A_orig—the amount of flavonoid before the digestion process.

2.7. Statistical Analysis

All experiments were conducted in triplicate batches with three individual samples. The mean values of the analysis were reported as the final results with standard deviation. ANOVA test, Duncan’s multiple range test, and Statistica.13 were used to analyze the data. The level of significance was established at p ≤ 0.05.

3. Results and Discussion

3.1. Evaluation of Dihydroquercetin Effect on Fermentation Processes

Acidification kinetics for the dough samples are presented in

Table 1. Kinetics of acidification during fermentation.

Samples	pH		TTA
	0 h	2 h	0 h	2 h
Control	5.69 ± 0.05	5.17 ± 0.02	0.90 ± 0.10	1.50 ± 0.10
Experimental sample 0.05	5.65 ± 0.03	5.30 ± 0.03	0.80 ± 0.10	1.25 ± 0.10
Experimental sample 0.07	5.65 ± 0.03	5.27 ± 0.02	0.80 ± 0.10	1.25 ± 0.10
Experimental sample 0.1	5.63 ± 0.04	5.40 ± 0.02	0.75 ± 0.20	1.20 ± 0.10

. No statistically significant differences were found between the control and experimental samples of freshly prepared dough in the values of pH and titratable acidity. The dough was characterized by almost identical pH values immediately after production (about 5.7). After 2 h of fermentation, a slight difference in pH values was observed: the samples with dihydroquercetin had pH values about 3–5% higher than the control sample. The TTA data confirmed the tendency of acidification displayed by the pH. In particular, a decrease in pH values corresponded to an increase in TTA, and, for the experimental test samples, the titratable acidity was slightly lower than for the control sample. Thus, use of dihydroquercetin had some inhibitory effect on yeast development. This effect was noted in several other works and was called “phenolic stress” [40,41,42].

The results are the mean values ± standard deviation of the four determinations (performed in duplicate for two independent productions).

The results of the cup test counts are shown graphically in Figure 2. No statistically significant differences were observed.

Both dough samples presented showed a decrease in pH at the end of fermentation. This can be attributed to activation of yeast in the fermentation process, when sugars are transformed into carbon dioxide and ethanol [43]. Heterofermentative reactions are taking place where oxygen is used to produce mainly acetic acid and lactic acid in different quantities depending on the temperature during proofing time. A variation in pH is an indicator of yeast activity. Yeast needs warm and humid conditions to thrive, with a relative humidity ranging from 50% to 90% being ideal for gas production [43]. Some studies show that polyphenols can reduce yeast activity. However, this effect is dose-dependent [40,41,42,44]. The amount of dihydroquercetin applied probably does not reach a toxic dose for yeast and does not adversely affect the fermentation process.

Generally, the amount of yeast increased during fermentation; after 2 h of fermentation, this microbial group counted at 10⁸ CFU/g.

3.2. Chemical Composition and Antioxidant Properties of Dough

The amount of dihydroquercetin in the dough samples at the beginning of fermentation and after 2 h is shown in Figure 3. According to the studies, the amount of dihydroquercetin decreases during dough kneading and fermentation.

The average level of dihydroquercetin retention in the freshly prepared dough ranged from 75–82% and was slightly higher in the samples with 0.07 and 0.1 mg/100 g dihydroquercetin added. After 2 h of fermentation, the amount of dihydroquercetin remained in the range of 70–75%. Dihydroquercetin losses may occur due to various factors, including the destructive effect of mechanical stirring during dough kneading and interaction of dihydroquercetin with flour proteins, starch, and yeast. Formation of complex compounds of flavonoids with proteins, starch, and dietary fiber has been noted in several studies [10,45]. Such interactions with macromolecules can have both positive and negative effects on the further bioavailability of flavonoids and their bioactive properties. Flour macromolecules can either act as a protective coating for flavonoids or reduce their antioxidant properties.

Figure 4 shows the total flavonoid content of the dough immediately after kneading and at the end of the fermentation process. The flavonoid content of the control dough ranged from 5.7 to 8.4 mg EQ/100 g. As expected, the control dough sample had lower flavonoid content than the dough with dihydroquercetin added. Moreover, the increase in the total flavonoid content of the dough was directly proportional to the added amount of dihydroquercetin.

The 2 h fermentation process resulted in an increase in flavonoid content regardless of the dough sample analyzed. Flavonoids in plant raw materials are usually found in conjugated and bound forms [3,46,47]. An increase in total flavonoids may occur due to the action of endogenous and microbial enzymes during fermentation, resulting in conversion of some flour flavonoid compounds to free forms. Tian, Chen, Tilley, and Li [3] reported that, during fermentation, yeast activity can partially degrade the phenolic-carbohydrate complex, resulting in an increase in free phenolic acids. On the other hand, yeast metabolism can consume flavonoids or transfer some of them into complexes with other macromolecules in the dough matrix. In general, 2 h fermentation can be recognized as favorable for growth of flavonoids [3,46,47,48,49,50].

The values of the antioxidant activity of the dough are shown in Figure 5. Immediately after kneading, the values of the antioxidant activity of the dough were directly proportional to the quantitative content of flavonoids. The highest values were characteristic of dough with 0.1 mg of dihydroquercetin added both at the beginning and end of fermentation (92.1–101.3%). In the present study, a significantly moderate correlation was established between the total flavonoid content and antioxidant activity of the dough (r = 0.476). Flavonoids’ effect on antioxidant activity depends on many factors, including the interaction processes of flavonoids with the food matrix and transition of flavonoids into forms with reduced bioactivity [46,49]. The study showed that dihydroquercetin in the food dough matrix withstands the fermentation process without significant losses.

3.3. Chemical Composition and Antioxidant Properties of Bread

Quantitative preservation of dihydroquercetin in finished bread is a key factor in formation of antioxidant properties of the product.

According to the obtained results (Figure 6), loss of dihydroquercetin in the technological process is quite significant (36.7–42.2 ± 0.4% on average).

Taken together, loss of dihydroquercetin in the crust and subcrust layers is, on average, 12 to 17% higher than in the crumb regardless of the amount of the additive used.

The results also agree with the results of similar studies conducted in the field of bakery products enrichment with polyphenolic substances [10,50].

Dihydroquercetin losses can be caused by active interaction of an antioxidant with macromolecules of a food matrix of bread. Being a substance of polyphenolic nature, dihydroquercetin is prone to formation of complex compounds, primarily with proteins and starch. These processes are based on both covalent and Van der Waals interactions. In addition, being a toxic component for yeast, dihydroquercetin can activate enzymatic processes aimed at its conversion, which can also lead to a decrease in the residual amount of the antioxidant in the finished product.

Table 2. Average values (±SD, n = 4) of the total flavonoids and antioxidant activity of bread.

Samples	Total Flavonoid Content, mg EQ/100 g		AOA, % (DPPH)
	Crumb	Crust and the Layer under the Crust	Crumb	Crust and the Layer under the Crust
Control	26.0 ± 0.5	22.4 ± 1.0	10.5 ± 0.5	12.9 ± 1.0
Experimental sample 0.05	53.0 ± 1.0	46.7 ± 1.2	42.1 ± 0.5	46.6 ± 1.0
Experimental sample 0.07	61.3 ± 1.0	54.1 ± 1.2	54.5 ± 1.0	58.4 ± 0.5
Experimental sample 0.1	69.5 ± 1.3	59.5 ± 0.5	61.5 ± 0.5	64.7 ± 0.5

shows that introduction of dihydroquercetin to bread led to a significant increase in content of flavonoids. The number of flavonoids in the crust was higher than that in the crumb in both control and enriched samples.

Antioxidant profiles of bakery products can arise from a variety of sources, including native polyphenols in wheat flour, incorporated phenolic compounds, intermediates, or products of thermal decomposition of phenolic compounds and polyphenol–starch/protein complexes [50].

Antioxidant activity values rise with an increase in amount of dihydroquercetin added, and increases in values of antioxidant activity with an increase in amount of added dihydroquercetin were observed in all the studied samples. The antioxidant activity of bread with dihydroquercetin ranged from 42–64%. There was a significantly moderate correlation between the amount of dihydroquercetin and the antioxidant activity of the test (r = 0.498–0.568). The highest values of antioxidant activity were observed in the crust of bread ES-0,1 added. Interestingly, a direct correlation of bread antioxidant activity with amount of flavonoids has not been established.

Thus, introduction of dihydroquercetin to wheat bread leads to a significant increase in its antioxidant properties. Similar results of antioxidant activity increase when the bread was enriched with plant ingredients were obtained with buckwheat bread, rice bran bread, and bread with amaranth flour [50,51].

The obtained results showed that introduction of dihydroquercetin to wheat bread is an effective way of increasing the flavonoids content and antioxidant capacity of the final product. Use of dihydroquercetin can be considered as a promising approach to functional bread production. It should be noted that quality and biological effect of enriched bread are influenced by many factors, including digestibility and bioavailability of phenolic substances, which generally requires additional research.

3.4. Potentially Bioaccessible and Bioavailable Dihydroquercetin from Digestion

Potential bioaccessibility is an important index to evaluate the nutritional value of a bioactive component: the portion of an ingested component that is potentially available for absorption is dependent on digestion and release from a food matrix. Considering further absorption through the intestinal wall, the potential bioavailability indicates a compound that is ready to reach systematic circulation [38,39,52]. As presented in Figure 7, no significant difference was observed in the potentially bioaccessible amount of dihydroquercetin in all the bread samples with dihydroquercetin fortification. The average loss of dihydroquercetin during digestion was 25%.

This result indicated a possible interaction between bread matrix and dihydroquercetin, enhancing the potential of dihydroquercetin to be absorbed. Dihydroquercetin could interact with wheat protein (gluten network) and starch during bread making [53], most probably via hydrogen bonding and hydrophobic interactions as a flavonoid [6,53]. The protective effect of the bread matrix on flavonoids in the process of digestion and other studies has been shown.

Most of the studies presented in the literature show that protein–flavonoid and starch–flavonoid interactions occur due to formation of non-covalent hydrophobic bonds, which can subsequently be stabilized by hydrogen bonds. Non-covalent interactions include hydrophobic, van der Waals interactions, formation of hydrogen bridges, and ionic interactions. They are weaker than covalent interaction and always reversible.

Some studies have examined the effect of protein–flavonoid and starch–flavonoid interactions on bioavailability of flavonoids. Recent studies describe both positive and negative effects.

The results of Ribnicky et al. showed that flavonoids adsorbed on soy protein isolate were more bioavailable and bioactive [6].

Several studies also indicate that proteins are good carriers of flavonoids in the gastrointestinal tract. These complexes may have several effects on activity of flavonoids. They can protect flavonoids from oxidative degradation. At the same time, the association between flavonoids and proteins/starch can influence the antioxidant activity of flavonoids, as was found for associations created between flavonoids and milk proteins in vitro. In fact, due to protein–flavonoid interactions, antioxidant activity of flavonoids was masked [6,51,52].

4. Conclusions

The results of this study have shown that introduction of dihydroquercetin in the amount of 0.05–0.1 g per 100 g of flour does not adversely affect the metabolic processes of Saccharomyces cerevisiae yeast cells. At the same time, dihydroquercetin itself withstands the fermentation process with losses of 20–25%.

Expediency of bread enrichment with dihydroquercetin for formation of antioxidant properties of the product has been demonstrated. The antioxidant properties of bread with dihydroquercetin are significantly higher compared to the control sample, 3.5–4 times on average depending on the dosage of biologically active substances. At the same time, it was found that loss of dihydroquercetin in the technological process of bread production is very significant, about 40%, which also indicates the need to find a solution to this problem.

A possible solution to provide beneficial properties of bread enriched with polyphenolic antioxidants may be to use them in an encapsulated form. Implementation of this approach is a perspective for future research.