Bacterial cellulose synthesized in Erythrina mulungu extract: structural and bioactive properties

Abstract

Bacterial cellulose is a biopolymer valued for its purity, biocompatibility, and versatility, but its production typically depends on synthetic media, limiting sustainability and hindering the incorporation of functional compounds during biosynthesis. The effects of plant-derived extracts on the physicochemical and biological properties of bacterial cellulose remain poorly understood. This study evaluates the use of an aqueous extract of Erythrina mulungu as an alternative culture medium capable of producing and functionalizing bacterial cellulose simultaneously. Over 20 days, hydrated membranes were obtained with an average productivity of 186.41 g/L. Infrared spectroscopy confirmed characteristic cellulose functional groups, while X‑ray diffraction revealed reduced crystallinity in mulungu‑derived membranes (46%) compared to saline‑derived membranes (72%). Antimicrobial assays showed inhibition of Escherichia coli and Staphylococcus aureus. Antioxidant activity reached 14.70% for the extract, 10.14% for mulungu‑derived cellulose, and 1.50% for saline‑derived cellulose. The extract enhanced functional properties, supporting applications requiring antimicrobial and antioxidant performance.

Keywords:
biopolymer membranes; natural culture medium; phenolic compounds; antioxidant activity; antimicrobial properties

1. Introduction

Cellulose is one of the most abundant natural polymers. It is formed by repeating β‑D‑glucopyranose units linked by β-(1→4) bonds and contains many hydroxyl groups along its chain[¹,²]. Although mainly produced by plants, it can also be synthesized by fungi, seaweeds, and some bacteria[³].

Bacterial cellulose (BC) stands out for its high crystallinity, purity, mechanical strength, water-holding capacity, biocompatibility, biodegradability, and non-toxicity[⁴,⁵]. These features, along with the possibility of structural modification, support its use in the medicine, food, and pharmaceutical industries; packaging; paper; textiles; water treatment; and electronics [⁶-⁹].

BC can be functionalized by adding substances that introduce new properties. The most common modification strategies are in situ (during synthesis) and ex situ (after production) [⁶]. Natural compounds have gained prominence as modifiers due to their functional potential and sustainability, especially those derived from medicinal plants[¹⁰,¹¹].

Erythrina mulungu is a Fabaceae species native to the caatinga, whose bark and leaves contain alkaloids, flavonoids, terpenoids, and phenolic compounds responsible for its antioxidant and antimicrobial activities[¹²]. When incorporated into the bacterial cellulose matrix, these metabolites can interact with the hydrogen‑bonding network, reducing crystallinity, altering fibrillar organization, and introducing new functional groups detectable by spectroscopy. Such interactions may also impart antioxidant and antimicrobial properties to the cellulose, enabling the production of functionalized membranes without additional modification steps. These physicochemical and biological changes expand the material’s potential for applications requiring enhanced bioactivity, such as active packaging, biomedical devices, and cosmetic formulations[¹³,¹⁴].

In this study, bacterial cellulose was produced using an aqueous extract of Erythrina mulungu as an alternative culture medium, to produce membranes with antimicrobial and antioxidant activity. The extract was characterized by High-Performance Liquid Chromatography (HPLC), total phenolic content, Fourier-transform infrared spectroscopy (FTIR), antioxidant assays, and antimicrobial tests. The BC membranes were analyzed by X‑Ray Diffraction (XRD), FTIR, Thermogravimetry (TG), Scanning Electron Microscopy (SEM), and antimicrobial and antioxidant assays.

2. Materials and Methods

2.1 Production and treatment of bacterial cellulose

2.1.1 Bacterial strain, plant raw material, and reagents

Gluconacetobacter hansenii was obtained from the Culture Bank of the Catholic University of Pernambuco, and Erythrina mulungu bark was purchased from a natural products store in Recife (PE, Brazil). The reagents used for microbial cultivation and analyses included peptone, agar, yeast extract, glucose, ethyl alcohol, DPPH, ascorbic acid, calcium chloride, acetic acid, sodium carbonate, Folin–Ciocalteu reagent, acetonitrile, gallic acid, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, and ferrous sulfate, all from standard commercial suppliers.

2.1.2 Culture medium and strain activation

The conservation medium followed Hestrin and Schramm[¹⁵]. For activation, 100 mL of medium was sterilized (121 °C, 20 min), melted, poured into sterile tubes, and solidified at 45°. Cells were inoculated with a platinum loop and incubated at 30 °C for 48 h. Cultures were stored at −2 °C and subcultured every 30 days.

2.1.3 Preparation of the aqueous extract

The extract was prepared by infusing 6 g of dried E. mulungu bark in 250 mL of distilled water ( 24 g/L) at 80 °C for 20 min. After cooling and filtration, it was stored at 4 °C.

2.1.4 Production of cellulose membranes

The culture medium consisted of the E. mulungu extract supplemented with sucrose (48 g/L). Membranes were cultivated in Roux flasks containing 400 mL of medium under static conditions at 25 ± 2 °C for 20 days.

The control saline medium followed patent BR1020200124005[¹⁶], containing glucose and mineral salts (ammonium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride, and ferrous sulfate), all analytical grade. Cellulose produced from the mulungu extract is referred to as BC Mulungu, and that from the saline medium as BC Salts.

2.1.5 Washing, purification, and drying

After cultivation, membranes were washed with distilled water, immersed in E. mulungu extract, and sterilized by gamma irradiation (25 kGy). They were dried at 45 °C for ~24 h and stored in a desiccator before characterization.

2.2 Characterizations

2.2.1 Characterization of the Erythrina mulungu aqueous extract

Phenolic compounds were identified by high-performance liquid chromatography (HPLC) using a Shimadzu UFLC system with a Phenomenex C‑18 column (250 × 4.6 mm). Mobile phases were water (A) and acetonitrile (B). The gradient started at 7% B, increased to 50% over 36 min, remained until 70 min, returned to 7% at 80 min, and ended at 90 min. Flow rate was 0.4 mL/min, column temperature 30 °C, detection at 280 nm, and injection volume 50 µL.

The Folin determined total phenolic content–Ciocalteu method[¹⁷]. Extract concentration (500 µg/mL) was obtained by drying 1 mL of extract at 60 °C to constant mass. The reaction mixture contained distilled water (8 mL), Folin–Ciocalteu reagent (0.5 mL), sample (0.5 mL), and saturated sodium carbonate (1 mL). After 60 min in the dark, absorbance was measured at 720 nm. Analyses were performed in triplicate, using PA ethanol as the blank and gallic acid as the standard.

Fourier‑transform infrared spectroscopy was performed on the dried extract using a Perkin Elmer Spectrum 400 with ATR, collecting spectra from 550–4000 cm⁻¹ (4 cm⁻¹ resolution, 16 scans).

Antimicrobial activity was evaluated by agar disk diffusion against Escherichia coli and Staphylococcus aureus. Filter paper discs (5.2 mm) impregnated with extract were placed on plates inoculated with 0.1 mL of bacterial suspension (10⁻⁴ McFarland). Plates were incubated at 30 °C for 48 h, and inhibition zones were measured.

Antioxidant activity was assessed by the DPPH method[¹⁸]. A DPPH solution (2.5 mg/100 mL in ethanol) was mixed with 100 µL of extract and kept in the dark for 60 min. Absorbance was measured at 515 nm, and inhibition was calculated using Equation 1.

$\text{\%}\text{DPPH inhibition}=\frac{\text{Abscontrol}-\text{Abssample}}{\text{Abscontrol}}\text{x}100$ (1)

2.2.2 Characterization of bacterial cellulose membranes

Fourier identified functional groups of the BC membranes‑transform infrared spectroscopy coupled with attenuated total reflectance, using spectra collected from 550 to 4000 cm⁻¹. Surface morphology was examined by scanning electron microscopy, enabling visualization of fibrillar organization, porosity, and possible structural modifications induced by the alternative culture medium.

Crystallinity was assessed by X‑ray diffractio, and the crystallinity index was calculated using the Segal method[¹⁹], providing information on the degree of structural order and the influence of the extract on cellulose microfibril arrangement. Thermal stability was evaluated by thermogravimetryusing a Mettler Toledo TG 2 Star System. Approximately 20 mg of each membrane were heated from 30 to 800 °C at 10 °C/min under a nitrogen atmosphere to prevent thermo‑oxidative degradation.

Antimicrobial activity was determined by the agar disk diffusion method using circular membranes (1 cm diameter) placed on nutrient agar plates inoculated with Escherichia coli and Staphylococcus aureus. Plates were incubated at 30 °C for 48 h, and inhibition zones were measured to assess the bioactive potential of the membranes.

Antioxidant activity was evaluated by the DPPH radical scavenging assay. Membrane fragments (1 × 1 cm) were extracted in 50% ethanol for 24 h, and 0.1 mL of the extract was mixed with 3.9 mL of DPPH solution. After 1 h in the dark, absorbance was measured at 515 nm, and antioxidant activity was calculated using Equation 1.

3. Results and Discussions

The hydrated and dry yields were calculated from membrane mass. BC Mulungu produced 186.41 g/L (hydrated) and 5.35 g/L (dry), while BC Salts reached 346.5 g/L and 17.35 g/L. These differences reflect the influence of medium composition: the saline medium, rich in simple sugars and minerals, favors bacterial metabolism and cellulose secretion, whereas the mulungu extract contains complex phytochemicals that may slow growth but contribute functional properties. Figure 1 illustrates the BC production process using the Erythrina mulungu extract, from extract preparation to the final dry membrane.

Figure 1
Production of bacterial cellulose using the aqueous extract of Erythrina mulungu: (a) extract; (b) membrane formation; (c) wet membrane; and (d) dry membrane.

3.1 Characterization of the aqueous extract of Erythrina mulungu

3.1.1 High-Performance Liquid Chromatography (HPLC)

The chromatogram obtained for the aqueous extract of mulungu is shown in Figure 2, in which the main peaks corresponding to the compounds present in the extract can be observed. Specific alkaloids and phenolic compounds were identified in the chromatogram.

Figure 2
HPLC chromatogram of the aqueous extract of Erythrina mulungu.

The peak at 5.9 minutes corresponds to minor phenolic compounds, while the main peak at 9.24 minutes is typical of common phenols such as chlorogenic and caffeic acids[²⁰]. Peaks between 16 and 30 minutes (16.96, 25.73, 30.19 min) indicate erythrin alkaloids like erysothrine and related derivatives, consistent with their higher hydrophobicity. The peak at 44.91 minutes suggests less polar alkaloid forms, and the late signal at 75.72 minutes likely reflects residual compounds or cellulose‑derived fragments retained longer in the column[²¹,²²].

3.1.2 Determination of total phenolics

The concentration of phenolic compounds in the aqueous extract of Erythrina mulungu, determined by the Folin–Ciocalteu method, was 38.18µg/mL or 7.64mg GAE/g of dry extract, a value considered moderate and relevant for antioxidant and bioactive applications[²³]. Da Silva Mota et al.[²⁴] reported 27.0mg GAE/g in hydroethanolic extract of the same species, a result compatible with this study, considering the greater extraction efficiency with mixed solvents.

3.1.3 Fourier transform infrared spectroscopy

Infrared spectroscopy was conducted to identify the main characteristic absorption bands of the aqueous extract of Erythrina mulungu. The resulting spectrum of the extract is presented in Figure 3.

Figure 3
Infrared spectrum of the aqueous extract of Erythrina mulungu.

The FTIR spectrum of the Erythrina mulungu extract (Figure 3) showed bands consistent with the HPLC profile. The broad signal near 3300 cm⁻¹ corresponds to O–H stretching of phenolic compounds, while the 2935 cm⁻¹ band reflects aliphatic C–H groups typical of flavonoids and alkaloids. Peaks at 1612 and 1381 cm⁻¹ indicate aromatic C=C stretching, and the 1183–1023 cm⁻¹ region shows C–H bending and C–O stretching associated with oxygenated groups and glycosidic linkages in erythrin alkaloids[²⁵]. Altogether, the spectrum supports the presence of phenolic and alkaloid constituents, confirming the extract’s chemical complexity.

3.1.4 Antimicrobial activity

After the incubation period, it was found that the aqueous extract of Erythrina mulungu presented a slight inhibition of the growth of the bacteria under study, as the formation of the inhibition halo for the two types of bacteria under analysis could be verified. As illustrated in Figure 4, inhibition halos were observed for both bacterial strains. The images relating to the antimicrobial activity test can be seen in the figures below.

Figure 4
Antimicrobial activity of the aqueous extract of Erythrina mulungu against (a) Escherichia coli and (b) Staphylococcus aureus.

Measurement of the inhibition halos yielded values of 9.75 ± 0.10 mm for Escherichia coli and 8.5 ± 0.06 mm for Staphylococcus aureus. These results indicate that both bacterial strains exhibited resistance to the aqueous extract of Erythrina mulungu, as the inhibition halo diameters were less than 14 mm.

3.1.5 Antioxidant activity

The antioxidant activity was evaluated using the DPPH radical inhibition method (Section 2.2.1), resulting in 14.70% inhibition for 100 µL of the Erythrina mulungu aqueous extract. As reported by Ramos et al.[²⁶], phenolic compounds are mainly responsible for the antioxidant activity of E. mulungu due to their ability to neutralize free radicals. The relatively low inhibition observed here may be related to the limited solubility of nonpolar compounds, such as flavonoids and alkaloids, which are known contributors to antioxidant activity but are poorly extracted in water.

Although this study focused on the aqueous extract as the culture medium, exploratory tests were also performed with the ethanolic extract. When used as the sole medium, the ethanolic extract did not support membrane formation, suggesting that its composition or residual ethanol affected bacterial viability. These results indicate that, while ethanol is more efficient at extracting antioxidant compounds, its direct use as a culture medium is unsuitable, being more appropriate as a supplement. This highlights the importance of balancing solvent extraction efficiency with microbial compatibility in bacterial cellulose production[²⁶].

These results, together with the chemical profile identified by HPLC and FTIR, reinforce that the phenolic and alkaloid compounds present in the extract not only influence BC yield but also confer relevant bioactive properties, even if at moderate intensity.

3.2 Characterization of the produced membranes

3.2.1 Fourier Transform Infrared Spectroscopy

The characteristic bands of BC can be observed in Figure 5, which shows the infrared spectrum of bacterial cellulose produced using the aqueous extract of Erythrina mulungu.

Figure 5
Infrared spectra of BC Salts and BC Mulungu membranes.

Figure 5 shows the FTIR spectra of BC produced in saline medium and in Erythrina mulungu extract. Both samples display the typical cellulose bands (O–H at 3690–2981 cm⁻¹ and C–H at 2974–2831 cm⁻¹). In the mulungu‑derived BC, additional signals appear between 1750 and 1028 cm⁻¹. The weak 1740–1750 cm⁻¹ band suggests esterified or conjugated C=O groups[²⁷,²⁸], while bands at 1652–1647 cm⁻¹ (asymmetric COO⁻), 1430–1412 cm⁻¹ (symmetric COO⁻), and 1300–1028 cm⁻¹ (aromatic vibrations) indicate phenolic and alkaloid compounds from the extract[²⁹]. These features confirm the incorporation of E. mulungu metabolites into the BC network.

3.2.2 X-ray diffractometry

In Figure 6, the XRD pattern of bacterial cellulose obtained from the aqueous extract of Erythrina mulungu is shown, in comparison with BC Salts.

Figure 6
XRD patterns of BC Salts and BC Mulungu membranes.

Figure 6 shows the XRD patterns of bacterial cellulose samples obtained in salt medium and in aqueous extract of Erythrina mulungu. Both profiles exhibit broad diffraction peaks, typical of semicrystalline materials such as bacterial cellulose, with reflections at 2θ ≈ 14.6° (plane 110), 17.1° (plane 110), and 22.7° (plane 200), corresponding to the crystalline structure of cellulose type I[³⁰].

The similarity between the BC Salts and BC Mulungu diffractograms indicates that the extract did not alter the cellulose I crystalline pattern. However, the crystallinity index decreased from 72% (BC Salts) to 46% (BC Mulungu), showing that the extract promoted a more amorphous structure. This reduction may result from interactions between extract metabolites and the cellulose hydrogen‑bonding network[³¹,³²]. Lower crystallinity can enhance flexibility, porosity, and active incorporation, which are desirable for food and biomedical applications[³³]. The decrease is also consistent with the lower thermal stability observed in TG analyses, reinforcing the relationship between structural organization and thermal resistance.

3.2.3 Thermogravimetry

The thermograms of the produced membranes are shown in Figure 7, and the degradation temperature results can be observed in Table 1.

Figure 7
TG (a) and DTG (b) curves of BC Salts and BC Mulungu membranes.

Table 1
TG parameters for BC Salts and BC Mulungu membranes.

Films	1st Stage (Ton – Toff – Tmax)	2nd Stage (Ton – Toff – Tmax)	3rd Stage (Ton – Toff – Tmax)
BC Salt	31.33 – 188.33 – 161.73	188.33 – 252.33 – 214.72	290.33 – 553.00 – 324.96
BC Mulungu	49.33 – 140.33 – 117.31	161.17 – 258.17 – 203.09	272.83 – 599.83 – 325.35

As shown in Figure 7 and Table 1, the bacterial cellulose samples exhibited three main mass‑loss stages: (i) moisture evaporation near 100 °C, (ii) cellulose decomposition with depolymerization and carbonaceous residue formation, and (iii) thermo‑oxidative degradation above 300–350 °C[⁴,²⁹,³⁴]. The second stage, occurring around 200–300 °C, corresponds to the breakdown of β‑1,4‑glycosidic bonds, consistent with typical BC degradation.

In this study, Ton, Tmax and Toff were obtained directly from the DTG curves, which provide more precise identification of the onset, maximum and end temperatures of each event, avoiding extrapolation from TG data.

Although Tmax values were similar between samples, BC produced with the mulungu extract showed a lower Ton, indicating earlier degradation and reduced thermal stability compared to BC Salts. This behavior is associated with its lower crystallinity, since more crystalline regions enhance thermal resistance[³⁵-³⁸]. Although the lower stability may limit certain industrial applications, it favors greater flexibility and porosity, which are desirable characteristics for biomedical and food‑related uses. Mass losses for each stage and the final residue at 600 °C are presented in Table 2.

Table 2
TG parameters of BC Salts and BC Mulungu membranes.

	Weight Loss (%)			Residue (%)
Samples	1° Stage	2° Stage	3° Stage
BC Salts	17.99	18.14	22.30	34.95
BC Mulungu	8.26	27.05	33.83	26.55

The final residue corresponds to the non‑volatile fraction, mainly ash and inorganic compounds resistant to 600 °C under nitrogen[³⁹]. BC from the aqueous extract showed lower mass loss in the first stage (8.26%) than BC Salts (17.99%), likely due to reduced moisture. In later stages, it exhibited higher mass losses and a smaller final residue (26.55% vs. 34.95%), indicating lower thermal stability, consistent with its reduced crystallinity.

3.2.4 Scanning electron microscopy

SEM analysis revealed that the microstructural morphology of BC Mulungu (Figure 8a) consists of an interconnected, three-dimensional network of cellulose nanofibrils, forming a porous and fibrous architecture. The membranes, previously sterilized by gamma irradiation to ensure purity and integrity, displayed a clean, nanofibrous arrangement, free of visible contaminants, highlighting the effectiveness of the treatment in preserving the intrinsic structure of bacterial cellulose. This morphology reflects the hierarchical organization of cellulose chains during the membrane formation process[⁴⁰]. Moreover, the observed architecture closely resembles that of the BC membrane produced using the salt medium (Figure 8b), confirming that the alternative cultivation strategy successfully promoted the synthesis of bacterial cellulose nanofibrils with comparable structural features.

Figure 8
Micrographs of (a) BC Mulungu and (b) BC Salts membranes.

3.2.5 Antimicrobial activity

The antimicrobial activity test was conducted for both membrane samples. Following the incubation period, inhibition of microbial growth was observed for both bacterial strains, as demonstrated in Figure 9.

Figure 9
Antimicrobial activity of BC Salts against (a) Escherichia coli; and (b) Staphylococcus aureus; and of BC Mulungu against (c) Escherichia coli; and (d) Staphylococcus aureus.

Post‑incubation analysis confirmed the formation of clear inhibition halos for all samples. BC Salts produced halos of 24.75 ± 0.10 mm against Escherichia coli and 39.25 ± 0.96 mm against Staphylococcus aureus. BC Mulungu showed larger halos, 31.50 ± 0.51 mm and 46.50 ± 0.24 mm for E. coli and S. aureus, respectively, demonstrating stronger antimicrobial activity.

The activity observed for BC Salts is attributed to metallic compounds in the saline medium, which can disrupt bacterial membranes and promote cell death[⁴¹]. The higher inhibition values obtained for BC Mulungu indicate that bioactive compounds from Erythrina mulungu enhanced the antimicrobial performance of the membranes, reinforcing the extract’s relevance as an effective culture medium.

Direct comparison with the literature is limited because no studies were found using the same concentration of aqueous extract. Still, previous works consistently report antimicrobial and antioxidant activities in Erythrina species, attributed mainly to flavonoids and alkaloids[⁴²-⁴⁵], supporting the coherence of the activities observed here. Moreover, the inhibition halos obtained for membranes produced with Erythrina mulungu extract exceeded values commonly reported for conventional bacterial cellulose, indicating that incorporating plant‑derived metabolites can be an effective strategy for functionalizing cellulose‑based membranes.

3.2.6 Antioxidant activity

The antioxidant activity of BC Mulungu was 10.14%, while BC Salts showed only 1.5%. The aqueous extract of Erythrina mulungu exhibited 14.70% activity, which decreased to 10.14% after incorporation into the BC membrane. This reduction reflects the partial adsorption of phenolic compounds into the cellulose matrix and their gradual release, yet the activity remains far higher than that of standard BC.

These results indicate that the phenolic compounds from the mulungu extract were effectively incorporated and retained in the BC structure, giving the membrane significant antioxidant functionality. In contrast, BC produced in the saline medium showed minimal activity, confirming the absence of bioactive compounds. Overall, the incorporation of mulungu extract generated a functionalized BC with clear bioactive potential and provides a basis for future optimization.

4. Conclusions

This study demonstrated that bacterial cellulose can be produced using an aqueous extract of Erythrina mulungu, generating membranes with good structural quality. FTIR confirmed the characteristic BC profile, while XRD showed a more amorphous structure in mulungu-derived membranes. Thermogravimetric analysis indicated lower thermal stability at higher temperatures but greater stability in the initial stages. Both membranes showed antimicrobial activity against Escherichia coli and Staphylococcus aureus, with stronger inhibition for BC produced in the mulungu extract. Antioxidant activity was also detected only in this membrane, confirming the incorporation of bioactive compounds. Overall, BC synthesized with E. mulungu presents combined antimicrobial and antioxidant functionalities, indicating potential for future technological and biomedical applications.

6. Acknowledgements

The authors gratefully acknowledge the support provided by the Foundation for the Improvement of Higher Education Personnel (CAPES), the Foundation for Science and Technology Support of the State of Pernambuco (FACEPE), the National Council for Scientific and Technological Development (CNPq), and the Center for Research in Therapeutic Innovation (Nupit).

• Data Availability:
  Research data is available upon request from the corresponding author.
• How to cite:
  Silva, G. S., Henrique, M. A., Alves Júnior, S., & Vinhas, G. M. (2026). Bacterial cellulose synthesized in Erythrina mulungu extract: Structural and bioactive properties. Polímeros: Ciência e Tecnologia, 36(3), e20260028. https://doi.org/10.1590/0104-1428.20250087

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Edited by

• Editor-in-Chief:
  Sebastião V. Canevarolo

Data availability

Research data is available upon request from the corresponding author.

Publication Dates

• Publication in this collection
  24 July 2026
• Date of issue
  2026

History

• Received
  29 Sept 2025
• Reviewed
  18 Apr 2026
• Accepted
  30 Apr 2026