Structural and functional variations of octenyl succinylated clustered amylopectin produced by distinctive glycogen branching enzymes

Abstract

Clustered amylopectins (CAPs) were produced using recombinant glycogen branching enzymes from Escherichia coli K12 (EcGBE) and Vibrio vulnificus (VvGBE). The CAPs were esterified with octenyl succinic anhydride (OSA), introducing novel functional characteristics. Vv-CAP demonstrated a reduced apparent molecular weight and a higher prevalence of both short (degree of polymerization (DP) ≤ 5, 13.3%) and medium branches (5 < DP ≤ 12, 58.2%) compared to Ec-CAP. FT-IR spectra confirmed the successful esterification of CAPs with OSA. ¹H-NMR analysis showed the degree of substitution (DS) for OSA-CAPs varied based on the CAP to OSA ratio (DS 0.021–0.058 for Ec-CAP and 0.011–0.066 for Vv-CAP). Esterification with OSA on shortened branches resulted in a slower digestion rate and increased resistant starch proportion. Additionally, this process enhanced the emulsifying properties of CAPs and improved the aqueous solubility of puerarin. OSA-modified CAPs are promising for pharmaceutical applications.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-025-01892-1.

Introduction

Glycogen branching enzyme (GBE), found in the glycoside hydrolase 13 (GH13) or 57 family (GH57), is one of the key enzymes in glycogen synthesis. GBE acts by cleaving an α-1,4-glycosidic linkage and transferring the released α-glucan onto the α-1,6 position of substrates like amylose, amylopectin, and glycogen, introducing branches (Gaenssle et al., 2021). GBEs in the GH13 family share three primary domains: a catalytic (β/α)8 barrel domain, an amino-terminal β-sandwich domain (N-domain), and a carboxyl-terminal domain (C-domain) (Palomo et al., 2009). GBEs are classified into type I and type II based on their amino acid sequences. In comparison to type II GBEs, type I GBEs found in Escherichia coli and Vibrio Vulnificus have an additional N-terminal stretch consisting of 100–150 amino acids. The N-domain of type I GBE includes the N1-domain of ~ 130 amino acids and the N2-domain containing carbohydrate-binding module 48 (CBM48) (Jung et al., 2020). Type I GBEs exhibit different branching patterns depending on the N1-domain, although the mechanism of action has not been elucidated (Jo et al., 2015).

Amylopectin is a highly branched polysaccharide composed of glucose units linked by α-1,4-linear and α-1,6 branched linkages. It serves as both an energy source in the human diet and additives in many foods. The molecular weight and degree of branching of amylopectin determine its functional properties, including water solubility and rheological properties. High molecular weight amylopectin has a large excluded volume promoting intermolecular interaction that reduce its solubility. Consequently, amylopectin is generally insoluble in water due to its substantial molecular weight but becomes soluble when broken down into smaller molecules by acids or enzymes (Raphael et al., 2011). GBEs play a crucial role in this process by cleaving an α-1,4 glycosidic bonds, followed by attaching the released segment onto the α-1,6 position of another linear chain to produce clustered amylopectin (CAP) with a lower molecular weight that is soluble in water.

Octenyl succinic anhydride (OSA) is a commonly used esterifying agent in the food industry for structurally modifying starches. It is permitted for use in foods at a maximum concentration of 3% based on dry starch weight (Siroha et al., 2022). During esterification with OSA, hydroxyl groups of starch are partially substituted with hydrophobic octenyl succinyl groups imparting hydrophilic starch with amphiphilic properties. OSA-modified starches (OSA starches) have been extensively studied for their ability to emulsify and encapsulate poorly soluble bioactive compounds (Sweedman et al., 2013; Wang et al., 2022). Zhang et al. (2018) investigated the effects of structural parameters, such as molecular size and chain length distributions, degree of branching, and degree of substitution, on the emulsifying properties of OSA-modified starches. Cheuk et al. (2015) utilized OSA-modified starch to stabilize rice bran oil emulsion incorporating coenzyme Q10. Gao et al. (2023) encapsulated doxorubicin hydrochloride (DOX) using OSA-modified starch granules grafted with folic acid for efficient delivery into the colon.

Therefore, it was hypothesized that the different branching patterns of two distinct bacterial GBEs would lead to the formation of clustered amylopectins, each exhibiting structural variations. These modifications are expected to confer diverse functional characteristics to amylopectin. Moreover, the functional properties of clustered amylopectins could be further enhanced by esterification with OSA, which introduce a hydrophobic segment. In the present study, amylopectin from waxy corn was modified with two different bacterial GBEs obtained from E. coli and V. vulnificus, respectively. The resulting products were esterified with OSA, and their functional attributes, such as digestibility, emulsifying ability, and the ability to solubilize hydrophobic bioactive compounds, were evaluated.

Materials and methods

Production of glycogen branching enzymes

E. coli MC1061 was used as the host for gene manipulation (Casadaban and Cohen, 1980). Plasmid p6xHis119, encoding glycogen branching enzyme from E. coli K12 (EcGBE) or V. vulnificus MO6-24/O (VvGBE), was utilized as a cloning vector as described in the previous study (Jung et al., 2020). The recombinant E. coli MC1061 cells were cultured in Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) supplemented with 50 μg/mL kanamycin at 37 °C for 16 h.

Isolation and purification of enzymes

Cells were collected by centrifugation at 6000×g and 4 °C for 20 min, then resuspended in 50 mM Tris buffer (pH 7.0) containing 300 mM NaCl and 10 mM imidazole. The suspension was lysed with ultrasonic processor (Q500, QSonica, Newtown, CT, USA) in an ice bath, followed by centrifugation and filtration. The soluble cell extract was purified using an AKTA pure protein purification system (Cytiva, Marlborough, MA, USA) equipped with a nickel-nitrilotriacetic acid (Ni–NTA) resin. The purified enzyme was stored at 4 °C prior to analysis.

Enzyme activity assay

The enzyme concentration was estimated using a Bradford assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard protein and the enzyme activity was assessed by Lugol’s iodine method (Tran et al., 2024). The enzyme solution was mixed with 1% amylose as a substrate in a 50 mM MOPS buffer (pH 7.5) and incubated at 30 °C for 10 min. The enzyme was inactivated by adding 50 μL of 0.1 N NaOH and neutralized with 0.1 N HCl. Then, 0.1 mL of the reaction mixture was added into 1 mL of iodine/potassium iodide solution, and the absorbance was measured at 620 nm. One unit of glycogen branching enzyme activity was defined as the amount of enzyme required to reduce 1 mg of amylose per minute under the specified assay conditions.

Synthesis and characterization of clustered amylopectin

The clustered amylopectin was synthesized by the method of Tran et al. (2024) with slight modifications. Fifty grams of amylopectin from waxy corn were dissolved in 700 mL DW and gelatinized by autoclaving at 121 °C for 15 min. The gelatinized amylopectin was cooled to room temperature and diluted in a 50 mM MOPS buffer (pH 7.5) containing 0.04% sodium azide, then pre-incubated at 30 °C. Enzymatic reaction was initiated by adding GBEs and continued for 72 h. After enzyme inactivation in boiling water for 30 min, the solution was centrifuged at 12,000 × g and 20 °C for 10 min. The supernatant was collected, mixed with two volumes of ethanol, and allowed to stand at − 20 °C for 12 h. The precipitate was isolated, washed with DW twice, and dried at 50 °C for 24 h. The synthesized clustered amylopectin (CAP) powder was stored in a desiccator prior to further use.

The apparent molecular weight distribution of the synthesized CAP was determined using a size exclusion chromatography system (LC-Forte/R YMCKOREA, Sungnam, Korea) equipped with a refractive index detector. Compounds were separated on a Bio-Gel P2 column (Bio-Rad). DW was used as a mobile phase at a flow rate of 1.0 mL/min. Pullulans (dextran, 6100 kDa; P-800, 805 kDa; P-400, 366 kDa; P-200, 200 kDa; P-100, 113 kDa; P-50, 48.8 kDa; P-20, 21.7 kDa; P-10, 10 kDa; P-5, 3.2 kDa) purchased from Showa Denko (Tokyo, Japan) were used as standards.

The side chain distribution of CAP was determined using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) after enzymatic hydrolysis of branched side chains. CAP, dissolved in a 50 mM sodium acetate buffer (pH 4.0), was pre-incubated at 40 °C. Enzymatic reaction was initiated by adding 50 U/mg isoamylase (Megazyme, Wicklow, Ireland) and continued for 72 h. Debranched fractions were analyzed by a DX-500 ion chromatography system (HPAEC, Dionex, Sunnyvale, CA, USA) equipped with a PDA (ED40, Dionex) and a CarboPac PA-1 column (4 × 250 mm, Dionex). For the detailed methods, please refer to the previous study (Jung et al., 2020).

Synthesis and characterization of octenyl succinylated clustered amylopectin

The octenyl succinylated clustered amylopectin was synthesized according to the method of Wei et al. (2023) with modifications. Different concentrations of CAP at 15%, 30%, and 45% (w/v) in DW were mixed with a 10% (v/w of CAP) octenyl succinic anhydride (OSA) solution at 35 °C. The reaction mixture was agitated constantly for 1 h at a pH of 8.5. After terminating the reaction by adding 1 N HCl, the reaction product was precipitated by adding ethanol, isolated by centrifugation (8000 rpm for 20 min), washed twice, and lyophilized. The resulting product was designated as octenyl succinylated clustered amylopectin (OSA-CAP). Octenyl succinylated maltodextrin (OSA-MD) was also synthesized under the same conditions for comparison. A commercial maltodextrin with DE of 10 (Daesang, Seoul, Korea) was used at 15%, 30%, and 45% (w/v).

The structure was determined using Fourier transform infrared (FTIR) spectroscopy. Each powder sample was mixed with potassium bromide (KBr) in a 1:100 ratio and pelleted. FTIR spectra were recorded using a Nicolet iS50 FTIR Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in the wavenumber range from 4000 to 400 cm⁻¹. The degree of OSA substitution (DS) for both OSA-CAPs and OSA-MD was determined using ¹H-NMR. Each powder sample (15 mg) was dissolved in 1 mL of deuterium oxide and transferred into an NMR tube. NMR spectra were recorded on a BRUKER AVANCE III 600 spectrometer equipped with a TCI Cryoprobe (Bruker Corporation, Billerica, MA, USA). The NMR spectrometer was operated at a Larmor frequency of 600 MHz at 40 °C. The degree OSA substitution was estimated using the following equation (Shih and Daigle, 2003):

$$\text{Degree of substitution}\left(\text{DS}\right)=\frac{{\text{I}}_{0.89}}{3\times {\text{I}}_{5.41}}$$

where, I is the integration value of the assigned peak.

In vitro digestibility

The digestion rates and extents of CAPs and OSA-CAPs were evaluated using two different enzymes: porcine pancreatic α-amylase (PPA) (Megazyme) and amyloglucosidase (AMG) (AMN06001, Novozyme, Denmark) (Jung et al., 2020). For PPA digestion, the process was initiated by adding 0.09 U/mL of PPA into a solution containing 1 mg/mL of either CAP or OSA-CAP, along with 0.2 mg/mL BSA and 1 mM CaCl₂, in a 50 mM MOPS buffer (pH 6.9). The digestion process conducted at 37 °C for 4 h. At specific time intervals during the digestion, an aliquot was withdrawn from the reaction mixture, and the resulting reaction product was quantified by the DNS assay.

In the case of AMG digestion, the process began with the addition of 0.1U/mL of AMG into a solution containing 1 mg/mL of either CAP or OSA-CAP in a 50 mM sodium acetate buffer (pH 4.5). The digestion was conducted at 55 °C for 4 h. At predefined time intervals during the digestion, an aliquot was withdrawn from the reaction mixture and the resulting reaction product (glucose) was quantified using the GOPOD assay.

Two phase exponential decay function was applied to the experimental data to evaluate the kinetics of digestion of CAPs and OSA-CAPs. The derived fitting parameters were used to estimate the reaction rates and digestible/resistant fractions. Two phase exponential decay function applied is as follows (Bello-Perez et al., 2019):

$$Y\left(t\right)=Y_{\infty }+Y_{\mathit{RDS}}{e}^{{-k}_{{\mathit{RDS}}^t}}+Y_{\mathit{SDS}}{e}^{{-k}_{{\mathit{SDS}}^t}}$$

where, Y_∞ corresponds to the equilibrium concentration. The terms k_RDS and k_SDS represent the digestion rates of rapidly digestible fraction (RDS) and slowly digestible fraction (SDS), respectively. Absolute values of Y_RDS and Y_SDS correspond to the RDS and SDS fractions, respectively. Resistant starch (RS) fraction was calculated by excluding the SDS and RDS fractions from the total starch.

Emulsifying ability

The emulsifying ability of CAPs and OSA-CAPs was evaluated by comparing the droplet size of oil-in-water (O/W) emulsions formulated with CAPs, OSA-CAPs, OSA-MD, and commercially available OSA-starch (Daesang Inc., Seoul, Korea) and sucrose fatty acid ester (IlshinWells Inc., Seoul, Korea), used as emulsifiers. To prepare the emulsions, a ternary mixture consisting of canola oil (10%), each emulsifier (0.25%), and DW were homogenized using a high-shear homogenizer (Polytron PT-2100, Kinematica AG, Switzerland) at 10,000 rpm for 1 min. Subsequently, the pre-emulsions were passed through a high-pressure homogenizer (Panda PLUS 2000, GEA, Niro Soavi, Italy) three times at 600 bar to further reduce the droplet size. The droplet size distribution of the emulsions was measured using photon correlation spectroscopy (PCS) with a Matersizer Hydro 2000 particle size analyzer (Malvern Instruments, Malvern, UK). The average droplet size was reported as the volume-weighted size distribution.

Solubilizing capacity for puerarin

Puerarin, with its limited aqueous solubility of 2.58 mg/mL (Xie et al., 2013), was utilized to evaluate the effectiveness of OSA-CAP as a solubilizer. Ten milligrams of puerarin were mixed with OSA-CAP at various mixing ratios (1:0, 1:1, 1:2, 1:3, and 1:4, w/w) in 1 mL DW and treated with a probe ultrasonic processor (QSonica) equipped with a microtip at 30% amplitude for 1 min. The mixtures were allowed to stand at room temperature for 30 min. The undissolved puerarin fraction was removed using a 0.2 μm syringe filter after centrifugation. The concentration of puerarin in the filtrate was determined by measuring the absorbance at 250 nm.

Results and discussions

Preparation of glycogen branching enzymes and synthesis of clustered amylopectin

Recombinant glycogen branching enzymes originated from E. coli K12 (EcGBE) and Vibrio vulnificus MO6-24/O (VvGBE) were successfully expressed in E. coli MC1061 cells and purified as presented in Supplementary Fig. 1 (Fig. S1). Both purified EcGBE and VvBGE were revealed as bands with a molecular weight of approximately 75 kDa on SDS-PAGE gel, which is consistent with a previous report (Wang et al., 2019).

Clustered amylopectin (CAP) was synthesized by reacting gelatinized amylopectin as a substrate with EcGBE (Ec-CAP) or VvGBE (Vv-CAP). The yield of Ec-CAP and Vv-CAP was 87.4% and 90.4%, respectively. The side chain length distribution peaks and normalized peak areas of the synthesized Ec-CAP and Vv-CP are presented in Fig. S2). Both enzymes exhibited branching activity on amylopectin but with different patterns. EcGBE slightly increased the peak areas of short side-chains (5 < DP ≤ 12), accompanied by the reduction of longer chains (DP ≥ 13), as observed in Fig. S2a and b. VvGBE transferred short side chains to the product much more than EcGBE (Fig. S2c). Moreover, VvGBE showed a unique branching pattern that forms a very short branch (DP 4) in the product, which was nearly absent in EcGBE. This result is consistent with a previous report (Jo et al., 2015). The branching patterns between EcGBE and VvGBE were compared by normalizing the peak areas (Table 1). In the Ec-CAP, the percentage of side chains with 5 < DP ≤ 12 increased to a less extent from 23.21 to 29.21%, accompanied by the reduction of those with DP ≥ 13 from 76.58% to 70.22%. On the other hand, the percentage of side chains with DP ≤ 5 and 5 < DP ≤ 12 in the Vv-CAP increased dramatically from 0.21% to 13.3% and 23.21% to 58.17%, respectively, while that of long side chains (DP ≥ 13) decreased to a great extent from 76.6% to 28.53%. The apparent molecular weight of both Ec-CAP and Vv-CAP was reduced by 10³ ~ 10⁵-fold compared to that of intact amylopectin (Table 1 and Fig. S3). The apparent molecular weight of Vv-CAP was approximately 1.5-fold lower than that of Ec-CAP, as reported in a previous study (Jung et al., 2020). Regarding the distribution of side chains, it can be deduced that Vv-CAP has a more homogeneous branching pattern, exhibiting a greater portion of shorter side chains (DP ≤ 12) in contrast to Ec-CAP, which has a larger portion of longer side chains (DP ≥ 13). Thus, the unique branching pattern of VvGBE seems to reduce the molecular volume of CAP compared to that of EcGBE. Vv-CAP, characterized by a higher proportion of shorter side chains, likely possesses a more compact structure. In this structure, the molecules are more densely packed, occupying a relatively smaller space. Such compactness leads to a reduction in the hydrodynamic volume, which is the volume occupied by the molecule in solution.

Table 1.

Side chain length distribution and apparent molecular weight in the clustered amylopectin synthesized by EcGBE and VvGBE

Sample	Side chain length distribution (%)			Apparent molecular weight (Dalton)
	DP ≤ 5a	5 < DP ≤ 12	13 ≥ DP
Amylopectin	0.21	23.2	76.6	7.0 × 107–5.7 × 109
Ec-CAPb	0.57	29.2	70.2	6.2 × 104
Vv-CAPc	13.3	58.2	28.5	4.0 × 104

Amylopectin was derived from waxy corn and its molecular weight was obtained from a previous report (Jackson et al., 1989)

^aDP: degree of polymerization

^bEc-CAP: clustered amylopectin synthesized by EcGBE

^cVv-CAP: clustered amylopectin synthesized by VvGBE

Synthesis and characterization of octenyl succinylated clustered amylopectin

Octenyl succinylated clustered amylopectin (OSA-CAP) was successfully synthesized through esterification between CAP and OSA. The FTIR spectra of OSA-modified Ec-CAP (OSA-Ec-CAP) and Vv-CAP (OSA-Vv-CAP) are shown in Fig. 1. Broad bands in the range of 3600–3000 and 2950–2850 cm⁻¹ in all spectra indicate the stretching vibrations of the –OH and alkane C–H groups, respectively. These bands are less useful in determining structural differences due to their abundance. The band at 1653 cm⁻¹ corresponds to the bound water interacting with the CAP. The appearance of a new band at 1724 cm⁻¹ in the OSA-Ec-CAP and OSA-Vv-CAP is attributed to the stretching vibration of the ester group (–COO–), indicating the formation of an ester linkage between CAP and OSA (Wei et al., 2023). Another new band at 1572 cm⁻¹ is attributed to the stretching vibration of the carboxylate (RCOO⁻) of OSA (Wang et al., 2010). From the results, it was confirmed that OSA was successfully attached to CAP. The formation of an ester linkage between CAP and OSA was further confirmed by ¹H-NMR spectroscopy as shown in Fig. S4. The peaks in the range of 3.49–4.0 ppm correspond to the hydrogen atoms at positions 2, 3, 4, and 5 of glucose units. The peak at 5.01 ppm indicates the α-1,6 linkage of amylopectin (Whitney et al., 2016). The peak at 0.8—0.87 ppm corresponds to the terminal methyl protons in the octenyl succinylated group. The peak at 5.2–5.6 ppm represents the equatorial proton of anhydroglucose unit (AGU) of amylopectin (Simsek et al., 2015). The peaks at 1.2 ppm correspond to the methylene group of the succinic anhydride (Whitney et al., 2016). The degree of substitution (DS) of OSA-Ec-CAP and OSA-Vv-CAP was estimated by comparing the integrated areas of the methyl proton peaks in the substituted octenyl succinylated group and is presented in Table 2. The DS tended to increase with an increasing concentration of CAP. In particular, DS increased dramatically at the CAP concentration of 45% in both OSA-Ec-CAP and OSA-Vv-CAP. The DS of OSA-Ec-CAP was higher than OSA-Vv-CAP at lower CAP concentrations (15 and 30%), but was lower at higher CAP concentration (45%). Based on the DS results, OSA-CAPs prepared at the CAP concentration of 45% were used as a solubilizing agent for enhancing the aqueous solubility of puerarin.

Fig. 1.

FTIR spectra of a OSA-MD b OSA-Ec-CAP, and c OSA-Vv-CAP: The percentage behind each product name indicates the concentration of CAP based on the total weight of the reaction mixture. OSA-MD, octenyl succinylated maltodextrin; OSA-Ec-CAP, octenyl succinylated clustered amylopectin synthesized by EcGBE; OSA-Vv-CAP, octenyl succinylated clustered amylopectin synthesized by VvGBE

Table 2.

Effect of clustered amylopectin concentration on the degree of substitution

OSA- CAP	CAP concentration (%)	Degree of substitution
OSA-Ec-CAPa	15	0.021
	30	0.022
	45	0.058
OSA-Vv-CAPb	15	0.011
	30	0.018
	45	0.066

Octenyl succinic anhydride (OSA) concentration was fixed at 10% (CAP weight basis, v/w)

^aOSA-Ec-CAP: octenyl succinylated clustered amylopectin synthesized by EcGBE

^bOSA-Vv-CAP: octenyl succinylated clustered amylopectin synthesized by VvGBE

In vitro digestibility

The digestion rates and extents in both CAPs and OSA-CAPs when subjected to the enzymes PPA and AMG are illustrated in Fig. S4. Under PPA treatment, all samples exhibited a rapid initial degradation, eventually reaching an equilibrium level. Ec-CAP revealed the highest digestion degree, followed by OSA-Ec-CAP, Vv-CAP, and OSA-Vv-CAP as depicted in Fig. S4a. After treatment with AMG, there was an initial sharp increase in digestion degree, similar to the behavior of PPA. However, unlike PPA, the digestion process continued at a slower rate over time. In this case, Ec-CAP had the highest degree, followed by Vv-CAP, OSA-Ec-CAP, and OSA-Vv-CAP (Fig. S4b). The extent of digestion was influenced by the structural characteristics of the CAPs and OSA-CAPs, as well as the specific enzymes used. The variation in digestion between Ec-CAP and Vv-CAP might be attributed to differences in different side chain branching patterns. VvGBE enzyme appeared to preferentially transfer shorter side chains compared to EcGBE as described in the side chain distribution and apparent molecular weight analyses. Additionally, the OSA modification significantly inhibited the hydrolysis of CAPs.

A two-phase exponential decay model was applied to access the digestion kinetics, and the derived parameters are listed in Table 3. The rate constant was notably higher in the presence of PPA compared to AMG. When subjected to PPA treatment, OSA-Ec-CAP exhibited the highest rate constant for the rapidly digestible fraction (RDS), followed by Ec-CAP, Vv-CAP, and OSA-Vv-CAP. On the other hand, for the slowly digestible fraction (SDS), Ec-CAP exhibited the highest rate constant, followed by Vv-CAP, OSA-Ec-CAP, and OSA-Vv-CAP. Following AMG treatment, the highest rate constant for RDS was observed in OSA-Ec-CAP, followed by Vv-CAP, Ec-CAP, and OSA-Vv-CAP. However, for SDS, the rate constants for Ec-CAP, Vv-CAP, and OSA-Ec-CAP were similar, while OSA-Vv-CAP showed a notably lower rate constant. It appeared that there was no strong correlation between the degree of hydrolysis and the rate constant for both PPA and AMG treatments. However, it was evident that the OSA modification led to a reduction in both the digestion degree and the rate constant of Vv-CAP. Regarding the proportions of RDS, SDS, and RS, Ec-CAP showed a balanced distribution among these fractions when treated with PPA. In contrast, Vv-CAP exhibited a higher proportion of RS compared to RDS and SDS. The OSA modification of CAP resulted in an increase in the RS proportion at the expense of both RDS and SDS fractions. Upon treatment with AMG, Ec-CAP demonstrated the highest fraction of RDS, accompanied by moderate SDS and a low RS fraction. On the other hand, Vv-CAP revealed a more balanced distribution, with a higher RS fraction compared to Ec-CAP. The OSA modification led to an increase in the RS fraction, while concurrently decreasing both RDS and SDS fractions.

Table 3.

Estimated digestion kinetic parameters and digestible fractions for clustered amylopectins and octenyl succinylated clustered amylopectins under various enzyme treatments

Enzyme	Samplesc	Rate constant (min−1)		Fractions (%)d			R2
		kRDS	kSDS	RDS	SDS	RS
PPAa	Ec-CAP	10.8577	0.0938	35.4	32.3	32.3	0.9784
	Vv-CAP	4.4534	0.0659	22.1	20.0	57.9	0.9728
	OSA-Ec-CAP	11.9423	0.0420	28.9	23.2	47.9	0.9628
	OSA-Vv-CAP	0.2105	0.0084	25.3	9.4	65.3	0.9950
AMGb	Ec-CAP	0.1163	0.0096	56.7	29.2	14.1	0.9969
	Vv-CAP	0.1345	0.0091	42.3	22.9	34.8	0.9946
	OSA-Ec-CAP	0.1629	0.0095	39.2	19.5	41.3	0.9961
	OSA-Vv-CAP	0.1451	0.0016	29.7	38.7	31.6	0.9954

^aPPA: porcine pancreatic α-amylase

^bAMG: amyloglucosidase

^cSamples: Ec-CAP, clustered amylopectin synthesized by EcGBE; Vv-CAP, clustered amylopectin synthesized by VvGBE; OSA-Ec-CAP, octenyl succinylated clustered amylopectin synthesized by EcGBE; OSA-Vv-CAP, octenyl succinylated clustered amylopectin synthesized by VvGBE

^dFractions: RDS rapidly digestible starch, SDS slowly digestible starch, RS resistant starch

Among the tested samples, OSA-modified Vv-CAP exhibited the highest proportion of resistant starch (RS) after both enzymatic treatments. Since RS is known to contribute to various health benefits such as improved glycemic response, enhanced gut health, and increased satiety, OSA-Vv-CAP may be considered the most promising candidate in terms of health-promoting potential. Therefore, despite its relatively lower digestion rate, the enhanced RS formation of OSA-Vv-CAP suggests that it could be advantageous for developing functional food ingredients with improved physiological effects.

Emulsifying ability

The emulsifying ability was assessed by measuring and comparing the droplet sizes in O/W emulsions, formulated with CAPs and OSA-CAPs, OSA-MD, and commercially available OSA-starch and sucrose fatty acid ester as emulsifiers. The average droplet size varied with the type of emulsifiers used (Fig. 2). Emulsions prepared with Ec-CAP and Vv-CAP had comparatively larger droplet sizes than the others, suggesting these CAPs possess lower emulsifying abilities. In contrast, the decrease in average droplet size for emulsions prepared with OSA-modified Ec-CAP and Vv-CAP indicates that structural modifications with OSA can enhance the emulsifying abilities of CAPs. Typically, the glucose units that make up the entire CAP structure are highly hydrophilic. However, introducing a hydrophobic chain to these hydrophilic CAPs through OSA modification adds amphiphilic properties, making them suitable for localization at the oil–water interface. The average droplet sizes of emulsions prepared with OSA-modified CAPs were similar with those prepared with commercially available OSA-starch and sugar fatty acid ester, although it was larger than the one prepared with OSA-MD.

Fig. 2.

Average droplet size of oil-in-water (O/W) emulsions formulated using CAPs and OSA-CAPs: Ec-CAP-E, O/W emulsion formulated using Ec-CAP; Vv-CAP-E, O/W emulsion formulated using Vv-CAP; OSA-Ec-CAP-E, O/W emulsion formulated using OSA-Ec-CAP; OSA-Vv-CAP-E, O/W emulsion formulated using OSA-Vv-CAP; OSA-MD-E, O/W emulsion formulated using OSA-MD; OSA-starch-E, O/W emulsion formulated using commercial OSA-starch; Sugar ester-E, O/W emulsion formulated using sugar fatty acid ester

Solubility enhancement of puerarin

Puerarin is one of the biologically active isoflavones abundant in many plants and herbs. Various biological activities, such as vasodilatory, cardioprotective, anti-diabetic, anti-inflammatory, anti-osteoporotic, and antioxidant activities have been reported (Zhou et al., 2014). However, its low water solubility limits the application in foods and lowers oral bioavailability upon consumption. Thus, the water solubility of poorly soluble puerarin should be enhanced to guarantee its practical food applications and oral bioavailability. For this purpose, the synthesized OSA-CAPs were used as a solubilizer to improve the water solubility of puerarin as shown in Fig. 3. The maximum water solubility of pure puerarin was 2.14 mg/mL. The water solubility of puerarin tended to increase with an increasing OSA-Ec-CAP concentration, and the maximum solubility increased by 2.1-fold at the OSA-Ec-CAP concentration of 40 mg/mL. However, the aqueous concentration of puerarin gradually decreased regardless of the OSA-Ec-CAP concentration during the period of storage for 5 days. Similarly, the concentration of puerarin gradually increased with increasing OSA-Vv-CAP concentration, and the maximum water solubility of puerarin increased by 2-fold at the OSA-Vv-CAP concentration of 40 mg/mL. Furthermore, no remarkable changes in the aqueous concentration of puerarin were observed during the period of storage for 5 days after solubilization.

Fig. 3.

Effects of the concentration of a OSA-Ec-CAP and b OSA-Vv-CAP on the aqueous solubility and stability of puerarin: The concentration of puerarin was fixed at 10 mg/mL: PRN, pure puerarin; PRN/OSA-Ec-CAP, puerarin with OSA-Ec-CAP at a specified mass ratio; PRN/OSA-Vv-CAP, puerarin with OSA-modified Vv-CAP at a specified mass ratio

In this study, we demonstrated that the OSA-modified CAPs not only effectively improved the solubility and stability of puerarin but also modified the digestibility and emulsifying properties of CAPs. The concentration-dependent solubility enhancement achieved using both EcGBE- and VvGBE-derived CAPs with OSA suggests potential applications in enhancing the delivery and bioavailability of bioactive compounds. These findings indicate the considerable potential of OSA-modified CAPs in food and pharmaceutical applications, particularly for the improved formulation and functionality of poorly soluble compounds like puerarin. Furthermore, no remarkable changes in the aqueous concentration of puerarin were observed during the period of storage for 5 days after solubilization.

These results indicate that OSA-Vv-CAP was more effective than OSA-Ec-CAP not only in enhancing the initial solubility of puerarin but also in maintaining its solubilized state during storage. This superior solubilizing and stabilizing performance suggests that OSA-Vv-CAP may serve as a more promising delivery matrix for poorly soluble bioactives like puerarin in functional food and nutraceutical applications.

Supplementary Information

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Funding

This work was funded by National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST), 2021R1A2C2006195, Jong-Tae Park, National Research Foundation of Korea (NRF), 2022R1A4A1033015, Jong-Tae Park.

Data availability

Data will be made available on request.

Declarations

Conflict of interest

The authors have not disclosed any competing interests.