Effect of alum treatment on the mechanical and antibacterial properties of poly-γ-glutamic acid nanofibers

Abstract

Poly-γ-glutamic acid (γ-PGA) is a natural polymer that is commonly found in the viscous filaments of fermented soybean (natto). γ-PGA is known for its superior biodegradability, biocompatibility, water retention, and ability to adsorb metal ions. We used an oxazoline-containing polymer (OXA) as a cross-linking agent and succeeded in preparing γ-PGA nanofibers by electrospinning with a water solvent system. The non-woven γ-PGA/OXA sheet (90/10 wt%) was treated with alum to improve both the mechanical and antibacterial properties by metal adsorption. The absorption of water for the alum-treated γ-PGA/OXA non-woven sheets decreased by half compared to the untreated sheets. The tensile modulus did not decrease, and the strength largely improved, even if the webs absorbed moisture. The webs endured high-pressure steam sterilization and exhibited antibacterial properties.

Keywords

Electrospinning poly-γ-glutamic acid alum antibacterial

Poly-γ-glutamic acid (γ-PGA) is a polyamino acid commonly found in the viscous filaments of fermented soybean (natto). It is a type of biopolymer produced by the action of microbes such as Bacillus species.¹^–³ γ-PGA possesses high hydrophilicity capacity for water retention and has often been used as a component in cross-linked or copolymerized gels.⁴^–⁹ If γ-PGA is converted to a fiber mat, it is useful for biomedical applications.

Spinning fibers of γ-PGA by electrospinning has been performed,¹⁰^–¹³ but it is costly, harmful, and dangerous to the environment since organic acids or fluorine compounds must be used as solvents. We developed a nanofiber fabrication method from γ-PGA–Na by electrospinning using a water solvent system. The electrospun γ-PGA non-woven nanofibers were readily dissolved by absorption of moisture from air. To retain the fiber morphology, an oxazoline-containing polymer (OXA) was used as a cross-linker, which produced non-woven nanofibers that maintained their fibrous forms in water.¹⁴ Unfortunately, the strength of the γ-PGA/OXA webs decreased in water. Another problem observed was area shrinkage. When the γ-PGA/OXA blend (blend ratio = 90/10 wt%) sample was moistened at 23 ± 0.5°C and 73 ± 2% relative humidity (RH) for 20 hours, the area shrinkage was 19%. Further improvement is thus required to make γ-PGA into a polymer that can be used under various environmental conditions.

In addition to cross-linking, other characteristics of γ-PGA such as its metal adsorption capability^8,15^–¹⁸ were also considered to enhance the mechanical strength. Alum is commonly used in food additives and as an ingredient in lotion, and is expected to form metal bridges as a precursor to non-woven γ-PGA/OXA nanofibers. Figure 1 shows the chemical structure expected upon treatment of γ-PGA/OXA with alum. Another advantage to alum is that it might impart a sterilization effect on the γ-PGA/OXA non-woven nanofiber.

Figure 1.

Molecular structures of γ-PGA, OXA, and cross-linked γ-PGA/OXA treated with alum.

In this study, the effect of alum treatment on the mechanical and antibacterial properties of non-woven γ-PGA/OXA was investigated in order to produce a non-woven sheet made from polyamino acid-based fibers.

Experimental

Preparation of γ-PGA/OXA nanofiber non-woven sheet

The γ-PGA/OXA nanofiber sheet was produced according to our previously developed method.¹⁴ The γ-PGA HM-Na⁺form (M_w ≈ 1.3 × 10⁶ Da; carboxyl group content: 6.62 mmol/g; manufactured by Vedan Co) was used as the starting material. The cross-linking agent was the oxazoline-containing polymer OXA (EPOCROS WS-700; M_w = 4 × 10⁴; aqueous solution with 25 wt% non-volatile matter; oxazoline group content: 4.50 mmol/g; Nippon Shokubai Co). Ethanol, hydrochloric acid, and alum (potassium sulfate aluminum) were all JIS special grade reagents manufactured by Kanto Kagaku Co.

To produce the electrospinning solutions, an aqueous γ-PGA solution and an aqueous OXA solution were mixed under agitation at a solid matter concentration of 8 wt%. In Table 1, the blending condition of the spinning solution is listed. The spinning solution consisted of water/ethanol /1 M hydrochloric acid/γ-PGA,OXA. Electrospinning was carried out using an NS-LAB200S electrospinner (Elmarco Co)¹⁹ at an electrode distance of 140 mm and a voltage of 75 to 80 kV at 30°C and 15% RH. In this electrospinning system, the solution was electrospun 10 times onto non-woven polypropylene (PP) (30 g/m²), which was moving at a winding velocity of 0.1 m/min. The obtained γ-PGA/OXA web on the substrate material was then heated in a vacuum dryer at 120°C for 1 hour for cross-linking. After the heat treatment, the γ-PGA/OXA web was easily peeled off the PP non-woven sheet by hand, producing 17 ± 2 g/m² of web (denoted as PGA90).

Table 1.

Blending condition for the electrospinning solution

γ-PGA/ OXA	PGA-Na: OXA = 1 : X
Weight ratio^a	Molar ratio^b	Concentration (wt%)	Water (wt%)	Ethanol (wt%)	1 M HCl (wt%)
90/10	0.076	8	45	27	20

Non-volatile matter.

Carboxyl/oxazoline group of the polymer unit.

Pulp consisting of cellulose raw materials (Kimwipes, 21 g/m²) made by Kimberly Clark Co and cotton plain cloth (Canequim No.3 JIS-L0803, 97 g/m²) were used as reference samples for comparison of the moisture and water absorption characteristics.

Alum treatment

A 10 mL solution of alum [AlK(SO₄)₂·12H₂O] in water was prepared at the concentrations of 100, 50, 30, 10, and 5 mmol/L. Sample (PGA90, 20 mg) was immersed in the solution for 0.5, 1, and 6 hours. The samples were then washed three times with 20 mL of distilled water then dried.

Another sample was immersed in the 50 mmol/L alum water solution for 1 hour. After washing by water, the sample was immersed in ethanol for dehydration. This sample was denoted as PGA90M.

PGA90 was dissolved in water and the weight loss of the sample was calculated using equation (1)

(1)

where W_I is the dry weight after alum treatment (mg) and W₀ is the initial weight of the electrospun web.

Morphological observations

The morphology of the sample was examined using a scanning electron microscope (SEM; 3D Real Surface View Microscope VE-8800, KEYENCE Co) at 20 kV accelerating voltage. Samples were sputter coated with gold. The fiber diameter was calculated from an average of 30 fibers in the image.

Area shrinkage after alum treatment

The samples (40 mm × 40 mm) were immersed in distilled water or an alum water solution (concentration: 50 mmol/L) for 0.5, 1, and 6 hours. The size of each sample was measured first, then after immersing in solution and drying. The area retention was calculated by equation (2)

(2)

where A_W,_D is the area of the sample after immersion followed by drying (mm²) and A_i is the initial area before immersing in the solution (mm²).

Moisture absorption/desorption characteristics

A sample conditioned at 23 ± 0.5°C and 30 ± 2% RH (sample W₀) was placed in a polypropylene frame (40 mm × 40 mm, 0.2 mm thickness, 5 mm width) and allowed to absorb moisture in an environment of 23 ± 0.5°C and 73 ± 2% RH for 20 hours. Samples were then transferred to desorb moisture at 23 ± 0.5°C and 30 ± 2% RH for 20 hours. During the experiment, the change in weight was monitored over time. Moisture regains, M1 and M2, were calculated as follows

(3)

(4)

where

w₀ = initial weight of sample W₀ conditioned at 30 ± 2% RH,

w₁ = weight after absorbing moisture at 73 ± 2% RH for 20 hours,

w₂ = weight after desorbing moisture at 30 ± 2% RH for 20 hours.

Three specimens were examined and the reproducibilities of results as expressed as CV% were less than 5% for M1 and 17% for M2. The accuracy of the weight balance was ± 0.01 mg.

Water absorbency and retentivity characteristics

Water absorbency was detected by two different methods. One method involved immersion of the samples in water, while the other required placement of a droplet of water on the samples to investigate the water diffusion.

Method 1

A 10 mg sample was weighed and immersed in distilled water at 23 ± 0.5°C for 30 minutes after which the sample was withdrawn from the water. Water droplets were removed by light wiping, and then the weight of the sample was determined as follows

(5)
where S_I and S₀ are the weights (mg) of the wet and dry samples, respectively.

Method 2

A sample with a polyethylene frame was placed on a hot plate (the sample surface temperature was adjusted to 30°C) under ambient conditions of 20 ± 0.5°C and 30 ± 1% RH. Water (10 µL, 20°C) was placed on the center of the sample by a syringe. The change in the surface temperature where the water was being applied was monitored over time using infrared thermography (TH9260 Thermotracer, NEC Avio Infrared Technologies) for 5 minutes. In this period, the temperature change characterized the water absorption/diffusion and vaporization behavior. Additionally, for water retention/vaporization behavior, 10 µL of distilled water was dropped on the sample surface and the weight change of the sample was monitored for 1 hour. Water absorbency was calculated as follows

(6)
where W_H is the weight of dropped water (mg) and W_M and W₀ are the wet and initial weights of the web (mg), respectively.

In the above experiments, at least three specimens were examined.

Tensile tests

The tensile properties of the sample were determined using the KES-G1S microtensile tester (Kato Tech. Co). The specimen dimension was 10 mm in width and the gauge length was 10 mm. A crosshead speed of 12 mm/min was used. The tensile tests were carried out at 27 ± 3°C under three different humidity conditions, 30, 75, and 100% RH, and in water. The strength and the elongation at the break point of the specimen and the modulus were determined from an average of 10 specimens.²⁰

Antibacterial tests

The antibacterial tests were designed based on the test method of 1902 JIS L (textiles and the bacteria liquid absorption method of the antibacterial effect mutatis mutants) with some modification. Differences between the JIS method and this study are listed in Table 2. The sample quantity was 40 mg, which was one-tenth of the weight used in JIS L 1902. The bacterial inoculation amount was chosen to be one-half that of JIS L1902. For the preprocessing of the sample for sterilization, two methods were examined instead of using JIS L 1902 because the γ-PGA/OXA non-woven sheet was not strong enough to survive the JIS test conditions. In one method, the sample was first immersed in 70% ethanol then transferred to the sterilized saline. To determine the effect of the acidity of alum on the antibacterial activity, further treatment was performed. The treated sample was immersed in a NaOH water solution at pH 10 for approximately 3 hours until the sample surface became neutral.
Table 2.
Antibacterial test procedures

Sample weight(mg) JIS L1902 Test condition

400 40

Bacteria concentration (counts/ml) 1 ∼ 3 × 10⁵ Staphylococcus aureus: 1.0 × 10⁵

Klebsiella pneumoniae: 6.4 × 10⁵

Sterilization method High pressure steam High pressure steam

(121°C 20 min) Dip in 70% ethanol and saline

Dip in 70% ethanol and alkari solution

Bacteria inoculation (ml) 0.2 0.1

Extraction saline (ml) 20 5

Two bacteria were tested: Staphylococcus aureus as an aerobic Gram positive bacterium (Staphylococcus aureus NBRC12732) and Klebsiella pneumoniae as an aerobic Gram negative bacterium (Klebsiella pneumoniae NBRC 13277). The bacteria were inoculated on a sample and placed in an environment of 37 ± 2°C for 18 hours. The bacteria liquid was then extracted with 5 mL saline and the number of living bacteria in the culture was measured.

PGA90 was not strong enough for the antibacterial test, so the constituents of PGA90 were tested as follows. Namely, 8 wt% of γ-PGANa solution and the spinning solution used for making PGA90 (as listed in Table 1) were used. Each solution was impregnated into paper (Kimwipes) in a 2 : 1 weight ratio then heated at 120°C for 1 hour to complete the cross-linking. The obtained samples were denoted as PGA/raw and PGA/es, respectively, and were used for the antibacterial tests in the same manner as the alum-treated sample. The sterilization activity value (L) was calculated using equation (7)

(7)
where Ma is the count of bacteria on the standard cloth just after bacteria inoculation and Mc is the count of bacteria in the sample after 8 hours of culture.

Results and discussion

Effect of alum concentration and immersion time on the dissolution in water

In Figure 2, the weight loss of the PGA90 samples in the six different concentrations of alum is plotted against immersion time. PGA90 without alum treatment dissolved approximately 25% in water. On the other hand, weight losses of the alum-treated non-woven γ-PGA/OXA samples were all smaller than that of the untreated sample. As expected, a carboxyl group of γ-PGA was bridged with an aluminum ion of the alum water so as to decrease the solubilization of γ-PGA. An increase in the immersion time decreased the solubility, due to the adsorption of metal ions on γ-PGA. Weight loss reached a nearly constant value after the sample was immersed for almost an hour.
Figure 2.
Weight loss of γ-PGA webs (PGA90) with alum treatment. Alum concentrations: (a) 100, (b) 50, (c) 30, (d) 10, and (e) 5 mmol; and (f) without alum.

Shrinkage of sample

Figure 3 shows the fiber morphology observed when the sample (PGA90) was immersed at different alum concentrations for 1 hour followed by drying. When the alum concentration was lower than 30 mmol/L [see (b) and (c) in Figure 3], remarkable shrinkage of samples occurred just after dipping in water. This shrinkage was not observed when PGA90 was immersed in more concentrated alum solutions.
Figure 3.
SEM images of γ-PGA webs before treatment (a) and after alum treatment for 1 hour with various concentrations of alum: (b) 5 mmol/L, (c) 10 mmol/L, (d) 30 mmol/L, (e) 50 mmol/L, and (f) 100 mmol/L.

In Figure 4, area retentions of PGA90 are compared when immersed in 50 mmol/L alum and distilled water. When the PGA90 was immersed in distilled water, it shrank immediately then returned to its initial size upon absorption of water. When this sample was dried, it began to shrink until the area retention became 21%. On the other hand, when the PGA90 was immersed in an alum water solution (50 mmol/L), the shrinkage gradually increased and area retention reached 36%. This alum-treated sample shrank further during drying, resulting in 26% of area retention. The weight of the sample (PGA90M) increased from 17 g/m² to 39 g/m² after the alum treatment. The fiber diameter of the PGA90M, which is the PGA90 treated in a 50 mmol/L alum water solution for 1 hour, increased from 160 nm to 200 nm by absorption. The dimensional stability and the solubility in water were found to be controlled by this alum treatment.
Figure 4.
Shrinkage of γ-PGA web with treatment: (a) 50 mmol/L alum and (b) water after drying.

Characteristics of the alum-treated samples

Moisture regain during absorption and desorption is shown in Figure 5 for PGA90, PGA90M, and pulp as reference. Moisture regains after 20 minutes absorption (M1) and desorption (M2) are also listed in Table 3. The absorbency of the alum-treated sample (PGA90M) decreased to 1/10 and became similar to the moisture absorbency of pulp. Because a carboxyl group of γ-PGA was used for linking to metal in the alum treatment, the water uptake of the fiber itself became low. When a water droplet was applied on the sample surface, the water tended to sink into the hydrophilic fibers or diffuse between the fibers. Figure 6 shows the temperature gradient on the sample surface over 5 minutes. The center area of the surface indicates the water drop temperature. Water diffused slowly in PGA90, partly dissolving it and turning it into a film-like structure. In the case of PGA90M, water diffused faster. This phenomena of PGA90M is observed in the temperature area of 23°C (see at ten seconds in Figure 6), that was almost the same as the areas observed in pulp and cotton samples.
Figure 5.
Moisture regain of γ-PGA webs: (a) PGA90, (b) PGA90M, and (c) pulp.

Figure 6.
Thermographs of γ-PGA webs after water absorption: (a) PGA90, (b) PGA90M, (c) pulp, and (d) cotton fabric.

Table 3.
Moisture regain of γ-PGA web with alum treatment

Moisture regain (%)
Water absorbency (%)

M1 M2 M1–M2

PGA90 53.7 6.3 47 2500 ± 210

PGA90M 6.2 1.5 4.7 1100 ± 580

Pulp 5.2 1.0 4.2 730 ± 70

In Figure 7, changes of water absorbency during drying are shown for four samples. Water absorbency of PGA90 decreased slowly, because water did not penetrate into the fibers and remained on the surface. In the case of PGA90M, water absorbency occurred much faster.
Figure 7.
Drying ability of γ-PGA webs affected by water droplet: (a) PGA90, (b) PGA90M, (c) pulp, and (d) cotton.

In Figure 8, the initial tensile modulus, stress, and strain at break are shown under three humidity conditions (30, 75, and 100% RH) as well as in water. When both PGA 90 and PGA90M were conditioned at 30% RH, the samples were fragile and showed small breaking strain. The difference between the two samples was more evident in the modulus and stress at break at humidities of 75 and 100% RH. The strength of PGA90M under 100% RH was 11 times larger than that of PGA90. When PGA90 was conditioned in the ambient humidity of 75 and 100% RH, the sample was partly dissolved. Moreover, PGA90 in water had become a gel-like fiber for water absorption.¹⁴ On the other hand, the moisture absorption of PGA90M maintained the fiber structure. As for the increase of the stress by the moisture absorption of PGA90M, it was considered that absorbed water acted on the hydrogen bonding between the fibers. The stress–strain curve of PGA90M in water showed that, after the yield point, the stress gradually increased with the increase of strain, with the breaking strain of 130% being achieved. This high strain was partly caused by its loose non-woven structure. Thus, the alum treatment was confirmed to increase the strength of PGA90M.
Figure 8.
Tensile properties of γ-PGA webs: (a) PGA90 and (b) PGA90M.

Effect on antibacterial properties with various sterilization treatment by alum treatment samples

For the antibacterial test (JIS L 1902), samples must be strong enough to survive the pretreatment by high-pressure steam sterilization. Figure 9 shows the sample morphologies observed before and after high-pressure steam sterilization of PGA90 and PGA90M. The PGA90 shrank remarkably after steam sterilization. When PGA90 was processed for the antibacterial test, the sample could not hold its shape because of hydrolysis, whereas PGA90M could maintain its shape throughout these processes.
Figure 9.
SEM images and extraction conditions of γ-PGA web: (a) PGA90 and (b) PGA90M. The B images were before steam sterilization, the O images were after steam sterilization, and the E images were after incubation with bacteria.

In Table 4, sterilization activity values (L) are shown for the three preprocesses. In the case of both high-pressure steam treatment and saline, the sterilization activity values of nanofiber webs (PGA90 and PGA90M) were positive, i.e. they possessed antibacterial properties. However, when the sterilization preprocessing was carried out in water at close to neutral pH (i.e. alkali), the sterilization activity values became negative for the Klebsiella pneumoniae. The cell walls of the Klebsiella pneumoniae are thin, but they have adventitia consisting of phospholipids and lipopolysaccharides, which might generate tolerance to the sterilization solution. The pH of the environment for growing Klebsiella pneumoniae is also 6.0–8.0, which corresponds to the pH range after neutralization. In effect, no difference in antibacterial properties due to alum treatment was observed between PGA90 and PGA90M. In light of these results, further experiments were carried out to investigate the antibacterial properties of PGA itself and also the PGA solution used for electrospinning.
Table 4.
Sterilization activity value obtained by antibacterial tests of γ-PGA webs

Sample Shape Preprocessing pH Staphylococcus aureus Klebsiella pneumoniae

PGA90 nanofiber web steam 4–5 >3.3 >4.1

PGA90M steam 3–4 >3.3 >4.1

PGA90 saline 4–5 >3.3 1.1

PGA90M saline 3–4 >3.3 2.1

PGA90 alkari 6.5–7 >3.9 −1.0

PGA90M alkari 7–7.5 1.7 −1.1

PGA/raw coating on pulp steam 6 −0.6 −1.8

PGA/es steam 4 >3.6 >3.9

PGA/es saline 5–6 >3.6 1.2

PGA/es alkari 7.5 −0.4 −0.3

The sterilization activity value of PGA/raw after high-pressure steam treatment was negative, but that of PGA/es, which was impregnated in the electrospinning solution, was positive. This is most likely the influence of hydrochloric acid which is in the electrospinning solution. The sterilization activity value for Klebsiella pneumoniae decreased when the PGA/es was treated in saline even after neutralization of the sample. PGA/es did not show antibacterial properties for either Klebsiella pneumoniae or Staphylococcus aureus. The growing pH of the Staphylococcus aureus is in the range of 4.0–9.8 (optimal pH: 6.5–7.3) and PGA/es solution is also in this pH range. Staphylococcus aureus has a thick wall, but does not have adventitia, thus its tolerance for the sterilization solution may be low, and parameters other than pH could have an effect. It is also seen that the sterilization activity values of the nanofiber (PGA90) were positive in the pH around 7.5; on the other hand, that of PGA/es was negative as shown in Table 4. While the detailed mechanism is unknown, these studies indicate that the antibacterial properties exhibited by γ-PGA/OXA non-woven fabric must be due to the nanofiber morphology such as a cell blockage and not the acidity imparted by the alum treatment.

Conclusion

The effective improvement of tensile strength in γ-PGA nanofibers was achieved by immersing γ-PGA/OXA (90/10 wt%) web in a 50 mmol/L alum water solution for 1 hour. The tensile modulus of this sample did not decrease upon absorption of moisture, and the strength was improved 11-fold in 100% RH and 1.7-fold in water compared to the untreated samples. As a result of the alum treatment, γ-PGA/OXA nanofibers were able to endure high-pressure steam sterilization for the antibacterial test and antibacterial properties were also confirmed. This system produces γ-PGA/OXA nanofibers by electrospinning using a water system, which is safe and economical. Improvement of mechanical properties was also accomplished, enabling the use of this γ-PGA web as sheets made of hydrophilic polyamino acid-based fibers.

Sample weight(mg)	JIS L1902	Test condition
Bacteria concentration (counts/ml)	1 ∼ 3 × 10⁵	Staphylococcus aureus: 1.0 × 10⁵
	Klebsiella pneumoniae: 6.4 × 10⁵
Sterilization method	High pressure steam	High pressure steam
(121°C 20 min)	Dip in 70% ethanol and saline
Dip in 70% ethanol and alkari solution
Bacteria inoculation (ml)	0.2	0.1
Extraction saline (ml)	20	5

	Moisture regain (%)	Water absorbency (%)
PGA90	53.7	6.3	47	2500 ± 210
PGA90M	6.2	1.5	4.7	1100 ± 580
Pulp	5.2	1.0	4.2	730 ± 70

Sample	Shape	Preprocessing	pH	Staphylococcus aureus	Klebsiella pneumoniae
PGA90	nanofiber web	steam	4–5	>3.3	>4.1
PGA90M	steam	3–4	>3.3	>4.1
PGA90	saline	4–5	>3.3	1.1
PGA90M	saline	3–4	>3.3	2.1
PGA90	alkari	6.5–7	>3.9	−1.0
PGA90M	alkari	7–7.5	1.7	−1.1
PGA/raw	coating on pulp	steam	6	−0.6	−1.8
PGA/es	steam	4	>3.6	>3.9
PGA/es	saline	5–6	>3.6	1.2
PGA/es	alkari	7.5	−0.4	−0.3

Footnotes

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science and technology of Japan [grant number 21300269].

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	Moisture regain (%)			Water absorbency (%)
	M1	M2	M1–M2	Water absorbency (%)
PGA90	53.7	6.3	47	2500 ± 210
PGA90M	6.2	1.5	4.7	1100 ± 580
Pulp	5.2	1.0	4.2	730 ± 70