Gallic acid-fortified buckwheat Wantuo: characteristics of in vitro starch digestibility, antioxidant and eating quality

Abstract

Gallic acid (GA), presented in various plant sources, is increasingly used as a nutritional food ingredient due to its prominent bioactive. In this work, common buckwheat Wantuo (BWT, a Chinese traditional starch gel food) was fortified with 1,3,5% (w/w) GA and assessed for physicochemical properties of flour as well as in vitro starch digestibility, antioxidant and eating quality of BWT. The results clearly showed that the hydration, pasting properties as well as gel microstructure and texture of gel were influenced with addition of GA, while the color of flours showed no significantly change. Hydrogen bonds interaction between GA and starch, more hydrophilic groups exposure and more acid hydrolysis of the starch were thought to be main reasons. Furthermore, combined with structural analysis of starch, the significantly decreased rapidly digested starch (8.62%)/slowly digested starch (12.90%) and increased resistant starch (78.48%) in BWT with 5% addition amount can be mainly due to digestive enzymes inhibition, formation of V-type conformation and alteration in the local structure of starch-phenol-enzyme complex. Meanwhile, the antioxidant activity of BWT-GA improved, where as its texture properties softened due to suppressed starch retrogradation. This study demonstrated the potential use of polyphenol as food ingredient to improve the nutritional properties and eating qualities of starch gel food.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05614-x.

Introduction

Buckwheat Wantuo (BWT) is a traditional Chinese food since Western Jin Dynasty period, and is liked by the consumers in northwest China (Fig. S1). The main raw material of BWT is common buckwheat (CB), which is rich in proteins, vitamins, dietary fibers, minerals and flavonoids. However, during BWT production, there are problems caused by the higher proportion of starch. Rapidly digested starch (RDS) in BWT would cause spikes in postprandial blood glucose that are followed by a heightened insulin response, which may impose a great risk like obesity, cardiovascular disease, and type II diabetes to the consumer (He et al. 2021). Meanwhile, as a starch gel food, the eating quality and shelf life of BWT are directly influenced by starch retrogradation rate during cooling and storage (Wu et al. 2009). Hence, using modern food technologies to reduce the enzymatic hydrolysis rate of starch and to regulate starch retrogradation behavior in traditional starch gel food is an economic and effective way to solve the present situation.

The common techniques for ameliorating the digestibility of starch are physical, chemical (Hu et al. 2022) and biological (Zhang et al. 2019) modifications. In contrast to above modifications methods, exogenous addition is another simple, safe and cheap strategy (Alvarez-Poblano et al. 2020). Recently, Wang et al. (2022) systematically investigated the influence on buckwheat starch digestion via exogenous addition of polyphenols (quercetin and rutin), and proved that the presence of quercetin and rutin significantly slowed down starch digestion by altering starch structure in bound forms and inhibiting digestive enzyme activity.

In fact, extensive research has shown that polyphenols, as annexing agents widely used in food production, usually not only impact the digestibility of product, but also impact the physicochemical properties via reversible/irreversible interactions with food macromolecules, including proteins, starch, dietary fiber, and lipids (Sęczyk et al. 2021). In particular, phenolics–starch interaction occurred in starch gel food (like BWT) can significantly retarded retrogradation of starch gel. Gallic acid (GA, hydroxybenzoic acids) is one of the simplest and the most representative phenolic acids, which is widely found in plant foods, such as fruits and tea (Gutierrez et al. 2020). Due to its high antioxidant and anti-inflammatory activity as well as multiple pharmacological effects, GA has been widely used in food field. Furthermore, exogenous addition of GA is proved to be an effective way to inhibit starch digestion via the binding/dipole–dipole interaction between GA and α-amylase/α-glucosidase as well as the interaction between GA and starch (Aleixandre et al. 2021). To the best of our knowledge, there has been no study profiting exogenous polyphenol addition on Chinese traditional starch gel cereal product.

Hence, the purpose of this work was to fortify BWT with GA to evaluate the effects of exogenous polyphenol addition on physicochemical properties of mixing flour as well as in vitro starch digestibility, antioxidant and eating quality of BWT.

Materials and methods

Materials

CB grains (Fagopyrum esculentum Moench, Shaanxi, China, total starch 79.14%, protein 3.90%, crude lipid 0.34%, ash 0.34%, crude fiber 0.72%). DPPH Assay, ABTS⁺ Assay and GA were purchased from Shanghai Yuanye biotech Co., Ltd. The α-amylase (10065, 30 units/mg), pepsin from porcine gastric mucosa (P7000, 800–2500 units/mg) and porcine pancreatin (P7545, 200 units/mg) were purchased from Sigma-Aldrich (Shanghai, China). α-amyloglucosidase (3260 units/mL) and glucose oxidase-peroxidase (GOPOD) agent were purchased from Megazyme Company, Ireland. Other chemical regents used were analytical grade.

Sample preparation

CB grains were ground into flour by using Quadrumat Junior small laboratory mill (Brabender Inc., Duisberg, Germany), and passed through a 100-mesh sieve. The flour was preserved in plastic ziplock bags until use.

The common buckwheat-polyphenol flour (BPF) was prepared by mixing an appropriate amount of GA with CB flour in varying ratios (1%, 3% and 5% w/w). The BPF with 1%, 3% and 5% GA were recorded as GA-1, GA-3 and GA-5, respectively.

16.667 g BPF (wet basis) above was added into a flat bowl (φ = 11.5 cm, 3.5 cm height) containing 50 mL distilled water (moisture content 75%). After mixing evenly, the bowl was put in a steam cooker with normal atmospheric pressure for 15 min and then placed in the refrigerator (4 °C) for 30 min to obtain BWT. The BWT with 1%, 3% and 5% GA were named as GA-1-WT, GA-3-WT and GA-5-WT, respectively. Some samples were frozen with liquid nitrogen, lyophilized for 48 h and then powdered by ball milling with liquid nitrogen to obtain counterpart BWT freeze-dried powder.

Color measurement

The color parameters of samples were measured by using a CM-5 chromameter (Konica Minolta Co., Tokyo, Japan) and described by the L*—a*—b* color space. The colorimeter was calibrated with standard white tiles before the test, and 3 values of L*, a* and b* were displayed for each measurement. Each sample was measured three times and the average value was taken.

Hydration properties

The water absorption index (WAI), water solubility index (WSI) and swelling power (SP) of common BPF were measured as follows.

1.0 g BPF (dry basis) denoted as W₁ was weighed into pre-weighed 50 mL centrifuge tubes with 25 mL ultrapure water was added. The tube along with the sample was maintained in water bath at the temperature of 90 ℃ for 30 min, and was shaken by hand 10 s per 10 min. The tube was cooled at room temperature and then was centrifuged at 4200 rpm for 15 min. The supernatant was transferred into a known weight (W₂) aluminum plate for drying to a constant weight (W₃) at 105 ℃ and the tube with sediment was weighted as W4. The analysis was done in duplicate. The WAI, WSI, SP were calculated by following formulas:

$$\text{WAI}\left(\text{g}/\text{g}\right)=\frac{W4-W1}{W0}$$ 1

$$\text{WSI}\left(\text{g}/100\text{g}\right)=\frac{W3-W2}{W0}\times 100$$ 2

$$\text{SP}\left(\text{g}/\text{g}\right)=\frac{W4-W1}{W0-\left(W3-W2\right)}$$ 3

Pasting properties

Pasting properties of BPF samples were analyzed using a Rapid Visco-Analyzer RVA-4500 (Perten instruments, Australia). Each sample was suspended in 25 mL ultrapure water, and suspension was adjusted to 14% (wet basis). A suspension was equilibrated at 50 °C for 1 min, heated at a rate of 6 °C/min to 95 °C, maintained at 95 °C for 3.5 min, cooled to 50 °C at a rate of 12 °C/min, and then preserved at 50 °C for 2 min. The whole process took 13 min. The rotating speed for the paddle was 960 rpm for the first 10 s, and kept 160 rpm for the remainder of the analysis.

SEM of gel section

The morphology of gel section with different polyphenol exogenous addition content was observed by a scanning electron microscope (TM3000, Hitachi, Japan). All samples were spread on conductive double-sided adhesive and coated with gold. The images were taken with 1500 × magnifications.

Texture properties of gel and BWT

The texture properties were evaluated using texture profile analysis (TPA) by texture analyzer (TA-XT2i, Stable Microsystems, U.K.). Gelatinized buckwheat samples prepared in RVA were kept for 24 h at 4 °C for determination of gel texture parameters. The gels were texted at a pretest of 2 mm/s, test speed and post-test speed of 1 mm/s, time interval of 5 s and shape variable of 65% using a P6 probe. The reference method proposed by Wang et al. (2019) was used to measure BWT texture properties with shape variable of 30%.

Total phenol content (TPC) and antioxidant activity

TPC

1.0 g BWT powder without polyphenol or 0.2 g sample with exogenous polyphenol was dissolved in 30 mL 70% (v/v) methanol solution. The solution was kept in a water bath at 65 °C for 2 h with shaking and filtered when hot. The resulting extracting solution was used in TPC and antioxidant activity experiments. TPC was determined by Folin-ciocalteu reagent according to Singleton et al. (1999) and expressed as mg of GA equivalents (GAE) per 100 g of BWT powder at 760 nm.

ABTS radical cation (ABTS.⁺) scavenging activity assay

The scavenging activity of ABTS radical cation (ABTS.⁺) was determined as follows. 200 μL extracting solution was added to 4.0 mL working ABTS.⁺ reagent. The hybrid solution was incubated at room temperature for 30 min, protected from light. A standard curve with different concentrations of Trolox was then plotted at 734 nm and the ABTS.⁺ of the samples was calculated according to the standard curve as μmol TE/100 g.

DPPH radical scavenging activity

The scavenging activity of 1,1-diphenyl-2-pycrylhydrazyl (DPPH) was determined as follows. Add 1 mL extracting solution to 4.5 mL 0.1 mmol/L DPPH methanol solution and the cultures incubated at room temperature for 30 min, protected from light. A standard curve with different concentrations of Trolox was then plotted at 517 nm and the DPPH radical scavenging capacity of the samples was calculated according to the standard curve as μmol Trolox equivalents (TE)/100 g.

Ferric-reducing antioxidant power (FRAP) assay

1 mL extracting solution was added to 4.0 mL working FRAP reagent. The hybrid solution was incubated at room temperature for 30 min, protected from light. A standard curve with different concentrations of Trolox was then plotted at 593 nm and the FRAP of the samples was calculated according to the standard curve as μmol TE/100 g.

XRD

The long-range ordered structure of BWT samples were analyzed by an X-ray diffractometer (D8, Bruker, Germany) with a scanning rate of 2°/min from 5 to 40° (2θ). The XRD patterns were analyzed by Jade 6.0 to calculate the relative crystallinity (RC) and the relative peak area at 17 and 20°.

FTIR

The short-range structure of BWT and control samples were determined by a FTIR spectrometer (SP2, PE, US) from 4000 to 600 cm⁻¹ with a resolution of 4 cm⁻¹ and an accumulation of 64 scan. The ratio of 1022/995 cm⁻¹ was calculated by OMNIC 8.2 software.

Digestibility

In vitro starch digestion of BWT was measured according to the method of Sun et al. (2019) and contained three digestion phases: simulated oral, gastric and pancreatic phases. The procedure is as follows.

2.5 g BWT samples were added to a conical flask with 30 mL distilled water, and incubated in a shaking water bath (130 rpm) at 37 °C. The oral phase was initiated by adding 0.1 mL of 10% α-amylase solution and terminated after 1 min by adding 0.8 mL of 1 M aqueous HCl. Then, the gastric phase was initiated by adding 1 mL of 10% pepsin solution. After 30 min, 2 mL of 1 M NaHCO₃ and 5 mL of 0.2 M Na maleate buffer (pH 6) were added to stop the reaction. The pancreatic phase was initiated by adding 1 mL of 5% pancreatin (0.2 M Na maleate buffer) and 0.1 mL amyloglucosidase. 1 mL of the reaction solution was taken into a centrifuge tube containing 4 mL ethanol at 0, 20, 60, 90, 120 and 180 min, and centrifuged at 4000 r/min for 10 min. 0.1 mL of the supernatant was taken to measure the glucose content by using a D-glucose (GOPOD) assay kit.

An Exponential fitting was used to describe the kinetics of BWT starch hydrolysis and the first-order equation can be written as following:

$$C=C_{\infty }\left(1-{\text{e}}^{-k\text{t}}\right)$$

where C (%) is the glucose concentration at t (min), C_∞ (%) is the equilibrium concentration, k is the kinetic constant, t is hydrolyzed time.

The area under the BWT starch hydrolysis curves (AUC) was calculated as following:

$$\text{AUC}={\text{C}}_{\infty }\left({\text{t}}_{\infty }-{\text{t}}_0\right)-\left({\text{C}}_{\infty }/k\right)\left[1-{\text{e}}^{-\text{k}({\text{t}}_{\infty }-{\text{t}}_0)}\right]$$

where t_∞ is the final reaction time, t₀ is the initial reaction time.

The AUC of white bread was used as the standard, and the hydrolysis index (HI) was calculated as the ratio of integral area of the AUC from the BWT compared to that of white bread. The predicted glycemic index (pGI) was estimated by the equation below:

$$\text{pGI}=39.71+0.\text{549HI}$$

The proportions of RDS, slowly digested starch (SDS) and resistant starch (RS) of all samples were calculated by the method of Englyst et al. (1992).

Sensory evaluation

The BWT samples were evaluated by 15 volunteers (9 females and 6 males aged between 20 and 30), recruited at university through a sensory acceptance analysis. Samples were analyzed immediately after preparation. A BWT of each sample was randomly placed on a white plastic plate coded with four-digit random numbers. Water was used for rinsing the mouth between each sample. Assessors were required to evaluate appearance (color, glossiness and structural integrity), texture (viscosity, springiness and hardness), taste according to Table S1.

Statistical analysis

Statistical analysis of the data was performed using SPSS software (Version 22.0, SPSS Inc., Chicago, IL, US). Significant differences (p < 0.05) among these data were determined by Duncan's multiple range test.

Results and discussion

Color analysis

The color parameters of BPF samples and control group were different as presented in Table S2. After adding GA, L* value (brightness) of BPF samples increased. Yet, the addition amount of GA does not significantly affect the brightness. Meanwhile, as exogenous addition of GA, b* (± , yellowness/blueness) of BPF increased, which is consistent with results of Zhang et al. (2018). These results suggest that exogenous addition can influence the color of BPF by its own color and interaction with food components in buckwheat.

Hydration properties analysis

The effect of exogenous polyphenol addition on WAI, WSI and SP of BPF is shown Table S3. Adding GA could increase WAI and SP, which may be ascribe to the hydrogen bonds interaction between GA and starch by competitively preventing the formation of cross-linking in starch molecules and facilitating the moisture penetrating into starch granules as well as the binding between starch and water (Amoako and Awika 2016). Importantly, after continuously adding GA, WSI of samples showed a significant augmentation, which might be ascribed to the addition of polyphenol increased starch dissolution by exposing more hydrophilic groups in starch and/or decreasing continuity of starch gel as well as decreasing its ability to encapsulate small molecules (Han et al. 2020). In addition, the influence of GA addition was presumed to be related to more acid hydrolysis of the starch and more leaching of amylose (Builders et al. 2014).

Pasting properties analysis

The pasting curves and the pasting parameters of all BPF samples are presented in Fig. S2 and in Table S4, respectively. As shown in Fig. S2, the pasting curve shapes of all buckwheat flour samples were quite similar, but the pasting parameters showed significant differences. From Table S4, adding GA could reduce the pasting viscosity of GA series samples, which might be the due to the inhibitory effect of GA on starch gel network formation by influencing the interaction between starch chains. At the same time, as GA content rose, the breakdown and setback value of samples became higher and lower, respectively, which indicated worse ability of resisting shearing and cold paste stability. In addition, the lower pasting peak time and temperature should be ascribe to the accelerated degradation via the interaction between polyphenol molecular and damaged starch granules during heating process (Kan et al. 2022). In brief, addition of GA would make the gelatinization of starch granules more rapidly and easily by influencing the interaction between starch chains which led to lower viscosity during heating process and worse gel stability during cold process. This phenomenon was consistent with the results of hydration properties above.

Gel section microstructure

The microstructural characteristics of BPF gel sections were observed via SEM micrographs (Fig. 1). From Fig. 1a., there was a dense and continuous gel section in CB with heterogeneous distribution of pores. As the addition of polyphenol, the number of the pores in gel section of all samples increased. Like the work by Chen et al. (2020), the BPF samples with GA added showed a looser gel structure and a higher proportion of large pores. To a certain extent, the gel network structure of GA-5-WT collapsed. The looser and porous structure might be contributed to the water-holding capacity of GA dispersed in the starch gels, which could inhibit water evaporation and help preserve the microstructure (Han et al. 2020).

Fig. 1.

The SEM images of gel section. a common buckwheat; b–d GA-1, GA-3, GA-5. GA-1: BPF with 1% GA addition; GA-3: BPF with 3% GA addition; GA-5: BPF with 5% GA addition

Structural properties of BWT

XRD

XRD measurements were performed in order to evaluate the effects of polyphenols on the crystalline structure of BWT. The XRD patterns, RC and characteristic peak areas of all samples were presented in Fig. 2. and Table 1, respectively. According to previous study, the peak at ~ 17° was the characteristic peak of the B-type structure due to retrogradation of starch, while the peak at ~ 20° was often the characteristic peak of the V-type structure due to the formation of amylose–lipid and/or amylose-phytochemical complex compounds (Wu et al. 2009). As observed in Fig. 2., the peak area of 17° gradually decreased and almost disappeared with increase in GA content, suggesting the significant inhibition by GA adding for starch retrogradation of BWT. From long-range order point of view, compared with control sample, the BWT samples with polyphenol exogenous addition showed lower RC which indicated the formation of double helices in starch had been subject to limitations by polyphenol-starch interaction. The reduction of long-range order also illustrated that polyphenol exogenous addition could inhibit the starch retrogradation in BWT samples.

Fig. 2.

The XRD spectrum of BWT mixing with GA. BWT: buckwheat Wantuo; GA-1-WT: BWT prepared with GA-1; GA-3-WT: BWT prepared with GA-3; GA-5-WT: BWT prepared with GA-5

Table 1.

Structural characteristics of BWT samples determined by XRD and FTIR

Samples	RC/(%)	17° peak area/(%)	20° peak area/(%)	1022/995 cm−1
BWT	22.60	10.10	39.90	1.63 ± 0.04a
GA-1-WT	22.18	9.80	48.70	1.59 ± 0.01ab
GA-3-WT	20.53	3.50	49.10	1.57 ± 0.01b
GA-5-WT	20.26	0.20	49.40	1.63 ± 0.01a

BWT: buckwheat Wantuo; GA-1-WT: BWT prepared with GA-1; GA-3-WT: BWT prepared with GA-3; GA-5-WT: BWT prepared with GA-5; RC: relative crystallinity

Data are expressed as the mean ± standard deviation. Values in the same column with different letters are significantly different at p < 0.05

Meanwhile, the peak area of 20° significantly increased after adding polyphenols, suggesting V-type crystalline conformation of starch in BWT increased continuously due to polyphenols tightly complexed inside the cavity of amylose helices by hydrophobic interaction (Zhu 2015). According to previous literature, GA had more hydrogen bond acceptor and donor sites and was easily to form V-type complex with amylose (Zhu et al. 2021a). Moreover, the proportion of V-type complex could obviously influence the digestibility of BWT.

FTIR

Figure 3. showed the FTIR spectra of BWT and control samples. We could clearly find the characteristic peaks of exogenous GA (standard) and physical mixture of GA and CB flour (GA-CB-PM) control sample could be assigned to the OH stretching vibration of benzene ring (3200–3550 cm⁻¹), in-plane bending OH vibration (1365 cm⁻¹), C=C stretching vibrations of the benzene ring (1450 -1600 cm⁻¹) and C–O/C–C stretching vibrations (1200–1300 cm⁻¹) in Fig. 4a. (Božič et al. 2012). However, the characteristic peaks above cannot be clearly observed in final BWT samples, which could be ascribe to lower additions of polyphenol, partial loss in preparation of BWT and in forming V-type complex as well as the poorer resolution of FTIR. From Fig. 3a., GA-CB-PM control sample showed both characteristic peaks of GA and starch with no significant shift in peak location, whereas the polyphenolic characteristic peaks disappeared and the peaks at 3500 cm⁻¹ blue shifted in BWT samples with exogenous polyphenol addition. All the above phenomena indicated there was no interaction between polyphenol and buckwheat only after simply physical mixing, while there were more hydrogen bonds between exogenous GA and starch in buckwheat as well as more V-type complex during cooking process.

Fig. 3.

The FTIR spectrum of BWT mixing with GA. BWT: buckwheat Wantuo; GA-1-WT: BWT prepared with GA-1; GA-3-WT: BWT prepared with GA-3; GA-5-WT: BWT prepared with GA-5; GA-CB-PM: physical mixture of gallic acid and common buckwheat flour

Fig. 4.

Changes in RDS, SDS and RS contents (a) and curves of in-vitro starch hydrolysis (b) BWT: buckwheat Wantuo; GA-1-WT: BWT prepared with GA-1; GA-3-WT: BWT prepared with GA-3; GA-5-WT: BWT prepared with GA-5

Meantime, as a strong weapon to determinate of the short-range ordered structure of starch, the enlarged FTIR spectras of 800–1200 cm⁻¹ were shown in Fig. 3b. In this range, the FTIR spectrum of BWT with exogenous polyphenol showed similar characteristic peaks with that of control sample, more precisely, similar with that of starch due to its pronounceable intensity. Obvious characteristic peaks at 1164 and 930 cm⁻¹ could be assigned to vibrations of the glucosidic C–O–C bond and the whole glucose ring as well as the skeletal mode vibrations of α 1 → 4 skeletal glycoside bonds, respectively (Dankar et al. 2018). Moreover, the characteristic peaks at 1047, 996 and 1022 cm⁻¹ can give valuable information about the crystalline and amorphous regions of starch for studying the evolution of the ordered and disordered structure. From ratio of 1022 cm⁻¹/996 cm⁻¹ in Table 1 and Fig. 3b., short range ordered structure of BWT with GA first increased and then decreased, indicating moderate GA could facilitate the formation of starch helical structure in BWT, while excess GA might destroy the interaction between starch chains and then decrease short range ordered structure of starch in BWT (Chi et al. 2018). The result of FTIR illustrated GA were capable of directly interacting with the starch in BWT via hydrogen bonding which might facilitate some degree of the formation of short double-helix chains. Moreover, this change in short range ordered structure was more greatly affected by the changes in GA content. In combination with the results from XRD characterization, exogenous addition of GA played two important roles for ordered helical structure: the promotion of starch-GA complex and reduction of starch rearrangement, which would delay starch digestion and inhibit starch retrogradation, respectively.

Eating quality of BWT

TPC

Buckwheat, especially tatary buckwheat contains abundant phenolic compounds which make it be an outstanding bioactive coarse cereal. The TPC of BWT samples were showed in Table S5 (The final result was expressed as mg of GAE in per 100 g of BWT). It had been determined that BWT samples have TPC of only about 4 mg GAE/100 g, which originated loss of water soluble phenolic or polyphenol compounds in the production process of BWT. The lower TPC of original BWT illustrated the need for exogenously adding to some extent. Meanwhile, TPC of in the GA-1-WT, GA-3-WT and GA-5-WT samples were about 626, 1704, 2907 mg GAE/100 g, respectively, which were almost increased proportionally with the increasing GA addition. This phenomenon indicated exogenously added GA could successfully access into BWT and retain. Meanwhile, compared with the actual addition amount of 1, 3, 5% GA, the measured TPC of BWT samples were slightly lower. This difference might be due to errors during determination and/or partially hard-to-extract GA which were tightly complexed with starch. The large number of polyphenols were mainly interacted with starch chains in BWT via weaker hydrogen bonding and could be easily extracted by methanol (Zhu 2015). This hard-to-extract GA might be released gradually throughout the digestion process which could effectively ameliorate the bioactive of BWT. Overall, the above data confirmed the presence of GA in BWT samples which was considered to have antioxidant activity.

Anti-oxidation activity

Antioxidant activity of phenolic compounds originate from a transfer based on hydrogen atoms or a single electron transfer through protons (Yan et al. 2020). From Table S5, the antioxidant activity of BWT samples were significantly enhanced with the increased amount of added GA, as GA are a group of polyphenols, which possess great radical scavenging activity. The ABTS assay is based on an electron transfer mechanism involving ABTS radicals while for the DPPH assay, the reaction between DPPH radicals and antioxidants is achieved through hydrogen atom and electron transfer mechanisms. Compared with DPPH and ABTS assay, FRAP assay is the only assay that directly measures antioxidants or reductants in a sample via combing all electron-donating reductants. Usually, rutin in natural buckwheat is mainly contributed to total antioxidant activity. In our study, exogenously added GA could provide comprehensively similar antioxidant activity via multimechanism. Otherwise, our study also proved that the antioxidation activity was correlated with TPC indeed.

Texture properties

The texture properties of BWT samples were shown in Table 2. The starch retrogradation in BWT were inhibited obviously after adding GA, hindering the gel network formation. Meanwhile, the GA-starch interaction via hydrogen bonding would restrict the bonding between starch chains which also destroying the gel network formation. The above two reasons led to the decreased texture properties of BWT after adding GA (Pan et al. 2019). Moreover, the hardness, cohesiveness, adhesiveness and resilience of BWT samples significantly decreased with the increased amount of added GA while the springiness and chewiness of BWT samples slightly decreased. In addition to the soft taste, the changed texture properties also bring about the amelioration in storage quality which might be worth pursuing in future research.

Table 2.

The texture properties of BWT mixing with GA

Samples	Hardness/(g)	Springiness/(g)	Cohesiveness	Adhesiveness (g/s)	Chewiness(g)	Resilience
BWT	395.19 ± 31.72a	0.96 ± 0.00a	0.84 ± 0.01a	332.75 ± 24.40a	318.32 ± 23.85a	0.48 ± 0.01a
GA-1-WT	290.19 ± 6.95b	0.94 ± 0.01b	0.80 ± 0.01b	232.27 ± 5.35b	218.68 ± 7.50b	0.42 ± 0.01b
GA-3-WT	263.28 ± 5.53c	0.94 ± 0.02ab	0.79 ± 0.04abc	209.16 ± 11.53c	195.90 ± 15.67b	0.42 ± 0.02b
GA-5-WT	218.19 ± 32.01d	0.95 ± 0.01ab	0.76 ± 0.01c	166.09 ± 26.62d	157.02 ± 25.34b	0.37 ± 0.01c

BWT: buckwheat Wantuo; GA-1-WT: BWT prepared with GA-1; GA-3-WT: BWT prepared with GA-3; GA-5-WT: BWT prepared with GA-5

Data are expressed as the mean ± standard deviation. Values in the same column with different letters are significantly different at p < 0.05

We also tested the gel texture properties of BPF samples for comparison (see table S6). The similar trends had been observed. Moreover, from the result of gel section microstructure, adding GA led the gel structure (pore numbers, size and arrangement) change significantly which always influenced the gel texture properties.

In-vitro digestibility

The curves of in-vitro starch hydrolysis and the digestibility, model parameters, calculated hydrolysis indices and predicted glycemic indices (pGI) of various samples were shown in Fig. 4. and in Table S7., respectively. According to difference in digestion time, starch is normally classified into RDS, SDS and RS (Englyst 1992). From Fig. 4a., presence of GA significantly decreased RDS content and correspondingly increased the SDS and RS content. For comparation, we also tested the in-vitro starch hydrolysis of CB flour. The proportion of RDS, SDS and RS were 41.14 ± 0.41%, 32.92 ± 0.54% and 25.94 ± 0.30%, respectively (Table S7). For comparation, the digestibility of original CB flour was measured according to Englyst method. Experimental results showed the RS fraction was significantly increased when CB flour was processed into BWT. This could be attributed to the dense and continuous gel network which can spatially hinder the contacting and binding between starch and enzyme for some degree (Chi et al. 2018). Combined with XRD analysis, the RS could be related to type 5 RS, which was mainly formed by amylose in cereal and lipids or other molecular like GA and with a V-type conformation (Qin et al. 2019). Exogenous addition with GA continuously increased the proportion of RS to 78.48 ± 0.71% while mildly decreasing that of RDS and SDS to 8.62 ± 0.19% and 12.90 ± 0.73%, respectively. This proved that exogenous addition with GA was an effective way to ameliorate the digestibility of BWT.

To predict the glycemic response, the in vitro kinetic of starch digestion was shown in Fig. 4b. and the estimated k and C_∞ are shown in Table S7. From Fig. 4b., the starch hydrolysis product of BWT samples increased gradually and reached a plateau at last, while the hydrolysis rate and the time to reach the plateau were different. For BWT, starch had a rapid hydrolysis rate was observed before 60 min, whereas the starch in GA-1-WT, GA-3-WT and GA-5-WT showed low hydrolysis rate until 180 min. The maximum hydrolysis extents (C_∞) of exogenous addition BWT samples, ranging between 20.4 and 40.0, were significantly lower than that of BWT (40.6). This result was in agreement with the RDS changes and further proved that exogenous addition of GA can reduce the starch digestibility. The kinetic constant (k) reflected the rate of hydrolysis in the early stage showed no significant difference. Generally speaking, the value of k was related to starch hierarchical structure, while the C_∞ was related to other factors like enzyme activity (Chi et al. 2018). Therefore, according to the results of C_∞ and k, the presence of GA in BWT samples influenced the starch digestibility via inhibiting the enzyme activity rather than by influencing starch gel structure.

Meanwhile, pGI values were also obtained from starch digestion curves and presented in Table S7. Obviously, exogenous addition of GA can help reduce pGI values of BWT products and must be more friendly to obesity group and type II diabetic patients. It is worth noting that control BWT had a pGI of around 64 (< 70) which can be divided in moderate GI food. This phenomenon can be due to the helical crystalline structure in starch gel retrogradation. Combining the ordered structure data, when the amount added was 1%,GA acted more like molecular chaperone and could assist the reassembly of starch (Chi et al. 2018) and thus enhanced the ordered structure of starch which led to a slight change of hydrolysis curve and pGI value. With the increase of GA addition, more V-type crystalline formed and the presence of excess GA would both significantly inhibit starch digestion which led to a persistently lowered pGI value. Meanwhile, the changing trends of pGI value were similar with those of RC and peak area at 17° in XRD analysis, which indicated the interaction between GA and starch would disturb the binding between starch and enzyme (Kan et al. 2022). In addition, previous literature believed GA would interact with enzymes during digestion via competing the active site of the enzyme with starch and form phenol-enzyme (Zhu et al. 2021b) or/and starch-phenol-enzyme complex (Yu et al. 2021) which also gave rise to change of structure and inhibited the digestion of starch in BWT samples.

Sensory evaluation

The results of the BWT sensory analysis were shown in Table S8. Exogenous addition of GA did not significantly affect the appearance and texture properties of BWT. All BWT samples had uniform color distribution, white glossiness, smooth surface without cracks, flat and symmetrical cross section, suitable springiness and hardness. Although the viscosity of samples increased slightly, as addition amount of GA increased, the samples was still smooth and did not stick to teeth. In addition, the taste score of the samples decreased gradually with the increase of GA content, which may be due to the own sour taste of GA (Diaz et al. 2022). Considering the Chinese dietary habits, BWT was usually eaten with vinegar and chili peppers, which could weaken the influence of the sour taste indeed. By comprehensive consideration, GA-3-WT had lower digestibility, favorable antioxidant activity and acceptable eating quality.

Conclusions

Exogenous GA with varying addition amount was used to fortify a traditional Chinese starchy food: common buckwheat Wantuo. The effect of exogenous addition GA on physicochemical properties of flour and eating quality were studied. Adding GA could make the flour brighter, less red and more yellow/blue, by exposing more hydrophilic groups, decreasing continuity of starch gel and creating more acid environment, the exogenous GA increased the value of WAI/WSI/SP. Meanwhile, the higher break down value and lower other pasting characteristic value showed addition of GA would make the gelatinization of starch granules more rapidly and easily. The looser and porous gel section structure can be observed after adding GA which can partly explain the decreasing gel texture properties. Furthermore, the softened BWT texture was related to suppressed starch retrogradation. Ordered structural changes of BWT proved the formation of double helices in starch had been subject to limitations and more V-type complex formed indeed after adding GA. Otherwise, more TPC and ameliorated antioxidant activity of BWT had been measured. It is worth noting that adding GA can obviously decrease the glucose release and increase proportion of RS as well as can be an effective way to obtain low GI starchy food.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BWT

Buckwheat Wantuo

GA

Gallic acid

RS

Resistant starch

RDS

Rapidly digested starch

SDS

Slowly digested starch

GOPOD

Glucose oxidase–peroxidase

BPF

Buckwheat-polyphenol flour

WAI

Water absorption index

WSI

Water solubility index

SP

Swelling power

FRAP

Ferric-reducing antioxidant power

pGI

Predicted glycemic index

CB

Common buckwheat

RC

Relative crystallinity

TPC

Total phenol content

GAE

GA equivalents

TE

Trolox equivalents

GA-CB-PM

Physical mixture of GA and CB flour

GA-1

BPF with 1% GA addition

GA-3

BPF with 3% GA addition

GA-5

BPF with 5% GA addition

GA-1-WT

BWT prepared with GA-1

GA-3-WT

BWT prepared with GA-3

GA-5-WT

BWT prepared with GA-5

Author contributions

DW: Formal analysis, Methodology, Investigation, Resources, Data curation, Writing-original draft, Writing-review & editing, Project administration. FG: Methodology, Investigation, Data curation, Writing-review & editing. HM: Methodology, Investigation. RX: Methodology, Investigation. WC: Writing-review & editing. XT: Writing-review & editing, Supervision.

Funding

This work is supported by National Natural Science Foundation of China (NSFC) (Grant Numbers:31901646).

Availability of data and materials

All data generated or analyzed during this study are included in this article and the supplementary material.

Declarations

Conflict of interest

The authors declare that they have no competing interest.

Ethics approval

Not applicable.