In vitro evaluation of new functional properties of poly-γ-glutamic acid produced by Bacillus subtilis D7

Abstract

We investigated the functionality of poly-γ-glutamic acid (γ-PGA), which is produced by Bacillus subtilis D7, for its potential applications in medicine and cosmetics. The γ-PGA had angiotensin-converting enzyme (ACE) inhibition activity. ACE inhibition activity was dependent on the γ-PGA concentration; the highest ACE inhibition activity was observed at 1.25 mg/l of γ-PGA. IC₅₀ (0.108 mg/ml) of the γ-PGA was lower than that of standard ACE inhibitory drug, N-[(S)-mercapto-2-methylpropionyl]-L-proline (0.247 mg/ml). The γ-PGA also had water-holding capacity and hygroscopicity. Furthermore, the γ-PGA inhibited growth of some pathogenic bacteria, including Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia and Esherichia coli. The γ-PGA exhibited a good metal adsorption capacity; Cr (VI) adsorption capacity of γ-PGA increased with decreasing pH, and the maximal adsorption was observed at pH 2. Our results suggest that γ-PGA may be expected to be widely applied in cosmetics, biomedical and environmental industries with the feature of being less harmful to humans and the environment.

1. Introduction

Poly-γ-glutamic acid (γ-PGA) is produced by a number of species of Bacillus. It is an unusual anionic polyamide that is made of d- and l-glutamic acid units connected by amide linkages between α-amino and γ-carboxylic groups. The γ-PGA is biodegradable, edible, water-soluble and non-toxic to humans and environment. Therefore, it has been suggested for use as biodegradable plastics (Tsutomu and Makoto, 2002), flocculants (Bajaj and Singhal, 2011), biological adhesive (Otani et al., 1996) and food additives (Lee and Kuo, 2011). For example, flocculating activity of the γ-PGA produced by Bacillus licheniformis CCRC 2826 (Shih et al., 2001) or Bacillus subtilis R23 (Bajaj and Singhal, 2011) has been reported. Some researchers also reported that the γ-PGA could adsorb basic dyes (Inbaraj et al., 2006).

Hypertension is a common progressive disease leading to several diseases such as cardiovascular disease, stroke and diabetes. Angiotensin-converting enzyme (AC) is a key component in rennin angiotensin aldosterone system which regulates blood pressure (Balasuriya and Rupasighe, 2011). Inhibition of ACE is a basic approach in the therapy of high blood pressure in humans. Therefore, screening of potential ACE inhibitors is expending broadly. ACE inhibitory peptides have been found in many different food proteins; milk casein, gelatin, corn gluten, fish proteins and Korean traditional fermented foods (Doenjang, Kanjang) (Ariyoshi, 1993). ACE inhibitory peptides derived from these food proteins have great potentials in the development of a novel functional food and drug for preventing hypertension and therapeutic purposes. If ACE inhibition activity of the γ-PGA was identified, it could be utilized as a new and harmless drug, since it is edible and nontoxic toward humans. However, there is no report on the ACE inhibition activity of the γ-PGA. On the other hand, exposure to external factor, i.e., air humidity, ultraviolet radiation and temperature, as well as endogenous factors, i.e. hormones, may disrupt skin balance between the water content and lipids on skin. In addition, there is common use of soaps, detergents and hot water disrupting skin balance (Dal’Belo et al., 2006). These factors may cause dry skin as well as atopic dermatitis on skin in severe case. Moisturizers not only increase the skin’s water content but also protect the skin and encourage an orderly desquamation process that makes the skin appear smoother. From the cosmetic point of view, therefore, the γ-PGA could offer a number of cosmetical advantages if its moisturizing effect would be characterized.

In this study, functionalities of the γ-PGA produced by B. subtilis D7 were screened; especially, ACE inhibition activity of the γ-PGA was investigated in detail to explore its possible use in the pharmaceutical industry. In addition, various properties of the γ-PGA, such as antimicrobial, moisturizing and heavy metal adsorbing activities, were also investigated. To the best of our knowledge, this is the first report highlighting the ACE inhibition potential of the γ-PGA.

2. Materials and methods

2.1. Bacterial strain and culture conditions

B. subtilis D7 used in this study was isolated from Korean traditional fermented food, Doenjang (Lee 2012). For the production of the γ-PGA, 100 ml of medium was dispensed into a 300-ml Erlenmeyer flask followed by inoculation with 1 ml of the strain D7 culture (1.0 × 10⁹ cells/ml) grown in nutrient broth at 30 °C and 200 rpm for 18 h. Cultivation was carried out at 30 °C and 200 rpm in a rotary shaker, and the γ-PGA yield and cell growth were checked at 24 h intervals. The medium used contained the following: 1% l-glutamate, 2% glucose, 0.5% yeast extract, 0.14% K₂HPO₄ and 0.04% MgSO₄·7H₂O (Yao et al., 2010). The pH was adjusted to 7.5 prior to sterilization.

2.2. Preparation of γ-PGA

The γ-PGA was purified by the method of Goto and Kunioka (1992). Briefly, cells were removed from the culture broth by centrifugation for 20 min at 17,418g and 4 °C. The culture supernatant was poured into 3 v of ice-cold ethanol and kept at 4 °C for 12 h. The precipitated γ-PGA was collected by centrifugation. For further purification, the precipitation step was repeated three times. The resulting γ-PGA was dialysed against distilled water, and finally freeze-dried for 2 days. Various concentrations of the freeze-dried γ-PGA were then used to determine functionality of γ-PGA.

2.3. ACE inhibition activity

ACE inhibition activity was measured using the method reported by Cushman and Cheung (1971). 100 μl of substrate solution (12.5 mM hippuryl-histidyl-leucine acetate) was incubated with 50 μl of sample for 5 min at 37 °C, and then 100 μl of the ACE solution was added and incubated for 30 min at 37 °C. The reaction was terminated by adding 250 μl of 1 N HCl. Hippuric acid was extracted with 1.5 ml of ethyl acetate, and then the mixture was vortexed for 15 s and centrifuged at 17,418g for 10 min to separate the ethyl acetate layer. A volume of 500 μl of the supernatant was transferred to another test tube and evaporated in dry block bath for 1 h at 90 °C. The extracted hippuric acid was redissolved in distilled water and absorbance was measured at 228 nm. N-[(S)-mercapto-2-methylpropionyl]-L-proline (Captopril) was used as positive control. ACE inhibition activity was calculated according to the following equation:

$$\text{ACE inhibition activity}(\%)=[1-\text{absorbance of sample/absorbance of control}]\times 100\text{.}$$

The inhibition concentration (IC₅₀) value was calculated from the plotted graph of inhibition activity versus the concentration of γ-PGA. A lower IC₅₀ value indicates the higher inhibition activity.

2.4. Moisturizing effect

Hygroscopy is the ability of a substance to attract and hold water molecules from the surrounding environment (Newman et al., 2008). A known concentration of the γ-PGA was put into the desiccator that maintains a high relative humidity (90%). The desiccator was then incubated at 30 °C. During the experiment, the change in the γ-PGA weight was monitored at regular intervals. Glycerol was used for control. The hydroscopicity of the γ-PGA was calculated as follows:

$$\text{Hygroscopicity}(\%)=[(W_1-W_0)/W_0]\times 100$$

where W₀ and W₁ were initial weight of γ-PGA and weight after moisture adsorption, respectively.

Water-holding capacity is the ability of a substance to retain moisture. A known concentration of the γ-PGA was hydrated by immersion in droplets of distilled water and weighed and then incubated at 30 °C. During the experiment, the change in the γ-PGA weight was monitored every day. The water-holding capacity of the γ-PGA was calculated as follows:

$$\text{Water-holding capacity}(\%)=[\text{weight after moisture desorption/initial weight of}\gamma -\text{PGA}]\times 100$$

2.5. Antimicrobial activity

Antimicrobial activity of the γ-PGA was evaluated using the paper disc method (De Beer and Sherwood, 1945). The microorganisms tested were Gram-negative bacteria (Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae and Pseudomonas aeruginosa), Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) and yeast (Candida albicans). The test strains were incubated in nutrient broth at 37 °C and 200 rpm for 24 h. The culture broth was then spread on a nutrient agar plate. The sterile paper disc (8-mm diameter) was placed on the agar plate and applied with 1% γ-PGA solution. The plate was incubated at 37 °C for 2 days and the inhibition zone was observed and calculated.

2.6. Adsorption of Cr (VI)

A volume of 20 ml of a Cr (VI) solution (100 mg/l) was prepared and adjusted to pH 2–7. The solution was agitated with 2 ml of 1% γ-PGA solution at 30 °C and 150 rpm for 24 h to achieve the equilibrium condition. Then sample was taken from each flask and centrifuged at 17,418g for 15 min. The supernatant was analysed for the Cr (VI) concentration. The Cr (VI) concentration in the solution was determined using the colorimetric method described previously (Clesscerl et al., 1958). Control experiment was carried out in the absence of γ-PGA. Standard curve was prepared using K₂Cr₂O₇.

All experiments were repeated three times unless otherwise indicated. The data presented in the tables and figures corresponded to mean values and standard deviation.

3. Results

3.1. Production of γ-PGA

Fig. 1 shows the γ-PGA production and cell growth by B. subtilis D7 in the medium used. The γ-PGA production and cell growth gradually increased and reached the maximum at 3 days, at which the γ-PGA production was 4.7 g/l. The medium became highly viscous due to the production of the γ-PGA along with cell growth.

Figure 1.

Time courses of γ-PGA production and cell growth by Bacillus subtilis D7 in the medium. Error bars (±S.D.) are shown when larger than the symbol. ● Cell growth and ○ PGA.

3.2. ACE inhibition activity of γ-PGA

The ACE inhibition activity of the γ-PGA is shown in Fig. 2. The ACE inhibition activity increased with increasing concentration of the γ-PGA. The highest ACE inhibition activity was 100% at 1.25 mg/l γ-PGA and remained constant thereafter. Inhibitory concentration (IC₅₀) value of the γ-PGA calculated from Fig. 2 was 0.108 mg/ml. The activity of standard ACE inhibitory drug (Captopril) was also concentration-dependent but its IC₅₀ value (0.247 mg/ml) was higher than that of the γ-PGA (Fig. 2).

Figure 2.

Angiotensin-converting enzyme (ACE) inhibition activity of γ-PGA and Captopril. Error bars (±S.D.) are shown when larger than the symbol.

3.3. Moisturizing effect of γ-PGA

We investigated some characters of the γ-PGA as a moisturizing agent. As shown in Fig. 3, the hygroscopicity of the γ-PGA increased with increasing the reaction time. Compared to control that contained glycerol instead of the γ-PGA, however, the hygroscopicity of the γ-PGA was low and 86.8% after 72 h. As shown in Fig. 4, the water-holding capacity of the γ-PGA was reduced as the reaction time increased up to 9 days at which the water-holding capacity was 56.9%. Similar values of the water-holding capacity after prolonged reaction were obtained (data not shown).

Figure 3.

Hygroscopicity of γ-PGA and glycerol at 30 °C and 90% relative humidity. Error bars (±S.D.) are shown when larger than the symbol. ● PGA and ○ glycerol.

Figure 4.

Water-holding capacity of γ-PGA. Error bars (±S.D.) are shown when larger than the symbol.

3.4. Antimicrobial activity of γ-PGA

The antimicrobial activity of the γ-PGA is shown in Table 1. The γ-PGA was found to inhibit both Gram-positive (L. monocytogenes and S. aureus) and Gram-negative (L. monocytogenes, S. typhimurium, K. pneumonia and E. coli) bacteria; S. aureus was highly inhibited by the γ-PGA. One percent of γ-PGA had an antibacterial activity against these bacteria, but 0.1% γ-PGA had not (data not shown). Otherwise, the γ-PGA did not exhibit antimicrobial activity against pathogenic yeast, C. albicans.

Table 1.

Antimicrobial activity of γ-PGA.

Pathogenic microorganism	Inhibition zone (mm)a
Gram positive bacteria
L. monocytogenes (a)	11
S. aureus (b)	13
Gram negative bacteria
S. typhimurium (c)	12
K. pneumonia (d)	9
E. coli (e)	11
P. aeruginosa	0
Yeast
C. albicans	0

^aInhibition zone including paper disc (7 mm).

3.5. Heavy metal absorption of γ-PGA

The influence of pH on the adsorption of Cr (VI) by the γ-PGA is shown in Fig. 5. The absorbed amount of Cr (VI) increased from 5.73 mg/g to 9.12 mg/g with decreasing the initial pH from 7.0 to 2.0. The influence of the γ-PGA concentration (0.5–5 g/l) on Cr (VI) adsorption is also shown in Fig. 5. The percentage removal of Cr (VI) increased with increasing the γ-PGA concentration, whereas the amount of Cr (VI) adsorbed per gram of γ-PGA was reduced.

Figure 5.

Influence of pH and γ-PGA concentration on Cr (VI) adsorption. Error bars (±S.D.) are shown when larger than the symbol. ● Cr adsorbed and ○ removal efficiency.

4. Discussion

The γ-PGA has been known for more than 80 years and many researchers have investigated the production of γ-PGA by bacteria. Attractive properties of γ-PGA, which are water-soluble, edible, safe and environmentally friendly, make it of interest for applications in the fields of medicine, foods and bioremediation.

ACE plays an important role in the regulating renin–angiotensin system of blood pressure. In the rennin angiotensin system, ACE cleaves the dipeptide portion of angiotensin I from the C-terminal and produces a potent vasopressor angiotensin II. Angiotensin II induces the release of aldosterone, which causes the retention of sodium ions by kidney and elevates the blood volume and increased blood pressure (Sun et al., 2009). Therefore, the inhibition of ACE is a basic approach in the therapy of high blood pressure in humans. On the other hand, Korean soybean paste, Doenjang is a traditional fermented soybean food and similar to Japanese Miso. Doenjang not only provides nutritional value such as essential amino acids and fatty acids (Namgung et al., 2010) but also has functionalities such as antioxidant, ACE inhibition and fibrinolytic activities (Choi et al., 2007; Park et al., 2008). The quality and functionality of Doenjang are affected by dominant bacteria, B. subtilis and B. licheniformis, during fermentation. In this study, the γ-PGA produced by B. subtilis D7 isolated from Doenjang had the ACE inhibition activity and its IC₅₀ value was 0.108 mg/ml (Fig. 2). To date, the most potent ACE-inhibitory compounds reported were released from proteins of milk fermented with several strains of Lactobacillus. The ACE inhibition activity of the γ-PGA from B. subtilis D7 was higher than those reported by Leclerc et al. (2002) and Quiros et al. (2005) for whey fractions obtained from casein-enriched milk fermented by Lactobacillus helveticus (0.6–1.1 mg/ml) and peptide derived from caprine kefir (0.365 mg/ml). This result suggests that the ACE inhibition activity of Doenjang might be associated with some compounds of Doenjang, including the γ-PGA produced by Bacillus strains in Doenjang. In addition, as the γ-PGA had ACE inhibition activity, it could be used as a value-added component to reduce high blood pressure as therapeutic drugs. Even though there have been many researches on ACE inhibitory compounds, little is known about the ACE inhibition activity of the γ-PGA. Thus, this is the first report on the ACE inhibition activity of the γ-PGA.

Moisturizing effect is an important characteristic of a material intended for cosmetic application. Ben-Zur and Goldman (2007) reported that the γ-PGA had moisturizing effect and skin elasticity more than collagen and hyaluronic acid. Also, water retention of 0.2% γ-PGA hydrogel was observed to be similar to that of 5% glycerol. In this study, the hygroscopicity of the γ-PGA from B. subtilis D7 was 86.8% and its water-holding capacity was 56.9% (Figs. 3 and 4). These studies suggest that the γ-PGA could have potential material as alternative to collagen, hyaluronic acid and glycerol in cosmetic constituent (moisturizer).

Antimicrobial activity is an important characteristic of a material intended for food and biomedical applications. It was reported that γ-PGA-coated magnetite particles and chitosan-γ-PGA polyelectrolyte complex hydrogels showed antibacterial activity against E. coli and S. aureus (Tsao et al., 2010; Inbaraj et al., 2011). However, little is known about the antimicrobial activity of pure γ-PGA. In this study, the γ-PGA from B. subtilis D7 showed antibacterial activity against L. monocytogenes, S. typhimurium, S. aureus, K. pneumonia and E. coli; especially, antibacterial activity was higher against Gram-positive bacteria than against Gram-negative bacteria. Although we do not explain the exact mechanism of antibacterial activity of γ-PGA, a possible antibacterial mechanism was reported by Inbaraj et al. (2011). It has been well known that the proliferation of bacteria is associated with its ability to adhere onto material surfaces (bacterial adhesion) through non-specific interactions such as electrostatic, hydrophobic, van der Waals forces and specific interactions between the bacterial cell-membrane receptor and material surface. Inbaraj et al. (2011) reported that the growth of bacterial cells could be significantly reduced by materials that are hydrophilic and anionic (as the net surface charge of bacterial cell is also negative) and therefore the antibacterial activity of the γ-PGA may be due to the hydrophilic and anionic nature of the γ-PGA. Nevertheless, a further study is necessary to elucidate the exact mechanism of the antibacterial effect of the γ-PGA. On the other hand, the paper disc diffusion assay for the measurement of antimicrobial activity was used in this study. In this case, it is important to consider the relationship between the molecular weight of the γ-PGA and the rate of diffusion, which is reflected on significance of the results. Therefore, further studies are also needed to investigate how the diffusion rate of the γ-PGA through a paper disc is influenced by its molecular weight.

Some researchers reported that the γ-PGA could adsorb Hg (II), Cr (III), Ni (II) and basic dyes (Inbaraj et al., 2006; Yao et al., 2007). Chromium exists in water supplies in both the hexavalent and the trivalent state although the trivalent form rarely occurs in potable water. Hexavalent chromium has been shown to be carcinogenic by inhalation and is corrosive to tissue (Clesscerl et al., 1958). Therefore, we investigated a potential as an adsorbent for the removal of Cr (VI). As shown in Fig. 5, Cr (VI) was strongly adsorbed at a low pH and its adsorption was declined at a higher pH range. Better adsorption at low pH by the γ-PGA may be attributed to a large number of H⁺ ions present at low pH which in turn neutralize the negatively charged adsorbent surface, thereby reducing disturbance to the diffusion of chromate ions (Rao et al., 2002). As shown in Fig. 5, with increasing γ-PGA concentration, the Cr (VI) removal efficiency was enhanced, whereas the amount of Cr (VI) adsorbed showed a decline. This result may be attributed to the adsorption sites remaining unsaturated during the sorption reaction. In other words, a less commensurate rise in Cr (VI) adsorption could occur following the increase of the γ-PGA concentration, probably due to a lower adsorptive capacity utilization of the γ-PGA. In addition, the particle interaction at high γ-PGA concentration may result in desorption of some weakly bound Cr (VI) from the γ-PGA surface (Inbaraj et al., 2006). In this study, the highest adsorbed quantity of Cr (VI) by the γ-PGA was 21.4 mg/g (Fig. 5); indicating the lower absorbed quantity as compared with other γ-PGA studies as an adsorbent for the removal of heavy metal ion (Rao et al., 2002; Mark et al., 2006). Nevertheless, our result suggests that the γ-PGA may play a role in Cr (VI) adsorption as an adsorbent even though it has low adsorption.

In conclusion, the γ-PGA produced by B. subtilis D7 had novel functionalities such as ACE inhibition activity, antibacterial activity and moisturizing effect. These results suggest that the γ-PGA could be considered a potential candidate for moisturizer, therapeutic drug and antimicrobial agent in the fields of pharmaceutical, food and cosmetics industries.