Preliminary Study on the Influence of the Polyphenols of Different Groups on the Digestibility of Wheat Starch, Measured by the Content of Resistant Starch

Abstract

The scientific goals of this research were to examine the impact of various polyphenols from different groups on resistant starch development. Wheat starch was tested, and the polyphenols were added to starch after its pasting in the amount suggested in the literature as optimal—10 mg, and at twice and half the optimal, i.e., 20 mg and 5 mg. The most frequently consumed and most frequently occurring compounds in food products were selected for the proposed research: (1) phenolic acids—p-coumaric acid, ferulic acid; (2) flavanones—hesperidin, naringenin; (3) flavanols—(+)catechin, epigallocatechin gallate; (4) flavonols—quercetin, kaempferol; (5) anthocyanins—cyanidin-3-O-glucoside, delphinidin-3-O-glucoside. As a result, either the dose or the kind of polyphenolic compound had a statistically significant influence on the wheat starch digestibility (p < 0.05). However the observed impact was dose-dependent, and interestingly, higher amounts of RS were found in the case of the lowest dose applied (5 mg—4.76% of starch gel; mean = 2.94 ± 1.23 g·100 g⁻¹ dm) as compared to the other doses: 10 mg—9.09% of starch gel (mean = 1.58 g·100 g⁻¹ dm) and 20 mg—16.66% of starch gel (mean = 1.51 ± 0.90 g·100 g⁻¹ dm). Among all tested polyphenols added to wheat starch gels in an amount of 10 mg and 20 mg, epigallocatechin gallate was found to be the most effective compound (p < 0.05), while (+)catechin was most efficient in the dose of 5 mg (p < 0.05).

1. Introduction

Problems related to the effects of free radicals on the human body are the subject of numerous scientific investigations. It has been well-established that oxidative stress caused by an overabundance of reactive oxygen species (ROS) that remains in the organism is the root of the pathogenesis of a number of disorders [1]. Oxidative stress can be triggered by various external and internal factors, including elevated blood glucose and insulin levels, which in turn lead to an increased risk of chronic non-communicable diseases, including obesity and type 2 diabetes [2,3]. Type 2 diabetes is now a serious problem in many countries around the world and represents 90% of all types of this disease, while being one of the five leading causes of death in the human population [4]. In order to diminish the risk of these illnesses, it is suggested to provide foods rich in natural bioactive components, including antioxidants such as polyphenolic compounds, with the diet. One of the desired and expected effects of polyphenols is their effect on reducing the digestibility of starch by binding to starch and thus forming insoluble complexes that are resistant to the action of alpha-amylase [5]. Thus, polyphenols, by delaying starch digestion, may lower postprandial blood glucose levels, which in turn may have beneficial effects in the prevention of type 2 diabetes [6,7,8]. From a nutritional standpoint, the ability to control starch digestibility is an extremely important strategy for food design in the future [9]. So far, polyphenols have been found to affect starch digestion. Polyphenols interact with α-amylase mainly through hydrogen bonds between hydroxyl groups and the enzyme’s active site and through hydrophobic interactions between polyphenols’ aromatic groups and the enzyme [10]. Binding of flavonoids to amylase follows a static mechanism; flavonoids bind near the enzyme’s active site [11]. Polyphenols can also interact with amylose through hydrogen bonding and hydrophobic interactions to form indigestible complexes [12]. Starch also appears to promote interactions with high-molecular-weight polyphenols, such as tannins [13]. There are a number of studies on the effects of polyphenolic compounds found in raw materials of plant origin and added to foods to increase their functional value [5]. Various products enriched with polyphenols were characterized by their effect on reducing starch digestibility in vitro (bread—black rice extract; cookies—pomegranate peel, apple peel; wheat bread—green tea extract, grape seed extract, resveratrol, freeze-dried fruits of various wild shrubs, baobab fruit extract; muffins—mango waste products) [5,14,15,16]. In the literature, the data on which groups of polyphenolic compounds (phenolic acids, flavones, flavanones, flavanols, anthocyanins) reduce the digestibility of starch, as well as their functional properties, are insufficient. There are other bioactive compounds such as dietary fiber, fat, proteins in plant waste products or plant raw materials containing different groups of polyphenolic compounds that can also affect the above-mentioned parameters (starch digestibility). Hence, first of all, it is difficult to clearly determine what the actual effect of polyphenols is, and especially which groups of these compounds show the best effect. The proposed research and approach to the problem of starch digestibility are innovative and have not been presented in the scientific literature so far. The objectives of the proposed research were (i) to see if there are differences between different groups of polyphenols in reducing wheat starch digestibility, as measured by resistant starch (RS) content; (ii) to find the most effective group of polyphenols in this regard; and (iii) to find the most effective compound.

2. Materials and Methods

2.1. Preparation of Starch–Phenolic Complexes (Conjugates)

Five percent (5%) wheat starch (Sigma Aldrich, S5127, St. Louis, MO, USA) gels were prepared. In order to gelatinize, the starches were weighed into screw cup tubes, mixed with the proper amounts of distilled water, incubated in a water bath with shaker (ELPIN 357, Łódź, Poland) at 95 °C for 30 min, then later cooled down at 37 °C for a further 30 min. Out of approximately 8000 currently identified polyphenolic compounds [5], the most frequently consumed and most frequently occurring compounds in food products were selected for the proposed research: (+)catechin hydrate (Sigma Aldrich 22110, St. Louis, MO, USA), epigallocatechin gallate (Sigma Aldrich PHR1333, St. Louis, MO, USA), quercetin (Sigma Aldrich Q4951, St. Louis, MO, USA), kaempferol (Sigma Aldrich K0133, St. Louis, MO, USA), naringenin (Sigma Aldrich N5893, St. Louis, MO, USA), hesperidin (Sigma Aldrich H5254, St. Louis, MO, USA), trans-ferulic acid (Sigma Aldrich 128708, St. Louis, MO, USA), p-coumaric acid (Sigma Aldrich C9008, St. Louis, MO, USA), delphinidin-3-O-glucoside (myrtillin chloride) (Extrasynthese, 0938S, Genay, France), and cyanidin-3-O-glucoside (kuromanin chloride) (Extrasynthese 0915S, Genay, France). Each of the above-mentioned compounds was added separately to the starch after making gels, every time in the amount suggested in the literature as optimal—10 mg [17], which should be consumed with a daily diet, assuming 5 recommended servings of vegetables and fruit. The second suggested doses were amounts twice and half the optimal—20 mg and 5 mg, to investigate not only the differences between the different polyphenol groups, but also the effect of the maximum, optimal and minimum doses.

2.2. Methods

The content of dry matter (dm) in the wheat starch gels was determined with standard methods recommended by the Association of Official Analytical Chemists [18]. Moisture percentage was determined by the oven dry method (AOAC method 940.26) using a laboratory drier (SML 30/250, Zalmed, Warszawa, Poland), while RS content was determined by commercial kits (Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, Ireland) with the application of AOAC 2002.02 and AACC 32-40 standards. In short, samples were incubated in a shaking water bath (MEMMERT WNE 14, Schwabach, Germany) with pancreatic α-amylase (3 Ceralpha Units/mg) and amyloglucosidase (3300 U/mL) for 16 h at 37 °C in order to hydrolyze the starch into D-glucose. The reaction was terminated by the addition of ethanol, and the RS was recovered as a pellet upon centrifugation (MPW MED. INSTRUMENTS 351e, Warszawa, Poland). The precipitate was washed twice in aqueous ethanol (50% v/v) and centrifugated. The supernatants were carefully decanted, and the tubes were inverted on absorbent paper to drain excess liquid. RS in the pellet was dissolved in 2 M KOH by vigorously stirring in an ice-water bath over a magnetic stirrer. The solution was neutralized with acetate buffer, and the starch was quantitatively hydrolyzed to glucose with amyloglucosidase. D-Glucose was measured with glucose oxidase/peroxidase reagent (GOPOD), and this was a measure of the RS content of the sample. All values were calculated on a dry matter basis (g·100 g⁻¹ dm).

2.3. Data Analyses

The results were presented as ranges of at least three parallel repetitions with standard deviation around the mean. Multivariate analysis of variance was applied in order to assess the influence of different groups, polyphenolic compounds and doses on the resistant starch development. The Duncan test was used in order to test the significance of differences at a level of p < 0.05. All calculations were carried out using Statistica, v.13 software (Statsoft, Inc., Tulsa, OK, USA).

3. Results and Discussion

The dry matter content of the tested samples equaled 4.54 ± 0.1%. The resulting RS contents in wheat starch gels upon addition of 10 single polyphenols belonging to five different polyphenolic groups in doses of 5 mg (4.76% of starch gel), 10 mg (9.09% of starch gel) and 20 mg (16.66% of starch gel) were in the range of: 1.24–4.89 g·100 g⁻¹ dm, 0.34–2.86 g·100 g⁻¹ dm, and 0.31–3.4 g·100 g⁻¹ dm, respectively (

Table 1. Contents of RS in wheat starch (g·100 g⁻¹ dm) depending on the type and dose of the specific polyphenol standard added.

Dose	5 mg (4.76% of Starch Gel)	10 mg (9.09% of Starch Gel)	20 mg (16.66% of Starch Gel)
Wheat with an addition of:
(+)catechin	4.89 ± 0.0 f,3	2.30 ± 0.2 f,2	1.93 ± 0.1 b,1
epigallocatechin gallate	2.75 ± 0.4 e,1	2.86 ± 0.2 g,1	3.40 ± 0.3 e,1
hesperidin	3.52 ± 0.0 b,3	1.42 ± 0.0 b,2	1.10 ± 0.0 d,1
naringenin	1.91 ± 0.2 a,1	2.07 ± 0.1 c,1	1.99 ± 0.1 b,1
trans-ferulic acid	4.21 ± 0.2 c,3	2.09 ± 0.2 c,2	1.38 ± 0.0 a,1
p-coumaric acid	1.71 ± 0.0 a,2	0.34 ± 0.1 a,1	2.11 ± 0.0 b,3
delphinidin-3-O-glucoside (myrtillin)	1.84 ± 0.1 a,1	1.83 ± 0.0 e,1	1.32 ± 0.1 a,2
cyanidin-3-O-glucoside (kuromanin)	1.24 ± 0.0 d,1	1.09 ± 0.0 d,2	1.21 ± 0.1 a,1
quercetin	3.89 ± 0.2 c,3	1.41 ± 0.0 b,2	0.31 ± 0.0 c,1
kaempferol	3.46 ± 0.4 b,2	0.34 ± 0.1 a,1	0.39 ± 0.1 c,1
Mean values	2.94 ± 1.23 a	1.58 ± 0.82 b	1.51 ± 0.90 b
Wheat without an addition of polyphenols	0.27 ± 0.0	-	-

The results are presented as mean ± standard deviation (SD). The values with different letters in columns (depending on the type) and numbers in rows (depending on the dose) are significantly different at p < 0.05.

). Either the dose or the type of polyphenolic compound had a statistically significant influence on the starch digestibility of the tested samples (p < 0.05). Among all tested polyphenols added to wheat starch gels in the amounts of 10 mg and 20 mg, epigallocatechin gallate was found to be the most effective compound (p < 0.05), while (+)catechin was most efficient at the dose of 5 mg (p < 0.05). On the other hand, kuromanin, p-coumaric acid and kaempferol, as well as quercetin and kaempferol contributed to the lowest level of RS after addition to the starch gels in the amounts of 5 mg (4.76% of starch gel), 10 mg (9.09% of starch gel) and 20 mg (16.66% of starch gel), respectively (Table 1), (p < 0.05). The highest and lowest degrees of starch digestibility inhibition, as measured by the development of the resistant starch, varied between the three applied doses (5, 10 or 20 mg), being statistically significantly highest in the case of the 5 mg dose (mean = 2.94 g·100 g⁻¹ dm), (p < 0.05) (Table 1).

The results for RS contents in wheat starch gels upon addition of five different groups of polyphenols at the doses of 5 mg (4.76% of starch gel), 10 mg (9.09% of starch gel) and 20 mg (16.66% of starch gel) were in the range of: 1.54–3.68g·100 g⁻¹ dm, 0.7–2.48 g·100 g⁻¹ dm and 0.35–2.67 g·100 g⁻¹ dm, respectively (

Table 2. Contents of RS in wheat starch (g·100 g⁻¹ dm) depending on the type and dose of the polyphenol group standard added.

	5 mg (4.76% of Starch Gel)	10 mg (9.09% of Starch Gel)	20 mg (16.66% of Starch Gel)
Wheat with an addition of:
Flavanols ((+)catechin, epigallocatechin gallate)	3.60 ± 1.2 a	2.48 ± 0.3 c	2.67 ± 0.8 c
Flavanones (hesperidin, naringenin)	2.83 ± 0.9 a	1.74 ± 0.4 a	1.54 ± 0.5 a
Phenolic acids (trans-ferulic acid, p-coumaric acid)	3.14 ± 1.3 a	1.21 ± 0.9 a,b	1.62 ± 0.4 a
Anthocyanins (myrtillin, kuromanin)	1.54 ± 0.3 b	1.46 ± 0.4 a	1.27 ± 0.1 a
Flavonols (quercetin, kaempferol)	3.68 ± 0.4 a	0.70 ± 0.6 b	0.35 ± 0.1 b
Wheat without an addition of polyphenols	0.27 ± 0.0	-	-

The results are presented as mean ± SD. The values with different letters in columns are significantly different at p < 0.05.

). Among the tested groups of polyphenols, flavanols ((+)catechin, epigallocatechin gallate) were found to be most effective when added to the wheat starch gels in the amounts of 10 mg and 20 mg, while flavonols (quercetin, kaempferol) acted worst at these two doses. At the same time, anthocyanins (myrtillin, kuromanin) contributed to the lowest level of RS at the dose of 5 mg (p < 0.05), (Table 2).

In the literature, there is no information on the content of resistant starch from wheat starch gels to which single polyphenols from different groups ((+)catechin, epigallocatechin gallate, hesperidin, naringenin, trans-ferulic acid, p-coumaric acid, delphinidin-3-O-glucoside (myrtillin), cyanidin-3-O-glucoside (kuromanin), quercetin and kaempferol) were added separately in the amount suggested in the literature as optimal—10 mg [17]. The latter should be consumed with a daily diet, assuming 5 recommended servings of vegetables and fruit. The second and third suggested doses were amounts twice and half the optimal, i.e., 20 mg and 5 mg. This study has been conducted for the first time. It is known, however, that the RS content in wheat starch is low and equals between 0.2 and 0.3% [19], which was also confirmed in this study (Table 1 and Table 2). The content of RS was above the presented range and exceeded these initial values in most of the samples by more than 3–16 fold (Table 1).

Starch-rich foods such as white bread, pasta, rice, cornflakes and other cereal-based products became the major contributors to the energy intake of the human daily diet and constitute a basis of nutrition for the world’s population. In most of these foods, starch is rapidly digested and absorbed, which contributes to increased oxidative stress, high plasma glucose levels, increased insulin resistance and elevated hypertension [20], which consequently may favor weight gain and the development of type 2 diabetes, cardiovascular diseases and certain types of cancer, including colon tumors [3,21]. At the same time, for the better part of the global population, it is impossible to give up consuming too much of the starchy food products, which are available at any time at a decent price. Therefore, important future goals for food designers should be to project starch-rich food products characterized by a delay in starch digestion without alterations of the desired organoleptic characteristics and specific features of the food expected by consumers. Hence, any efforts to slow down the digestibility of starch are highly desirable. Recently, leading strategies proposed to achieve this goal include: (i) modulating starch structure by preserving crystalline structure, to promote resistant starch production or reduce the mobility of gelatinized starch gel; (ii) favoring mashing over milling, to preserve intact cell walls and select large particle size flours (iii) modulating the Maillard reaction by, for example, converting starch to melanoidins [9].

The results of the present study clearly indicated that either the dose or the type of the polyphenolic compound had a statistically significant impact on the development of resistant starch in wheat starch gels (Table 1 and Table 2, p < 0.05). However, the observed impact was dose-dependent, and interestingly, higher amounts of RS were found in the case of the lowest dose applied (5 mg—4.66% of starch gel; mean = 2.94 ± 1.23 g·100 g⁻¹ dm) as compared to the other doses: 10 mg—9.09% of starch gel (mean = 1.58 g·100 g⁻¹ dm) and 20 mg—16.66% of starch gel (mean = 1.51 ± 0.90 g·100 g⁻¹ dm), which might suggest that the optimal dose in this matter might be 5 mg instead of 10 mg. There is a lack of knowledge in the available literature concerning the impact of single polyphenolic compounds on wheat starch digestibility, measured by RS content. Generally, starch is composed of two types of polymers, namely amylose and amylopectin. Amylose is typically a linear polyglucan consisting of D-glucose units linked by glycosidic bonds, while amylopectin is a relatively larger moiety than amylose and is highly branched [22]. Amylose has long unbranched sections of glucose molecules linked together by sporadic branching points. Amylopectin has a high molecular weight with a large number of short-chain branching [20]. The starch of maize, rice, wheat and potato consists of about 70–80% amylopectin and 20–30% amylose. This genetically determined amylose-to-amylopectin ratio in starch has a great impact on its digestibility since amylose is less susceptible to gelatinization than the branched amylopectin. It has been evidenced so far that a greater amylose-to-amylopectin ratio contributes to lower starch digestibility, and hence, a lower glycemic index [20,23]. It is known, however, that amylose tends to retrograde into resistant starch, while amylopectin retrogrades into slowly digestible starch [9]. Upon the presence of water and high temperature, the starch granules swell, and the hydrogen bonds between amylose and amylopectin chains are broken down. Subsequently, amylose and amylopectin moieties are released from the starch granules, forming spaces that allow water to enter inside, consequently leading to the loss of the starch crystalline structure in a process called gelatinization. In this form, starch becomes more accessible to digestive enzymes and rapidly undergoes the process of digestion and absorption [14]. At the same time, there is a part of starch that resists enzymatic hydrolysis, passes undigested through the small intestine, and reaches the colon, where it becomes a substrate for microbial fermentation forming short-chain fatty acids (SCFAs). This fraction is generally called resistant starch [20,24]. From a nutritional point of view, this RS has a favorable, stabilizing effect on blood glucose levels and has potential to be a part of management and treatment programs to control type 2 diabetes [3,20]. Among the different factors affecting starch digestibility, both internal (the amylose-to-amylopectin ratio, dietary fiber, fat, protein contents, starch–macromolecule interactions) and external ones can be specified (processing: cooking, parboiling, soaking, cooling or retrogradation) [20]. Special attention has been paid to polyphenols as secondary plants metabolites that are characterized by many bioactive properties, including, among others, the possible influence on the starch digestibility [9,24,25,26]. On one hand, polyphenols may bind to starch granules, thus forming insoluble complexes that are resistant to the action of α-amylase [5]. This might partly explain the increase in the amount of RS after the addition of different polyphenols to the wheat starch gels (Table 1 and Table 2). However, little is known about the mechanisms by which polyphenols interact with starch, forming insoluble ligands. It has to be pointed out that the tested polyphenols were added to the starch after its pasting and gelatinization, which could limit the formation of hydrogen bonds between starch granules and polyphenols due to the fact that the starch has already been linked to the water molecules. On the other hand, it has been evidenced that polyphenols may directly act as inhibitors of α-amylase and amyloglucosidase [25,26,27,28]. However, their inhibitory properties strongly depend on their molecular structures (

Table 3. Inhibitory activity related to molecular structure [27].

Polyphenol	***** Excellent **** Very Good *** Good ** Satisfactory * Poor	-OH Groups ↑	-OCH3 Groups ↓	Double Bond Conjugated with Carbonyl Group ↑	Glycolysated Form ↓	Galloyl Moieties ↑	C = O Bond in Galloyl Moieties ↑	Additional Benzene Ring ↑
epigallocatechin gallate	*****	8	⁻	⁻	⁻	+	+	+
(+)-catechin	****	5	⁻	⁻	⁻	⁻	⁻	⁻
delphinidin-3-O-glucoside	****	6	⁻	⁻	+	⁻	⁻	⁻
quercetin	****	5	⁻	+	⁻	⁻	⁻	⁻
cyanidin-3-O-glucoside	***	5	⁻	⁻	+	⁻	⁻	⁻
hesperidin	***	3	⁻	⁻	⁻	⁻	⁻	⁻
kaempferol	***	4	⁻	+	⁻	⁻	⁻	⁻
naringenin	**	3	⁻	⁻	⁻	⁻	⁻	⁻
p-coumaric acid	**	1	⁻	+	⁻	⁻	⁻	⁻
trans-ferulic acid	*	1	+	+	⁻	⁻	⁻	⁻

Contribute to ↓ decrease or ↑ increase of the inhibitory actvity.

), and the differences between the resulting amounts of RS might be explained by the diverse chemical structures of the tested polyphenols. Generally, polyphenols can be classified as flavonoids and non-flavonoids [5,29]. Within the group of flavonoids are the following sub-groups: flavones, flavonols, flavanols, isoflavones, flavanones, and anthocyanins. The non-flavonoids are divided into phenolic acids, lignans, and stilbenes [5]. Flavonoids are composed of two aromatic benzene rings (A and B) and an oxygenated heterocycle ring (C), attached to ring A. Subgroups of flavonoids differ from each other in terms of the type of ring C, the substitution pattern of hydroxyl (-OH) and methoxy (CH₃) groups at positions R3, R5, R6, and R7 in the A–C ring system, as well as at positions R3′–R7′ in ring B, and the position of ring B [30]. Phenolic acids differ from flavonoids in terms of their chemical structures as non-flavonoid compounds. They consist of a benzene ring connected with the carboxylic acid group and at least one hydroxyl group [27,31]. Within the group of phenolic acids, there are two subgroups such as hydroxycinnamic acids and hydroxybenzoic acids [27]. In the literature, the following differences in chemical structures were mentioned as the most important in affecting the starch digestibility: (1) the presence of hydroxyl (-OH) groups, which take part in the formation of hydrogen bonds between -OH groups of phenolics and amino acids of the active site of the enzyme (such as Asp197 and Glu233); (2) flavonoids without substitution of -OCH₃ at -OH in their molecular structures, which are more likely to be effective inhibitors of α-amylase; (3) the 2,3-double bond in the ring C conjugated with the 4-carbonyl group enhances electron delocalization between ring C and A, forming a highly stable conjugated system at the active site of the α-amylase with strong inhibitory properties; (4) hydrogenation of 2,3-double bond decreases the inhibitory activity (flavanons and flavanols had weaker inhibiting properties than flavonols and flavones); (5) glycosylation on flavonoids decreases the inhibitory activity; (6) more galloyl groups, resulting in greater inhibitory properties as the galloyl groups provide three hydroxyl groups that might interact with the catalytic site of α-amylase [26,27,28,30]. Taking into account the above mentioned, the tested polyphenols in this study have been arranged according to their inhibitory activity (Table 3), from excellent to poor.

In the presented list, the first two polyphenols expected to have the strongest inhibitory activity, belonging to the flavanols group (epigallocatechin gallate and (+)catechin), were also the most efficient in this study in RS development at three doses (Table 1). This might result from a process of aggregation of hydroxyl groups in the structures of analyzed flavanols. Hydroxyl groups are crucial for the inhibitory activity of flavonoids against α-amylase and amyloglucosidase [27,28,30,32,33]. Hydrogen bonds between the -OH groups in position R6 or R7 of the A ring and position R4′ or R5′ of the B ring are favorable for interaction of the flavonoid ligand with the side chains of the catalytic center of α-amylase [27,30]. The tested anthocyanins (delphinidin-3-O-glucoside and cyanidin-3-O glucoside), although positioned quite high in the inhibitory activity list due to many hydroxyl groups [26], were relatively poorly efficient in RS formation at the three doses used in this study (Table 1 and Table 2). This might result from the fact that delphinidin-3-O-glucoside and cyanidin-3-O glucoside are glycosylated forms of anthocyanidins [34] that might decrease their binding affinity to α-amylase, mainly due to some changes in their spatial structures to bulky non-planar arrangements, limiting their affinity for hydrophobic sites of the enzyme [27,28]. Flavonols (quercetin, kaempferol) are characterized by good and very good inhibitory activities due to the several -OH groups they contain and additionally the 2, 3-double bond in ring C conjugated with a 4-carbonyl group (Table 3). However, in this study, they were most efficient in contributing to RS formation only at the 5 mg dose (4.76% of starch gel), and then the formation of RS was drastically lowered (Table 1 and Table 2). Flavanones (hesperidin, naringenin) are characterized by good and satisfactory inhibitory properties (Table 3) and favor the formation of RS to the greatest extent at the 5 mg dose, but at the higher doses (10 mg—9.09% of starch gel, 20 mg—16.66% of starch gel), the effect was not further observed (Table 1 and Table 2). The analyzed phenolic acids (trans-ferulic acid, p-coumaric acid) belong to hydroxycinnamic acids, which show much higher inhibitory activity against α-amylase than hydroxybenzoic acids [27,28,35,36]. They contain C=C double bonds conjugated with the carbonyl group, which can form a highly conjugated system, stabilizing the compounds when binding to the active site of α-amylase (Table 3) [27,28]. On the other hand, in comparison to the other polyphenolic groups, phenolic acids are characterized by rather mediocre inhibitory activity, mainly due to having one hydroxyl group and the fact that dehydroxylation and methylation may lower their inhibitory activity against α-amylase. Similarly to flavonols, the tested phenolic acids were most efficient in contributing to RS formation only at the 5 mg dose (4.76% of starch gel), and then the formation of RS was drastically lowered (Table 1 and Table 2). The possible explanations might be that polyphenol is able to reversibly bind with the active sites of α-amylase, competing with the substrate (i.e., starch) and that soluble polysaccharides also interact with polyphenols in the pursuit of the active site of the enzyme [27,28]. Another explanation might be that some polyphenols have low solubility in buffered solutions, and hence, their activity might not be fully visible [28].

4. Conclusions

In the present study, it was indicated that either the dose or type of polyphenolic compound could have a statistically significant impact on the development of resistant starch in wheat starch gels. However, the tested polyphenols were characterized by ambiguous effects in this matter. Most of them contributed to the highest development of RS at the lowest 5 mg dose (4.76% of starch gel) (hesperidin, (+)catechin, trans-ferulic acid, kuromanin, myrtillin quercetin and kaempferol), while p-coumaric acid addition resulted in the highest RS level at the 20 mg dose (16.66% of starch gel). The rest of the polyphenols (epigallocatechin gallate and naringenin) led to comparable amounts of RS independently of the dose. Among all of the tested polyphenols added to the wheat starch gels in the amounts of 10 mg (9.09% of starch gel) and 20 mg (16.66% of starch gel), epigallocatechin gallate was found to be the most effective compound (p < 0.05), while (+)catechin was most efficient at the dose of 5 mg (4.76% of starch gel) (p < 0.05). Further studies are needed that will fully enable exploring the influence of different polyphenols originating from various groups on other types of starch (e.g., potato, rice, maize) before and after gelatinization, expanding the range of analyses, namely by testing the amounts of the other starch fractions (i.e., rapidly digestible starch, slowly digestible starch) and conducting in vitro glycemic index evaluations.