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. 2020 Aug 31;5(36):22793–22799. doi: 10.1021/acsomega.0c01813

Bio-Based, Flexible, and Tough Material Derived from ε-Poly-l-lysine and Fructose via the Maillard Reaction

Kazunori Ushimaru 1, Tomotake Morita 1, Tokuma Fukuoka 1,*
PMCID: PMC7495479  PMID: 32954127

Abstract

graphic file with name ao0c01813_0006.jpg

We report a bio-based, soft, elastic, and tough material prepared from a mixture of ε-poly-l-lysine (ε-PL) and d-fructose. The obtained complex was insoluble in water, whereas its ingredients had high water solubility. This complex was likely formed via Schiff base formation and subsequent rearrangement reactions, that is, the Maillard reaction, because the reaction occurred between reducing sugars and cationic polyelectrolytes having primary and secondary amino groups. The progress of the Maillard reaction was investigated by proton nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. Mechanical properties of the complexes were evaluated by tensile testing, and the properties of the optimized complex [ε-PL/fructose = 60:40 (w/w), maximum stress = 27.9 MPa, strain at break = 46%, Young’s modulus = 741.6 MPa] resembled those of some petroleum-based plastics. Additionally, the ε-PL/fructose complex displayed antimicrobial activity against Bacillus subtilis. These ε-PL/fructose complexes have biological properties such as antimicrobial activity, low toxicity toward mammals, and biodegradability, which are attributable to the intrinsic nature of ε-PL, as well as enhanced mechanical properties and water resistance compared with pure ε-PL.

Introduction

Polymers, which are essential materials for today’s society, are used for the production of a wide variety of structural items. However, widespread use of polymers has led to societal issues, such as large-scale consumption of fossil fuels and plastic pollution in the ocean.1,2 Hence, bio-based polymers are of particular interest because they are obtained from renewable resources and are mostly biodegradable.3,4

ε-Poly-l-lysine (ε-PL), a representative commercial bio-based polymer, is a polyamide composed of amino acids (l-lysine) produced from sustainable resources such as sugars and glycerol by microbial fermentation by Streptomyces.5−8 A unique structural feature of ε-PL is the many cationic amino groups on the side chains, which provide this polymer with excellent antimicrobial activity and water solubility. ε-PL is used as a food preservative because of its high antimicrobial activity and low toxicity toward mammals.5,6,9−12

ε-PL can be considered a high-performance bio-based plastic because of the good stability of its amide bonds compared with ester bonds. For example, although polycaprolactam (nylon 6) and poly(ε-caprolactone) have comparable monomer structures aside from their chemical bonding (i.e. the C6 monomer unit linked by amide and ester bonds, respectively), thermal degradation onsets are clearly different (about 350 and 280 °C, respectively).13,14 This superior stability of the bio-based polyamide compared with polyester suggests new applications in harsh environments. The stability and feasible manufacture of ε-PL makes it a good bio-based polymer candidate for structural items.

Despite these many benefits of ε-PL, applications to structural items, for example, moldable materials, have rarely been reported. The low molecular weight of ε-PL leads to poor moldability, although it is thermoplastic when dry and has a glass transition temperature of 88 °C and melting point of 173 °C.15 These limitations have restricted the development of solid ε-PL to a soft hydrogel16,17 and a few other materials.18−21 To overcome the poor moldability, we increased the apparent molecular weight of ε-PL by forming a molecular complex via ionic interactions with an anionic detergent18 or a lignin derivative.19

To further develop ε-PL-based materials, we tried to improve our ionic complexes and create novel moldable materials from ε-PL via mechanisms other than ionic interactions. During the latter investigation, we unexpectedly found that a mixture of ε-PL with fructose formed a clear orange, soft, and elastic solid.

Herein, we report the mechanistic, chemical, and mechanical properties of the complex to aid the development of novel moldable materials from ε-PL. This complex was produced via the Maillard reaction and had good mechanical properties that resembled those of some petroleum-based plastics.

Results and Discussion

Preparation of the Cationic-Polyelectrolyte/Fructose Complexes

As noted above, we found that mixing and drying an 80/20 (w/w) ε-PL/fructose mixture, in the absence of other chemical reagents, formed a soft and elastic complex. The ε-PL solutions with and without fructose were almost colorless and could not be distinguished by their appearance before drying. After drying, the pure ε-PL became white and readily fractured into brittle fragments. However, the ε-PL/fructose mixture dried to an orange-colored, soft, and elastic sheet (Figure 1). This elastic sheet was insoluble in water, whereas the dried ε-PL without fructose could be redissolved in water.

Figure 1.

Figure 1

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Photographs of pure ε-PL and ε-PL/fructose mixtures before and after drying. The weight ratio of ε-PL to fructose was 80:20.

The color and insolubility of the mixture suggested that this phenomenon might be due to the formation of a Schiff base between amino groups and a reducing sugar, followed by cross-linking via rearrangement of the Schiff base. This reaction is well-known as the Maillard reaction.22−25 To investigate whether the Maillard reaction occurred in the ε-PL/fructose mixtures, we prepared a mixture of ε-PL with a nonreducing sugar (sucrose) instead of fructose. The addition of sucrose did not affect the color or mechanical features (Figure S1 in the Supporting Information). This result strongly indicated that the soft and elastic complexes formed from ε-PL and fructose through the Maillard reaction.

The Maillard reaction is a common reaction in many amine compounds. Hence, we also evaluated this phenomenon with other cationic polyelectrolytes. We prepared cationic-polyelectrolyte/fructose complexes using four cationic polyelectrolytes having different structures, that is, poly(allylamine) (PAA; primary amino group), linear poly(ethyleneimine) (linear PEI; secondary amino group), branched poly(ethyleneimine) (branched PEI; coexisting primary, secondary, and tertiary amino groups), and poly(diallyldimethylammonium chloride) (PDADMACl; quaternary ammonium group). These four cationic polyelectrolytes were mixed with fructose at the cationic-polyelectrolyte/fructose weight ratio of 80:20, and the solution was then dried in the same manner as the ε-PL/fructose mixture.

These cationic-polyelectrolyte/fructose mixtures also changed color, from clear or pale yellow to orange, except for the PDADMACl mixture. In that case, a soft and elastic sheet formed without the addition of fructose, and the appearance and mechanical features were unaffected by the addition of fructose (data not shown; the mechanical figures of merit of a pure PDADMACl sheet were reported previously26). This indicated the absence of reactivity between a quaternary ammonium group and fructose. The other cationic polyelectrolytes (PAA, linear PEI, and branched PEI) showed similar coloring (orange) and formed soft and elastic sheets but were brittle solids or sticky liquids in the absence of fructose or the presence of sucrose (Figure S1 in the Supporting Information). These results indicated that the orange coloring and alteration of mechanical features required the reaction of primary and/or secondary amino groups with a reducing sugar, that is, the Maillard reaction.

Verification of the Maillard Reaction

The Maillard reaction was also verified by proton nuclear magnetic resonance (1H NMR) spectroscopy. We selected PAA as the model compound because of its simple molecular structure compared with the other cationic polyelectrolytes used in this study. A 70:30 PAA/fructose solution was prepared at a low polymer concentration to avoid solidification and incubated at 40 °C for 48 h. The light brown reacted solution was analyzed by 1H NMR spectroscopy. Figure 2 shows the 1H NMR spectra of the PAA/fructose solution before and after incubation. The peaks at about 3.5–3.6 ppm (assigned as protons on C-1;27Figure 2b) disappeared after incubation. This suggested that a structural change occurred around the C-1 carbon, corresponding to the formation of a Schiff base during the initial step of the Maillard reaction (because this reaction involves the carbonyl group on C-2 next to C-1).28,29 Furthermore, small peaks appeared over the broad range of 3.2–4.0 ppm (particularly from 3.3 to 3.5 ppm). These new peaks would be assigned to the formation of new chemical bonds via the Maillard reaction.

Figure 2.

Figure 2

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1H NMR spectra of PAA/fructose mixtures before (black solid line) and after (red dotted line) reaction. (a) Full spectrum and (b) enlarged spectrum from 3.0 to 4.5 ppm with the assignment of peaks. The reaction was performed at 40 °C for 48 h. The PAA/fructose weight ratio was 70:30.

As a supplemental analysis, we analyzed the cationic-polyelectrolyte/fructose complex by Fourier transform infrared spectroscopy (FT-IR) with an attenuated total reflectance (ATR) accessory. The peaks around 950–1150 cm–1 in the spectrum of fructose (black solid line in Figure 3) disappeared and/or broadened in the ε-PL/fructose complex (blue dotted line in Figure 3). This change of FT-IR spectra suggests a structural change of fructose molecules in the ε-PL/fructose complex because a similar change was observed in the Maillard reaction of ε-PL and reducing sugars in aqueous solutions.30

Figure 3.

Figure 3

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FT-IR spectra of pure fructose (black solid line), pure ε-PL (red dashed line), and ε-PL/fructose complex (blue dotted line) after drying. The ε-PL/fructose weight ratio in the ε-PL/fructose complex was 80:20.

The color change to orange by addition of reducing sugars and no change of color and mechanical properties by the addition of a nonreducing sugar indicate the Maillard reaction in the cationic-polyelectrolyte/fructose mixtures. This is also supported by the 1H NMR and FT-IR analyses. This reaction enabled the formation of the soft and elastic complex from the dried mixtures.

Effect of Cationic Polyelectrolyte Structure on Mechanical Properties

In addition to the first-found ε-PL/fructose complex, we prepared three cationic-polyelectrolyte/fructose mixtures using PAA, linear PEI, and branched PEI instead of ε-PL. All of the samples were prepared at the cationic-polyelectrolyte/fructose weight ratio of 80:20, and tensile tests were performed to determine the mechanical properties of these complexes. Among the tested cationic-polyelectrolyte/fructose complexes, the linear PEI/fructose complex was slightly sticky compared with the others and was difficult to form into dumbbell-shaped tensile test specimens. This sticky property of the linear PEI/fructose complex resulted from insufficient cross-linking due to the lower reactivity of the secondary amino group with carbonyl compounds.31,32

Tensile tests were performed on all samples except the linear PEI/fructose complex; the results are listed in Table 1. Regarding the maximum strength and Young’s modulus, ε-PL/fructose and PAA/fructose complex showed higher strength and modulus compared with the branched PEI/fructose complex (Table 1). Both ε-PL and PAA have only primary amino group, whereas branched PEI contains primary, secondary, and tertiary amino groups. A primary amino group has higher reactivity, whereas secondary and tertiary amino groups have lower or no reactivity with carbonyl compounds.31,32 The high reactivity of primary amino groups in ε-PL and PAA would bring high cross-linking density; therefore, the strength and modulus of the ε-PL and PAA complexes are higher than those in the branched PEI complex.

Table 1. Mechanical Figures of Merit of Cationic-Polyelectrolyte/Fructose Complexesa.

cationic polyelectrolyte maximum strength (MPa) Young’s modulus (MPa) strain at break (%) toughness (MJ/m3)
ε-PL 15.0 ± 2.8 208.9 ± 49.3 89 ± 4 7.2 ± 1.4
PAA 16.0 ± 0.7 670.1 ± 40.0 5 ± 1 0.6 ± 0.1
branched PEI 0.8 ± 0.02 8.5 ± 0.2 9 ± 1 0.04 ± 0.003
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a

The cationic-polyelectrolyte/fructose weight ratio was 80:20. ε-PL: ε-poly-l-lysine; PAA: poly(allylamine); branched PEI: branched poly(ethyleneimine). All samples were equilibrated at 30 °C (50% relative humidity) for 5 days. All tested samples were obtained as a uniform sheet (∼1 mm thickness). Results are means ± standard deviation of at least three samples.

The ε-PL/fructose complex had superior toughness compared with the PAA and branched PEI complexes. In the previous study, the polyelectrolyte complex composed of ε-PL and lignosulfonate also had better toughness than those prepared from other cationic polyelectrolytes.14,18 The reason for the excellent toughness of the ε-PL/fructose and ε-PL/lignosulfonate complexes is not yet clear. We presume that the chemical structure of ε-PL, which is the same as that of nylon 6 (a tough polyamide used in engineering applications), except for the amino groups on the side chain, is responsible for the excellent toughness. Further studies are underway to clarify the chemical structure.

The ε-PL/fructose complex had good toughness, is completely bio-based, and would be suitable for medical and agricultural materials because of the low toxicity and biodegradability of its ingredients. We performed further analyses of ε-PL/sugar complexes because of their good mechanical properties, sustainability, and potential broad applications.

Optimization of the Sugar Component in the ε-PL/Sugar Complex

We prepared five ε-PL/sugar complexes using different reducing sugars (d-fructose, d-arabinose, d-glucose, lactose, and d-xylose) at the ε-PL/sugar weight ratio of 80:20 to evaluate the effect of the sugar component. In the cases of glucose and lactose, the solutions with ε-PL became orange but were sticky liquids after drying under the same conditions as the ε-PL/fructose complex. The ε-PL complexes made with fructose, arabinose, and xylose could be peeled from their poly(tetrafluoroethylene) (PTFE) molds, and tensile test specimens could be prepared from them (Table 2). These complexes showed various mechanical figures of merit from rigid (Young’s modulus > 200 MPa; fructose) to soft (Young’ modulus < 100 MPa and strain > 200%; xylose) depending on the sugar. The fructose complex had the highest toughness (7.2 MJ/m3; Table 2) and was used in the following experiments.

Table 2. Mechanical Figures of Merit of ε-PL/Reducing Sugar Complexesb.

sugar maximum strength (MPa) Young’s modulus (MPa) strain at break (%) toughness (MJ/m3)
fructose 15.0 ± 2.8a 208.9 ± 49.3a 89 ± 4a 7.2 ± 1.4a
arabinose 3.1 ± 0.3 14.0 ± 0.8 152 ± 15 2.5 ± 0.4
xylose 1.7 ± 0.2 7.3 ± 1.0 205 ± 17 1.8 ± 0.3
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a

The data are the same as in Table 1.

b

The ε-PL/sugar weight ratio was 80:20. All samples were equilibrated at 30 °C (50% relative humidity) for 5 days. All tested samples were obtained as a uniform sheet (∼1 mm thickness). Results are means ± standard deviation of at least three samples.

This variation in mechanical properties was likely due to differences in reactivity between each sugar and the amino groups. For example, the aqueous solution of lysine or ε-PL became colored more quickly with fructose than with glucose.28,30,33 This higher reactivity of fructose may have increased the cross-linking density, thereby resulting in higher Young’s modulus and low strain at break in the ε-PL/fructose complex compared with other complexes. The lower reactivity of glucose and lactose may explain why their solutions with ε-PL dried to sticky liquids under the same drying conditions.

Mechanical Properties of the ε-PL/Fructose Complexes by Fructose Content

We varied the ε-PL/fructose ratio to explore its impact on the mechanical properties of the ε-PL/fructose complex (Table 3). The 2.5 wt % fructose complex was turbid, yellowish-white, and too brittle to peel from its PTFE mold. The 5 wt % fructose complex formed a soft, elastic but slightly turbid sheet (Figure S2 in the Supporting Information). The yellowish color change suggested that the Maillard reaction occurred for 2.5 and 5 wt % fructose, but their greater turbidity and brittleness than those of the 10 wt % fructose (see Table 3) implied that unreacted ε-PL remained in these fructose-poor complexes. The ε-PL/fructose complexes became transparent orange at a fructose content exceeding 10 wt %. The maximum stress and Young’s modulus increased with increasing fructose content from 10 to 40 wt %. However, the strain at break decreased with fructose addition (Table 3). Similar hardening occurs in cross-linked materials, such as rubbers and gels, with increasing cross-linking density.34,35 More fructose (as a cross-linking reagent) accelerated the Maillard reaction and increased cross-linking of the complexes; higher cross-link density resulted in harder complexes. The greater efficiency of the Maillard reaction at a high fructose content was also supported by the color change of the complex; the orange color darkened with increasing fructose content (Figure S3 in the Supporting Information). A greater amount of colored aromatic compounds, which are typical products of the Maillard reaction,22−25 would be produced with higher fructose content. The maximum stress and Young’s modulus were lower at the highest fructose level (50 wt %). We presume that the excess of fructose and fewer amino groups in the complex prevented efficient formation of a cross-linked structure.

Table 3. Mechanical Figures of Merit of ε-PL/Fructose Complexes by Weight Ratio of ε-PL to Fructosed.

    mechanical properties of a dumbbell-shaped test piece
ε-PL fructose maximum strength (MPa) Young’s modulus (MPa) strain at break (%) toughness (MJ/m3)
100 0 a   a a
97.5 2.5 b   b b
95 5 3.0 ± 0.1 11.9 ± 2.3 95 ± 9 1.9 ± 0.1
90 10 2.5 ± 0.1 4.7 ± 0.2 119 ± 6 1.6 ± 0.1
80 20 15.0 ± 2.0c 208.9 ± 49.3c 89 ± 4c 7.2 ± 1.4c
70 30 22.7 ± 1.9 759.8 ± 120.4 49 ± 1 7.7 ± 0.8
60 40 27.9 ± 1.2 741.6 ± 77.3 46 ± 3 8.5 ± 0.1
50 50 22.5 ± 3.0 320.1 ± 61.3 52 ± 5 6.7 ± 1.0
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a

The sample spontaneously cracked, and no test piece could be prepared.

b

The sample was brittle and could not be peeled from the mold without damage.

c

The data are the same as in Table 1.

d

All samples were equilibrated at 30 °C (50% relative humidity) for 5 days. All tested samples were obtained as a uniform sheet (∼1 mm thickness). Results are means ± standard deviation of at least three samples.

The maximum strength (27.9 MPa) of the 60:40 ε-PL/fructose complex (Table 3) is comparable to that of petroleum-based plastics, such as high-density polyethylene (25 MPa),36 polystyrene (39 MPa),37 and poly(methyl methacrylate) (20 MPa).38 The Young’s modulus for this composition (741.6 MPa; Table 3) is very close to that of high-density polyethylene (756 MPa).36 The strain at break (46% for 60:40 ε-PL/fructose; Table 3) is also superior to that of some plastics [e.g., 3% for both polystyrene and poly(methyl methacrylate)]37,38 but less than that of soft plastics such as high-density polyethylene (730%).36 Improving the strain at break without reducing the maximum strength would expand the applications of this complex as a sustainable polymeric material.

To utilize the ε-PL/fructose complex, we should note about the molding process of the complex. This ε-PL/fructose complex had a glass transition temperature (Tg) around room temperature (Tg = 22.6 °C, determined from the dynamic mechanical analysis; the result is shown in Figure S4 in the Supporting Information) but did not reveal a melting point as far as we tried (data not shown). The lack of a melting point suggests that the ε-PL/fructose complex would not be suitable for a thermal molding process, and we regard this complex as a kind of hardening resin. This difference of moldability between the ε-PL/fructose complex and thermoplastics should be considered for suitable applications of this complex.

Antimicrobial Activity of the ε-PL/Fructose Complex

We expected that the ε-PL/fructose complex would inherit the antimicrobial activity of pure ε-PL.5,6,9−12 To verify this, two different bacteria were inoculated in liquid media containing the ε-PL/fructose complex, and bacterial growth was evaluated. Figure 4 shows that the growth of Bacillus subtilis (a typical Gram-positive bacterium) was inhibited in all tested samples containing 2.5–20 mg/mL of the ε-PL/fructose complex. This antimicrobial activity would be derived from a small amount of eluted ε-PL from the complex. The eluted ε-PL adsorbed on the bacterial cell and disrupted the cell as with the antimicrobial mechanism of pure ε-PL.7,11,12 Meanwhile, Escherichia coli (a typical Gram-negative bacterium) grew even under the highest concentration (20 mg/mL) of the ε-PL/fructose complex. This difference in growth could be explained by the higher resistance of E. coli than B. subtilis [the minimum inhibitory concentration (MIC) = 32 and 8 μg/mL, respectively], which was determined from a MIC assay of pure ε-PL by the broth microdilution method. In this experimental condition, the eluted ε-PL from the ε-PL/fructose complex would be less than the MIC of E. coli.

Figure 4.

Figure 4

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Antimicrobial activity assays of the ε-PL/fructose complex. (a) Photographs of culture media (2 mL) containing different amounts of the complex. (b) Optical density of these media at 600 nm. Open square: B. subtilis. Closed circle: E. coli.

Additionally, the growth of E. coli (MIC for pure ε-PL = 32 μg/mL) in the presence of 20 mg/mL of the complex (corresponding to more than 10,000 μg/mL of ε-PL) suggested that the elution of ε-PL from the ε-PL/fructose complex was very little. In other words, the complex was almost insoluble in the water-based media. To broaden the range of antibacterial activity including Gram-negative bacteria such as E. coli, controlled release of ε-PL would be necessary by optimization of cross-linking density and stability.

On the other hand, the high water solubility (i.e., low water resistance) of ε-PL is an additional barrier for its use in structural applications, but the above result suggests that the ε-PL/fructose complex is water-insoluble. The good mechanical properties and water resistance of this ε-PL/fructose complex will facilitate structural applications of this bio-based polymer.

Conclusions

We synthesized soft and elastic complexes composed of cationic polyelectrolytes, including ε-PL and reducing sugars. These complexes were prepared by simple mixing and drying of ε-PL and aqueous sugar solutions in the absence of other chemical reagents, such as organic solvents, condensation reagents, and cross-linkers. The appearance of the complex in various experiments strongly suggested that it formed via the Maillard reaction. The complexes had tunable mechanical properties that depended on their ingredients and composition; the Young’s modulus increased and the strain at break decreased, with increased cross-linking density. The mechanical figures of merit of the optimized complex (ε-PL/fructose at the weight ratio of 60:40) were comparable to those of some petroleum-based plastics.

In addition to these good mechanical properties, this ε-PL/fructose complex displayed antimicrobial activity against B. subtilis and water-resistance. The antimicrobial activity of the complex originated from the activity of ε-PL, but the water-resistance was contrary to the high water solubility of pure ε-PL. The coexistence of these properties in the complex is not possible in pure ε-PL.

The present cationic-polyelectrolyte/sugar complexes composed of bio-based ingredients would have great advantages for the sustainability and comfortability of modern society because of their facile preparation process, excellent mechanical properties, and unique features compared to those of pure cationic polyelectrolytes.

Experimental Section

Materials

ε-PL (25 w/v % aqueous solution, 25–35 mer, molecular weight 3000–4500 g/mol) was kindly gifted by JNC Corporation (Tokyo, Japan). PAA (15 w/v % aqueous solution, molecular weight 15,000 g/mol) and linear PEI (molecular weight 250,000 g/mol) were purchased from Polysciences, Inc. (Warrington, PA, USA). Branched PEI (molecular weight 10,000 g/mol) was obtained from Wako Pure Chemical Industries (Osaka, Japan). PDADMACl (molecular weight 200,000–350,000 g/mol) was purchased from Sigma-Aldrich (St. Louis, MO, USA). d-(−)-Arabinose was obtained from Tokyo Chemical Industry (Tokyo, Japan). Other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan). All reagents were used as received without further purification.

Preparation of Complexes

A cationic-polyelectrolyte/sugar complex was prepared by mixing aqueous solutions of cationic polyelectrolyte and sugar, followed by drying. Complexes containing ε-PL or branched PEI were prepared by mixing aqueous solutions of ε-PL or branched PEI (25 w/v %) with an aqueous sugar solution (25 w/v %; hot water was used for lactose). The mixture was cast into a PTFE (6 cm × 6 cm) or perfluoroalkoxy alkane (50 mm Petri dish) mold, dried in a humidity-controlled chamber (THR030FA; Advantec Toyo, Tokyo, Japan) at 30 °C (50% relative humidity), and peeled from the mold. To prepare the complexes from PAA or PDADMACl, powdered sugar was directly mixed with aqueous solutions of PAA (15 w/v %) or PDADMACl (20 w/v %), and the solution was then cast into the mold and dried as described above. Finally, linear PEI/sugar complexes were prepared by mixing a neutralized linear PEI solution (80 g/L of linear PEI dissolved in 0.68 mol/L of HCl and 0.24 mol/L NaOH; pH = 9–10 after the addition of the linear PEI) with powdered sugar. All complexes were equilibrated in a humidity-controlled chamber at 30 °C (50% relative humidity) for 5 days after demolding.

NMR Analysis

The PAA/fructose complex was prepared for 1H NMR analysis as follows: The PAA aqueous solution (15 w/v %) was lyophilized and redissolved in deuterium oxide [99.9 atom % containing 0.05 w/v % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt; Sigma-Aldrich] at 10 mg PAA/mL. Powdered d-(−)-fructose was added to the PAA solution at a PAA/fructose weight ratio of 70:30 and then incubated at 40 °C for 48 h in a sealed NMR tube. The incubated sample was analyzed using a Bruker AVANCE III instrument (400 MHz; Bruker, Karlsruhe, Germany) at room temperature before and after incubation.

FT-IR Analysis

Powdered d-(−)-fructose was mixed with ε-PL (25 w/v %) solution at a ε-PL/fructose weight ratio of 80:20. The mixture was cast into a PTFE mold and then dried in a humidity-controlled chamber at 30 °C (50% relative humidity). Infrared spectra of the dried complex (a homogeneous sheet, ∼1 mm thickness) was analyzed using an ALPHA II instrument (Bruker, Karlsruhe, Germany) with ATR option. Dried fragments of pure ε-PL and powdered fructose were also used as test samples separately. The measurement was performed under ambient conditions.

Mechanical Testing

The equilibrated complexes (∼1 mm-thick) were cut into dumbbell-shaped test specimens according to International Organization for Standardization (ISO) standard 37-3 [equivalent to Japan Industrial Standard (JIS) K-6251-8]. The cross-head speed for the test was 50 mm/min, and the test was performed using an EZ-LX tensile strength tester (Shimadzu, Kyoto, Japan) at room temperature. The toughness of the sample was determined based on the total area of the stress–strain curve and the volume of the specimen. Averages and standard deviations were calculated from at least three specimens.

Dynamic Mechanical Analysis

The ε-PL/fructose complex (ε-PL/fructose weight ratio of 80:20) was equilibrated at 30 °C (50% relative humidity) for 5 days after demolding and then cut into a strip-shaped sample (5 mm × 4 cm, ∼1 mm thickness). The sample was analyzed using a DMA Q800 instrument (TA Instruments, Delaware, USA) from −100 to 150 °C at the heating rate of 3 °C/min under air flow condition. The test was carried out at a frequency of 10 Hz. The glass transition temperature of the sample was determined from the peak top of loss modulus/storage modulus (tan δ).

Antimicrobial Activity Testing

The antimicrobial activity assay of the ε-PL/fructose complex was based on a published study39,40 with minor modifications. B. subtilis NBRC3134 (Gram-positive bacterium) and E. coli NBRC3301 (Gram-negative bacterium) were grown at 30 °C for 20 h on a reciprocal shaker (200 strokes/min) in Luria-Bertani broth (LB broth) consisting of 1 w/v % Bacto tryptone, 0.5 w/v % yeast extract, and 1 w/v % NaCl. The culture broth of these microorganisms was diluted to give 1.0 × 104 colony-forming units (CFU)/mL, and 2 mL of diluted bacterial cultures was then dispensed into sterilized test tubes. A piece of ε-PL/fructose complex (weight ratio of 80:20; 5–40 mg of complex in each tube) was placed into the test tubes and cultivated at 30 °C for 24 h in a reciprocal shaker (200 strokes/min). The antimicrobial activity of the ε-PL/fructose complex was evaluated by measuring the turbidity of the cultures at 600 nm using a spectrophotometer (V-630; JASCO, Tokyo, Japan).

The antimicrobial activity of pure ε-PL was determined by the broth microdilution method, which is a popular method for determining the MIC. Briefly, B. subtilis NBRC3134 and E. coli NBRC3301 were cultivated and diluted to 5.0 × 104 CFU/mL as above. An aliquot (40 μL) of a bacterial culture was inoculated into 160 μL of LB broth containing 320–0.31 μg/mL of ε-PL (1.0 × 104 CFU/mL of bacteria and 256–0.25 μg/mL of ε-PL at the final concentration) in a 96-well plate. Cultures were grown at 30 °C for 20 h on a microplate shaker at 600 rpm (M·BR-022UP; TAITEC, Saitama, Japan). The MIC was visually estimated based on the turbidity of the culture.

Acknowledgments

We express our gratitude to JNC Corporation for providing ε-PL. We are grateful to Dr. Ryota Watanabe for the dynamic mechanical analysis. This work was supported by JSPS KAKENHI grant number JP19K15645.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01813.

  • Photographs of pure cationic polyelectrolyte, polyelectrolyte/sucrose, and polyelectrolyte/fructose mixtures after drying; photographs of ε-PL/fructose complexes at ε-PL/fructose weight ratios of 95:5 and 90:10; photographs of ε-PL/fructose complexes at different ε-PL/fructose weight ratios; and dynamic mechanical analysis of ε-PL/fructose complexes at a ε-PL/fructose weight ratio of 80:20 (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c01813_si_001.pdf (297.1KB, pdf)

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