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Comparison of bacterial nanocellulose produced by different strains under static and agitated culture conditions
Carbohydrate Polymers 227 (2020) 115323
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Comparison of bacterial nanocellulose produced by different strains under static and agitated culture conditions
T
Hongliang Gao, Qian Sun, Zebei Han, Jiahe Li, Bowen Liao, Lulu Hu, Jie Huang, Chunjing Zou, ⁎ ⁎ Caifeng Jia, Jing Huang, Zhongyi Chang, Deming Jiang , Mingfei Jin School of Life Sciences, East China Normal University, Shanghai, 200241, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial nanocellulose Static and agitated culture Yield Crystallinity Beverage stabilizer
Bacterial nanocellulose (BNC) has many advantages over plant cellulose, which make it widely used in many fields, especially in the food industry. In this study, three strains including BCA263, BCC529, and P1 were selected for characteristics analysis of BNCs under static and agitated culture conditions. The BNCs produced under static culture condition were in the shape of uniform membrane, while BNCs produced under agitated culture were in form of small agglomerates and fragments. BCA263 and BCC529 strains were more suitable for static culture, while P1 strain was more suitable for agitated culture. BNCs produced under static culture condition exhibited higher crystallinity, stronger tensile strength, denser network structure, higher temperature resistance and good flame retardancy; while BNCs produced under agitated culture condition exhibited larger porous and lower crystallinity. Furthermore, BNCs produced under agitated culture condition were more suitable as a stabilizer of coffee milk beverage.
1. Introduction Cellulose has a large commercial use in paper, textile, cellulose nanofibrous mats, and pulp production units (Deng et al., 2010; Klemm, Heublein, Fink, & Andreas, 2005; Zhang et al., 2015). Plant cell walls are the major source of cellulose. However, this type of cellulose contains several impurities, including lignin, pectin, and hemicelluloses etc. Apart from plants, cellulose is also found in many microorganisms such as fungi, bacteria, and algae. Recent studies have revealed that cellulose can be produced by different bacteria, including Gluconacetobacter (formerly Acetobacter), Azotobacter, Rhizobium, Agrobacterium, Pseudomonas, Salmonella, Alcaligenes. (Esa, Tasirin, & Rahman, 2014; Shoda & Sugano, 2005). These bacteria produce a fine cellulose fibril with a width of 50–80 nm and a thickness of 3–8 nm. When several fibers entangle, a three-dimensional nano-network with pores is formed (Tabuchi, 2007). Therefore, bacterial cellulose (BC) is a fascinating polymer with a three-dimensional structure formed by nanofibers of pure cellulose, which is also named as bacterial nanocellulose (BNC) due to its nanostructure. The bacterium Acetobacter xylinum, also known as Gluconacetobacter xylinum or Komagataeibacter xylinus, has been shown to have the highest rate of BC production among all the BNC-producing bacteria types (Reiniati, Hrymak, & Margaritis, 2017). It has been observed that one cell can convert 108 glucose molecules
⁎
per hour into cellulose (Wang, Tavakoli, & Tang, 2019). BNC has many advantages over plant cellulose, including high purity, good water absorption, strong water retention, high tensile strength, good biocompatibility and biodegradability (Aydin & Aksoy, 2014; Bi et al., 2014; Islam, Ullah, Khan, Shah, & Park, 2017; Kuo, Teng, & Lee, 2015; Qiu & Netravali, 2014). Based on these characteristics, BNC has been widely used in many areas such as electronics, biomedicine, food industry and energy storage (Keshk, 2014; Torres, Arroyo, & Troncoso, 2019). Especially in the food industry, BNC is traditionally and widely used to make nata de coco as a better dietary fiber in Southeast Asia (Nugroho & Aji, 2015; Shi, Zhang, Phillips, & Yang, 2014; Tabuchi, 2007).It is also used in foods such as multifunctional food ingredients to control the properties of food or beverage as a thickener, stabilizer and texture modifier. (Esa et al., 2014, Tabuchi, 2007). In the biomedical field, BNC is widely studied as a highly biocompatible raw material for wound healing, artificial blood vessels, and tissue engineering (Czaja, Krystynowicz, Bielecki, & Brown, 2006; van Zyl & Coburn, 2019). There are two main methods to produce BNC by bacterial strains (Czaja, Romanovicz, & Brown, 2004). One is the static culture, which results in the accumulation of a gelatinous membrane of BNC is formed at the gas-liquid junction with the shape of the culture vessel (Rangaswamy, Vanitha, & Hungund, 2015; Trovatti et al., 2011). The
Corresponding authors. E-mail addresses: [email protected] (D. Jiang), [email protected] (M. Jin).
https://doi.org/10.1016/j.carbpol.2019.115323 Received 19 June 2019; Received in revised form 20 August 2019; Accepted 10 September 2019 Available online 11 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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contains the following constituents (%, w/v): sucrose, 5.0; peptone, 0.5; yeast extract, 0.9; K2HPO4, 0.3 ; MgSO4, 2.0; acetic acid, 0.2; The initial pH was adjusted to 6.0. The seed and fermentation medium for BCA263 strain contains the following constituents (%, w/v): sucrose, 5.0; corn steep liquor, 5.0; (NH4)2SO4, 0.33; K2HPO4, 0.13; MgSO4, 0.012; FeSO4, 0.0002; CaCl2, 0.0011; Na2MoO4, 0.00002; ZnSO4, 0.0001; MnSO4, 0.0001; and CuSO4, 0.0000032; The initial pH was adjusted to 5.0 (Cheng et al., 2009).
other one is agitated culture, where BNC is synthesized in deep medium by bacterial strain in a shake flask or a fermenter under aerated and agitated conditions, and BNC is dispersed in the fermentation broth in the form of fibrous suspensions, pellets or irregular masses (Aydin & Aksoy, 2014; Chao, Ishida, Sugano, & Shoda, 2015). Strains and culture conditions have a great influence on the microstructures and physiochemical properties of BNC. Changes in crystallinity and mass fraction of the Iα allomorph were observed in BNC produced by different strains and culture conditions (Deng et al., 2015). Some additives, such as agar, carboxymethylcellulose and microcrystalline cellulose were added into the fermentation medium in agitated culture show obviously changes on the yield and structure of BNC produced by Acetobacter xylinum (Cheng, Catchmark, & Demirci, 2009). Thus, screening strains with high BNC yields under static and agitated culture conditions has been a critical issue in BNC research for a long time. A series of BNC-producing strains have been isolated from different sources such as rotten fruits, vegetables and soils (Toyosaki et al., 2014). Among them, A strain isolated from kombucha show potential for commercial applications owing to its high phenotypic stability and sustainable production capacity of 7.56 ± 0.57 g/L under static culture and 8.31 ± 0.79 g/L under shaking culture (Zhang, Wang, Qi, Ren, & Qiang, 2018). Although many strains have been isolated for BNC production from fruit (Jahan, Kumar, Rawat, & Saxena, 2012; Rangaswamy et al., 2015), there are few studies on the comparison of the properties and application in food of BNC produced by different strains under different culture conditions, which is important for the improvement of the production of BNC and furthering their applications. In this study, the properties of macro- and micro-structure, yield, crystallinity, tensile strength and thermal stability of the BNCs produced by three different strains under static or agitated culture conditions were investigated. Komagataeibacter xylinus BCC529 (CICC 10529) and Acetobacter xylinum BCA263 (ATCC 53263) are the representative model strains with potential industrial application as shown in previous reports (Lida, Ohnishi, & Horinouchi, 2008; Lin, Li, Lopez-Sanchez, & Li, 2015). Gluconacetobacter xylinus P1 was isolated and stored in our lab. Finally, BNCs produced by static or agitated methods were added as a stabilizer to the coffee milk beverages, and the stability of coffee milk beverages with different BNCs was analyzed based on precipitation rate, suspension stability and zeta potential.
2.4. Culture conditions All bacterial strains were pre-culture in seed agar medium at 30 °C for 48 h. The slant culture was transferred into seed medium and cultured at 30 °C for 24 h under static condition. After that, the seed culture was filtered with sterile cotton, and the filtered culture was used as seed. In a static culture condition, the filtered seed culture (96 mL) was transferred into a container (52 cm × 33 cm × 7.5 cm) containing 1.2 L fermentation medium, and culture at 30 °C for 96 h. The produced BNCs were purified following the purification method below. The BNCs produced by three strains were represented by BCC529-S, P1-S and BCA263-S respectively, where S indicated static culture condition. In an agitated culture condition, the filtered seed culture (2.4 mL) was added to 30 mL seed medium, and cultured at 160 r/min at 30 °C for 18 h to obtain the second seed culture. 200 mL of the second seed culture were added into the 3 L fermenter containing 2.5 L of fermentation medium. Culture was carried at a speed of 300 r/min and a temperature of 30 °C for 96 h, and the aeration rate was 1.0 vvm. The produced BNCs were purified following the purification method below. The BNC produced by the three strains was represented by BCC529-A, P1-A and BCA263-A respectively, where A indicated agitated culture condition. 2.5. BNC production and purification Under static culture condition, both the fermentation broth and the BNC membranes poured into the centrifugal bottle, while under agitated culture condition BNCs which were fibrous suspensions and pellets also poured into the centrifugal bottle, only a few BNCs attached to the impeller was scraped into the fermentation broth with tweezers. Then the suspension was homogenized and centrifuged (9000 r/min, 5 min). The product was then washed twice with distilled water, and was boiled in 1% NaOH for 30 min. After cooled down to the room temperature, the product was centrifuged (9000 r/min, 5 min) and neutralized with 1% acetic acid. The product was continuously washed with distilled water to neutral. Finally, the BNC product was dried at 60 ºC to constant weight and weighed (Karahan et al., 2011).
2. Materials and methods 2.1. Materials All the chemical reagents used in this study, such as glucose, peptone, yeast extract, Na2HPO4, critic acid and corn steep liquor, were purchased from Chemical reagent of national medicine group, Shanghai, China. 2.2. Microorganisms
2.6. Fourier transform infrared spectroscopy (FT-IR)
Strain Komagataeibacter xylinus BCC529 (CICC 10529) was purchased from China Center of Industrial Culture Collection (CICC) (Beijing, China). Strain Gluconacetobacter xylinus BCA263 (ATCC 53263) was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Strain Gluconacetobacter xylinus P1 was isolated and stored in the Food and Microbial Technology laboratory, School of Life Science, East China Normal University at China.
The BNC samples produced by different strains under static and agitated conditions were freeze-dried on a plastic dish to form thin films. The infrared spectra of the films were measured using Fourier transform infrared spectrometer Spectrum 2000 (PE company, America). All spectra were recorded with a resolution of 2 cm−1 in the range from 4000 to 500 cm−1. Baselines for each sample spectrum were normalized using the spectrum software (Santos et al., 2015).
2.3. Culture media
2.7. X-ray diffraction (XRD)
The seed medium for BCC529 and P1 strains were Hestrin and Schramm (HS) medium with some modification (%, w/v) (Schramm & Hestrin, 1954): glucose, 2.0; peptone, 0.5; yeast extract, 0.5; Na2HPO4, 0.27; critic acid, 0.115; corn steep liquor, 1.0. The initial pH was adjusted to 6.0. The fermentation medium for BCC529 and P1 strains
The freeze-dried BNC films were placed in the X-ray holder (D/max2500, Japan). Scans were performed over the 5-40°, speed of 10°/min, and 2Ɵ range using steps of either 0.02° in width. The crystallinity of BNC was calculated according to the formula: CrlXRD=(I200-Iam)/ I200×100% (Santos et al., 2015). 2
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Fig. 1. The photo images of BNC produced by three strains under static (A) and agitated (B) culture conditions.
evaluated. The formula of stabilizers was composed of BNC 18.3%, xanthan gum 6.2%, carboxymethyl cellulose 12.1%, maltodextrin 6.4% (g/L). As a control assay, a suspension containing 1.5% coffee, 0.08% baking soda, 3% non-dairy creamer, 2.5% whole milk powder, 6% sugar, 0.05% sucrose ester and 0.15% monoglyceride in distilled water was prepared by heating at 60 °C for 20 min. Then a stabilizer containing different BNCs samples were added into a control suspension at concentration of 0.35% and the mix was sheared at a high speed of 10000 r/min for 10 min, the coffee milk beverage was filtered through a 180-mesh sieve and the solution was homogenized twice at 65–75 ºC and 30 MPa. Finally, the solution was packed in bottles and sterilized at 115 °C for 20 min. These coffee milk beverages were allowed to stand at room temperature for 24 h. During the time, the statuses of the beverages were observed, and photographs were taken. Determination of Precipitation rate: the coffee milk beverage was weighed (denoted as m), and then was centrifuged at 3000 r/min for 15 min. The precipitate was collected and weighed (denoted as m1). The precipitation rate was calculated according the formula: precipitation rate (%) = m1/m × 100%. Determination of suspension stability: the coffee milk beverage was centrifuged at 4200×g for 15 min. The supernatant was collected and measured its absorbance at 660 nm. Determination of Zeta potential: the coffee milk beverage was diluted, and then the ultrasonic treatment was conducted. After that, the zeta potential of the solution was measured by Dynamic Light Scattering Instrument DynaPro NanoStar (Wyatt, America).
2.8. Scanning electron microscope (SEM) The freeze-dried BNC films were sprayed with gold and observed by scanning electron microscopy EVO (Zeiss, Germany) operating at 10 or 15 kV to observe its microstructure (Cakar, Ozer, Aytekin, & Sahin, 2014). The diameter of BNC was calculated by software Image J to obtain the approximate nanometer range of the fiber filament. 2.9. Mechanical properties A texture analyzer (TA-XT, Stable Micro System, UK) was used to measure BNC mechanical properties according to ASTM D882 (Concepts, 2014). The purified BNC membranes obtained from static culture of three strains were cut into strips (2 × 7 cm). The initial grip separation and crosshead speed used were set at 40 mm and 1 mm/s, respectively. In each test, Tensile strength (TS) and elongation (E%) were calculated using Eqs. (1) and (2), respectively (Antoniou, Liu, Majeed, & Zhong, 2015). TS = F/(L × X)
(1)
E% = 100%×(l-l1)/l1
(2)
Where F is the tensile force (N), L the width of the film (mm), X the thickness (mm), l1 is the initial length of the film and l is the length of the film at breaking point. The slope of the curve in the linear elastic region was calculated to obtain Young’s modulus. 2.10. Thermogravimetric analysis (TGA)
3. Results and discussion
The TGA of the freeze-dried BNC films were carried out by Thermal Gravimetric Analyzer TG209 (Naichi, Germany) to test its heat resistance. The experimental conditions were 25–600 °C, 10 K/min, and N2 was fed at a flow rate of 30.0 mL/min (Cheng et al., 2009).
3.1. Morphology and yield of BNC In this study, the morphology and yield of BNC produced by three strains under static and agitated culture conditions were compared. It has been reported that the morphology of BNC are greatly affected by different cultivation conditions, such as static and agitated cultivation (Hestrin & Schramm, 1954; Toyosaki et al., 2014). All three strains under static culture conditions produced relatively uniform BNC
2.11. Capability to stabilize coffee milk The capability of BNC to stabilize a coffee milk beverage was 3
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the absorption peak near 1059 cm−1 was generated by COCee stretching vibration; 617 cm−1 may be the peak of some inorganic impurities (Fig. 3). These groups were basically consistent with the organic compound groups contained in the molecular formula of cellulose produced by other aerobic bacteria (Vazquez, Foresti, Cerrutti, & Galvagno, 2012). BNCs produced by agitated culture had a larger hydrogen-bonding vibration, which was the reason why it had stronger water retention. 3.3. XRD analysis of BNCs XRD analysis was used to compare the microstructural changes in BNC samples from different culture conditions and especially to estimate if the shaking causes any disturbance in the crystallization process. The XRD pattern had two main diffraction peaks at 14.5° and 22.7° for both BNC samples from static and agitated culture conditions, which was a characteristic peak of cellulose I (Fig. 4). The result indicated that the BNCs produced by three strains under different culture conditions all belonged to cellulose I, which was consistent with previous reports (Gea et al., 2011; Oh et al., 2005). The crystallinity was calculated, and the results were shown in supplement Table 1. The crystallinity of BNCs produced by three strains under static culture were significantly higher than those under agitated culture. Since the shearing force under agitated culture affected the polymerization of BNC (Singhsa et al., 2018), the degree of polymerization during the formation of BNC was reduced and the crystallinity was low under agitated culture condition.
Fig. 2. Yield of BNC produced by three strains under static and agitated culture conditions.
membranes which were consistent with previous report (Verschuren, Cardona, Nout, De Gooijer, & Van den Heuvel, 2000). Among them, the membranes of BCA263-S and BCC529-S were thinner and more transparent than P1-S (Fig. 1A). While BNCs produced by three strains under agitated culture condition showed forms of fibrous suspensions and pellets (Fig. 1B). BNCs produced under agitated culture condition appeared in forms of spherical shape and irregular granules that were well dispersed in culture medium, only a few BNCs were attached to impeller. The yield of BNC produced from BCA263, BCC529 and P1 under static condition was 3.9744 g/L, 2.4768 g/L and 1.4016 g/L respectively. While the yield of BNC produced from BCA263, BCC529 and P1 under agitated condition were 1.6992 g/L and 1.6608 g/L 1.7184 g/L respectively. The results demonstrated that the yield of BNC from BCA263 and BCC529 under static condition was higher than those under agitated condition, while oppositely from P1 (Fig. 2). Singhsa, Narain, and Manuspiya (2018) have also reported that the yield of BNC was mainly affected by types of BNC-producing strains and methods of fermentation. From the results, it can be concluded that strain BCA263 and BCC529 were more suitable for static culture. However, strain P1 was more suitable for agitated culture. Strain P1 might be a potential BNC-producing strain to apply to large-scale, agitated and aerated fermentation systems.
3.4. SEM imaging of BNCs In addition, the BNC produced by three strains under static and agitated conditions was observed using SEM. The results showed that BNCs obtained under static culture formed a denser network structure, while the network structure of BNC obtained by agitated culture was loose and porous (Fig. 5). It was consistent with the higher crystallinity of the BNC under static culture observed by XRD. Among them, BNC produced by BCC529 and BCA263 strains under agitated culture condition had larger pores than P1 strain associated with low crystallinity. The results indicated that the network structure of BNC was closely related to its crystallinity. The BNC produced under different culture conditions had a diameter of 20–40 nm via calculation, which belonged to the nanometer scale (Table 1).
3.2. FT-IR analysis of BNCs To further analysis of the effect of static and agitated culture conditions on the properties of BNC, BNCs produced by three strains were compared by FT-IR, X-ray diffraction, SEM, tensile test and TGA, respectively. FT-IR showed that the strong absorption peak of 3345 cm−1 was the stretching vibration absorption peak of eOH; 2896 cm−1 and 1400–1200 cm−1 were the stretching vibration absorption peaks of CeH; the absorption peak near 1658 cm-1 was the expansion of C]O; 1428 cm−1 was the deformation vibration peak of methylene (eCH2);
3.5. Mechanical properties of BNC membranes The Tensile strength (TS), elongation (E%) and young’s modulus were used to estimate mechanical properties of BNC membrane produced by three strains under static culture conditions. As shown in Table 2, there are little difference in tension strength among the three BNC membranes. The percentage elongations of the membranes of P1-S
Fig. 3. FI-IR spectrum of BNC produced by (A) BCC529, (B) P1, and (C) BCA263 strains under static (black curves) and agitated (red curves) culture conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 4
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Fig. 4. X-ray diffraction of BNC produced by (A) BCC529, (B) P1, and (C) BCA263 strains under static (black curves) and agitated (red curves) culture conditions. Table 1 Cross-section diameter of BNC produced by three strains under static and agitated culture conditions. Strain
BCC529
BCA263
Culture system diameter/nm
Static 29.13 ± 6.53
Agitated 29.51 ± 8.03
P1
Static 32.85 ± 9.27
Agitated 29.49 ± 5.86
Static 40.99 ± 9.63
Agitated 35.64 ± 8.10
Fig. 5. SEM images of BNC produced by three strains under static and agitated culture conditions. (A and B) BNC produced by BCC59 strain, (C and D) BNC produced by P1 strain, and (E and F) BNC produced by BCA263 strain, respectively.
3.6. TGA of BNCs
Table 2 Tensile test of BNC produced by three strains under static culture condition. Sample
Tension strength/MPa
E%/ %
Young’s modulus/ MPa
BCC529-S P1-S BCA263-S
0.235 ± 0.064 0.234 ± 0.044 0.264 ± 0.037
33.564 ± 6.266 12.118 ± 1.348 28.914 ± 2.082
0.722 ± 0.252 1.968 ± 0.527 0.92 ± 0.172
Using a thermogravimetric analyzer to detect the thermal stability of BNC, as shown in Fig. 6, with the increase of temperature, the quality of BNC was decreasing. At the beginning, free water was evaporated. BNC began to decompose after 200 °C, and the decomposition rate reached the maximum at around 300 °C, and the decomposition rate became stable after 350 °C (Fig. 6). The result indicated that BNCs produced by three strains under static culture condition were more resistant to high temperatures and had good flame retardancy, which may be due to its dense network structure (Supplement Table 2).
was the smallest, and the young’s modulus of the membranes of P1-S was the highest, which further proved that P1 strain was more suitable for agitated culture. BNC membranes produced under static culture condition by three strains had great stretchability, and they were preferably used as a supporting material in biomedical field such as wound healing, artificial blood vessels, and tissue engineering.
3.7. Capability to stabilize coffee milk Finally, BNC produced by different strains under different culture conditions was applied to coffee milk beverages to determine and 5
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Fig. 6. Thermogravimetric analysis of BNC produced by (A) BCC529, (B) P1, and (C) BCA263 strains under static (black curves) and agitated (red curves) culture conditions.
Fig. 7. (A)The state of the coffee milk beverage with BNC as a stabilizer was allowed to stand for 24 h (Control means no BNC added); (B) Determination of suspension stability of coffee milk beverages with different BNC; (C) Determination of precipitation rate of coffee milk beverages with different BNC.
suspension stability showed that the OD660 of P1-A, BCC529-A and BCA263-A were higher than P1-S, BCC529-S and BCA263-S respectively (Fig. 7B), indicated that BNC produced under agitated culture condition more suitable as a stabilizer using in coffee milk beverages. P1-S and P1-A have higher OD660 than others, indicating BNCs from P1 strain has high capacity of improving the stability of coffee milk beverages. The results of precipitation rate were opposite to suspension stability, but the conclusion was consistent. It is worth noting that the value of the control group was higher than all experimental groups, and P1-A was the lowest one (Fig. 7C). This results demonstrated that BNC produce by strain P1 under agitated culture condition was the best one as a
compare the capacity of BNC as a key component of stabilizer. The control group showed obvious stratification, and the addition of BCA263-S and BCC529-S in the experimental group also appeared stratification after standing for 24 h. However, the addition of P1-S, P1A, BCA263-A and BCC529-A in the experimental groups were not stratified (Fig. 7A). Since BNC produced under agitated culture condition showed larger pores and smaller crystallinity, it was no surprise that BNC produced under agitated culture condition more suitable as a stabilizer using in coffee milk beverages. The properties of coffee milk beverages with different BNCs were further analyzed, such as suspension stability, precipitation rate and Zeta potential. The results of 6
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online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115323.
Table 3 Determination of zeta potential of beverages with different BNCs. Types of BNC
Zeta potential/mV
CK BCC529-S BCC529-A P1-S P1-A BCA263-S BCA263-A
−42.6 −44.1 −46.5 −48.0 −52.8 −43.8 −44.8
± ± ± ± ± ± ±
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1.46 0.91 1.51 1.63 3.20 1.72 0.72
stabilizer using in coffee milk beverages among three strains. The results of precipitation rate and Zeta potential of P1-A confirmed our conclusion (Table 3).Taken together, it can be concluded that BNCs produced under agitated culture was more suitable as a stabilizer in beverages than those under static culture, and P1 strain in this experiment showed more advantages than the other two strains. 4. Conclusion In summary, BNCs from Komagataeibacter xylinus BCC529, Gluconacetobacter xylinus P1 and Acetobacter xylinum BCA263 under static and agitated culture conditions were compared. BNCs produced by three strains under static culture condition were uniform in film shape, while under agitated culture condition BNCs formed small clumps or fragments. BCA263 strain and BCC529 strain were more suitable for static culture, while P1 strain was more suitable for agitated culture. In comparison of properties, The FT-IR results showed that these groups of eOH, CH, CH2, COC, COeeee], were basically consistent with the organic compound groups contained in the molecular structure formula of cellulose produced by other aerobic bacteria, and the hydrogen bond generated by the agitated culture had a strong hydrogen-stretching vibration, indicating strong water retention of BNCs under agitated culture condition ; The XRD results showed that the crystallinity of BNC produced by the static culture of the three strains was higher than by agitated culture; the mechanical properties results showed that the percentage elongations of the membranes of P1S was smaller and the young’s modulus of the membranes of P1-S was higher than that of BNCs produced by BCC529 strain and BCA 263 strain; the image of SEM showed the network structure of the BNCs under static culture condition were denser than those under agitated culture condition. While both of them had diameters in the range of 20–40 nm belonged to nanostructure; TGA showed that NBCs produced by static culture condition were more resistant to high temperature and good flame retardancy. The addition of BNC can increase the stability of the coffee milk beverage, and BNCs produced by agitated culture was more suitable as a stabilizer in beverages. Taken together, BNC produced by P1 strain under agitated culture condition was promising for food industrial applications. Acknowledgements This work was supported by the National Natural Science Foundation of China (31670138) and Taixing Dongsheng Biotechnology Co. Ltd., China. Among many colleagues from the lab, we are especially grateful to Associate Prof. Yang, Xuexia and Professor Zhang, Qiang for their advice during this study. We thank Material science and Engineering School, Donghua University for the test of FTIR and X-ray diffraction. We thank Xie, Xiujuan from Taixing Dongsheng Biotechnology Co. Ltd for the beverage technical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the 7
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Trovatti, E., Silva, N. H., Duarte, I. F., Rosado, C. F., Almeida, I. F., Costa, P., et al. (2011). Biocellulose membranes as supports for dermal release of lidocaine. Biomacromolecules, 12(11), 4162–4168. Vazquez, A., Foresti, M. L., Cerrutti, P., & Galvagno, M. (2012). Bacterial cellulose from simple and low cost production media by Gluconacetobacter xylinus. Journal of Polymers and the Environment, 21(2), 545–554. Verschuren, P. G., Cardona, T. D., Nout, M. J., De Gooijer, K. D., & Van den Heuvel, J. C. (2000). Location and limitation of cellulose production by Acetobacter xylinum established from oxygen profiles. Journal of Bioscience and Bioengineering, 89 414e419. Wang, J., Tavakoli, J., & Tang, Y. (2019). Bacterial cellulose production, properties and applications with different T culture methods – A review. Carbohydrate Polymers, 21(9), 63–76. Zhang, T.-T., Zhou, P.-H., Zhan, Y.-F., Shi, X.-W., Lin, J.-Y., Du, Y.-M., et al. (2015). Pectin/lysozyme bilayers layer-by-layer deposited cellulose nanofibrous mats for antibacterial application. Carbohydrate Polymers, 117, 687–693. Zhang, W., Wang, X., Qi, X., Ren, L., & Qiang, T. (2018). Isolation and identification of a bacterial cellulose synthesizing strain from kombucha in different conditions: Gluconacetobacter xylinus ZHCJ618. Food Science and Biotechnology, 27(3), 705–713.
restoration. Carbohydrate Polymers, 116, 173–181. Schramm, M., & Hestrin, S. (1954). Factors affecting production of cellulose at the air liquid interface of a culture of Acetobacter-xylinum. Journal of General Microbiology, 11(1), 123–129. Shi, Z., Zhang, Y., Phillips, G. O., & Yang, G. (2014). Utilization of bacterial cellulose in food. Food Hydrocolloids, 35(1), 539–545. Shoda, M., & Sugano, Y. (2005). Recent advances in bacterial cellulose production. Biotechnology and Bioprocess Engineering, 10(1), 1–8. Singhsa, P., Narain, R., & Manuspiya, H. (2018). Physical structure variations of bacterial cellulose produced by different Komagataeibacter xylinus strains and carbon sources in static and agitated conditions. Cellulose, 25(3), 1571–1581. Tabuchi, M. (2007). Nanobiotech versus synthetic nanotech? Nature Biotechnology, 25(4), 389e390. Torres, F. G., Arroyo, J. J., & Troncoso, O. P. (2019). Bacterial cellulose nanocomposites: An all-nano type of material. Materials Science and Engineering C, 98(12), 77–129. Toyosaki, H., Naritomi, T., Seto, A., Matsuoka, M., Tsuchida, T., & Yoshinaga, F. (2014). Screening of bacterial cellulose-producing Acetobacter strains suitable for agitated culture. Bioscience, Biotechnology, and Biochemistry, 59(8), 1498–1502.
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Comparison of bacterial nanocellulose produced by different strains under static and agitated culture conditions
T
Hongliang Gao, Qian Sun, Zebei Han, Jiahe Li, Bowen Liao, Lulu Hu, Jie Huang, Chunjing Zou, ⁎ ⁎ Caifeng Jia, Jing Huang, Zhongyi Chang, Deming Jiang , Mingfei Jin School of Life Sciences, East China Normal University, Shanghai, 200241, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial nanocellulose Static and agitated culture Yield Crystallinity Beverage stabilizer
Bacterial nanocellulose (BNC) has many advantages over plant cellulose, which make it widely used in many fields, especially in the food industry. In this study, three strains including BCA263, BCC529, and P1 were selected for characteristics analysis of BNCs under static and agitated culture conditions. The BNCs produced under static culture condition were in the shape of uniform membrane, while BNCs produced under agitated culture were in form of small agglomerates and fragments. BCA263 and BCC529 strains were more suitable for static culture, while P1 strain was more suitable for agitated culture. BNCs produced under static culture condition exhibited higher crystallinity, stronger tensile strength, denser network structure, higher temperature resistance and good flame retardancy; while BNCs produced under agitated culture condition exhibited larger porous and lower crystallinity. Furthermore, BNCs produced under agitated culture condition were more suitable as a stabilizer of coffee milk beverage.
1. Introduction Cellulose has a large commercial use in paper, textile, cellulose nanofibrous mats, and pulp production units (Deng et al., 2010; Klemm, Heublein, Fink, & Andreas, 2005; Zhang et al., 2015). Plant cell walls are the major source of cellulose. However, this type of cellulose contains several impurities, including lignin, pectin, and hemicelluloses etc. Apart from plants, cellulose is also found in many microorganisms such as fungi, bacteria, and algae. Recent studies have revealed that cellulose can be produced by different bacteria, including Gluconacetobacter (formerly Acetobacter), Azotobacter, Rhizobium, Agrobacterium, Pseudomonas, Salmonella, Alcaligenes. (Esa, Tasirin, & Rahman, 2014; Shoda & Sugano, 2005). These bacteria produce a fine cellulose fibril with a width of 50–80 nm and a thickness of 3–8 nm. When several fibers entangle, a three-dimensional nano-network with pores is formed (Tabuchi, 2007). Therefore, bacterial cellulose (BC) is a fascinating polymer with a three-dimensional structure formed by nanofibers of pure cellulose, which is also named as bacterial nanocellulose (BNC) due to its nanostructure. The bacterium Acetobacter xylinum, also known as Gluconacetobacter xylinum or Komagataeibacter xylinus, has been shown to have the highest rate of BC production among all the BNC-producing bacteria types (Reiniati, Hrymak, & Margaritis, 2017). It has been observed that one cell can convert 108 glucose molecules
⁎
per hour into cellulose (Wang, Tavakoli, & Tang, 2019). BNC has many advantages over plant cellulose, including high purity, good water absorption, strong water retention, high tensile strength, good biocompatibility and biodegradability (Aydin & Aksoy, 2014; Bi et al., 2014; Islam, Ullah, Khan, Shah, & Park, 2017; Kuo, Teng, & Lee, 2015; Qiu & Netravali, 2014). Based on these characteristics, BNC has been widely used in many areas such as electronics, biomedicine, food industry and energy storage (Keshk, 2014; Torres, Arroyo, & Troncoso, 2019). Especially in the food industry, BNC is traditionally and widely used to make nata de coco as a better dietary fiber in Southeast Asia (Nugroho & Aji, 2015; Shi, Zhang, Phillips, & Yang, 2014; Tabuchi, 2007).It is also used in foods such as multifunctional food ingredients to control the properties of food or beverage as a thickener, stabilizer and texture modifier. (Esa et al., 2014, Tabuchi, 2007). In the biomedical field, BNC is widely studied as a highly biocompatible raw material for wound healing, artificial blood vessels, and tissue engineering (Czaja, Krystynowicz, Bielecki, & Brown, 2006; van Zyl & Coburn, 2019). There are two main methods to produce BNC by bacterial strains (Czaja, Romanovicz, & Brown, 2004). One is the static culture, which results in the accumulation of a gelatinous membrane of BNC is formed at the gas-liquid junction with the shape of the culture vessel (Rangaswamy, Vanitha, & Hungund, 2015; Trovatti et al., 2011). The
Corresponding authors. E-mail addresses: [email protected] (D. Jiang), [email protected] (M. Jin).
https://doi.org/10.1016/j.carbpol.2019.115323 Received 19 June 2019; Received in revised form 20 August 2019; Accepted 10 September 2019 Available online 11 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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contains the following constituents (%, w/v): sucrose, 5.0; peptone, 0.5; yeast extract, 0.9; K2HPO4, 0.3 ; MgSO4, 2.0; acetic acid, 0.2; The initial pH was adjusted to 6.0. The seed and fermentation medium for BCA263 strain contains the following constituents (%, w/v): sucrose, 5.0; corn steep liquor, 5.0; (NH4)2SO4, 0.33; K2HPO4, 0.13; MgSO4, 0.012; FeSO4, 0.0002; CaCl2, 0.0011; Na2MoO4, 0.00002; ZnSO4, 0.0001; MnSO4, 0.0001; and CuSO4, 0.0000032; The initial pH was adjusted to 5.0 (Cheng et al., 2009).
other one is agitated culture, where BNC is synthesized in deep medium by bacterial strain in a shake flask or a fermenter under aerated and agitated conditions, and BNC is dispersed in the fermentation broth in the form of fibrous suspensions, pellets or irregular masses (Aydin & Aksoy, 2014; Chao, Ishida, Sugano, & Shoda, 2015). Strains and culture conditions have a great influence on the microstructures and physiochemical properties of BNC. Changes in crystallinity and mass fraction of the Iα allomorph were observed in BNC produced by different strains and culture conditions (Deng et al., 2015). Some additives, such as agar, carboxymethylcellulose and microcrystalline cellulose were added into the fermentation medium in agitated culture show obviously changes on the yield and structure of BNC produced by Acetobacter xylinum (Cheng, Catchmark, & Demirci, 2009). Thus, screening strains with high BNC yields under static and agitated culture conditions has been a critical issue in BNC research for a long time. A series of BNC-producing strains have been isolated from different sources such as rotten fruits, vegetables and soils (Toyosaki et al., 2014). Among them, A strain isolated from kombucha show potential for commercial applications owing to its high phenotypic stability and sustainable production capacity of 7.56 ± 0.57 g/L under static culture and 8.31 ± 0.79 g/L under shaking culture (Zhang, Wang, Qi, Ren, & Qiang, 2018). Although many strains have been isolated for BNC production from fruit (Jahan, Kumar, Rawat, & Saxena, 2012; Rangaswamy et al., 2015), there are few studies on the comparison of the properties and application in food of BNC produced by different strains under different culture conditions, which is important for the improvement of the production of BNC and furthering their applications. In this study, the properties of macro- and micro-structure, yield, crystallinity, tensile strength and thermal stability of the BNCs produced by three different strains under static or agitated culture conditions were investigated. Komagataeibacter xylinus BCC529 (CICC 10529) and Acetobacter xylinum BCA263 (ATCC 53263) are the representative model strains with potential industrial application as shown in previous reports (Lida, Ohnishi, & Horinouchi, 2008; Lin, Li, Lopez-Sanchez, & Li, 2015). Gluconacetobacter xylinus P1 was isolated and stored in our lab. Finally, BNCs produced by static or agitated methods were added as a stabilizer to the coffee milk beverages, and the stability of coffee milk beverages with different BNCs was analyzed based on precipitation rate, suspension stability and zeta potential.
2.4. Culture conditions All bacterial strains were pre-culture in seed agar medium at 30 °C for 48 h. The slant culture was transferred into seed medium and cultured at 30 °C for 24 h under static condition. After that, the seed culture was filtered with sterile cotton, and the filtered culture was used as seed. In a static culture condition, the filtered seed culture (96 mL) was transferred into a container (52 cm × 33 cm × 7.5 cm) containing 1.2 L fermentation medium, and culture at 30 °C for 96 h. The produced BNCs were purified following the purification method below. The BNCs produced by three strains were represented by BCC529-S, P1-S and BCA263-S respectively, where S indicated static culture condition. In an agitated culture condition, the filtered seed culture (2.4 mL) was added to 30 mL seed medium, and cultured at 160 r/min at 30 °C for 18 h to obtain the second seed culture. 200 mL of the second seed culture were added into the 3 L fermenter containing 2.5 L of fermentation medium. Culture was carried at a speed of 300 r/min and a temperature of 30 °C for 96 h, and the aeration rate was 1.0 vvm. The produced BNCs were purified following the purification method below. The BNC produced by the three strains was represented by BCC529-A, P1-A and BCA263-A respectively, where A indicated agitated culture condition. 2.5. BNC production and purification Under static culture condition, both the fermentation broth and the BNC membranes poured into the centrifugal bottle, while under agitated culture condition BNCs which were fibrous suspensions and pellets also poured into the centrifugal bottle, only a few BNCs attached to the impeller was scraped into the fermentation broth with tweezers. Then the suspension was homogenized and centrifuged (9000 r/min, 5 min). The product was then washed twice with distilled water, and was boiled in 1% NaOH for 30 min. After cooled down to the room temperature, the product was centrifuged (9000 r/min, 5 min) and neutralized with 1% acetic acid. The product was continuously washed with distilled water to neutral. Finally, the BNC product was dried at 60 ºC to constant weight and weighed (Karahan et al., 2011).
2. Materials and methods 2.1. Materials All the chemical reagents used in this study, such as glucose, peptone, yeast extract, Na2HPO4, critic acid and corn steep liquor, were purchased from Chemical reagent of national medicine group, Shanghai, China. 2.2. Microorganisms
2.6. Fourier transform infrared spectroscopy (FT-IR)
Strain Komagataeibacter xylinus BCC529 (CICC 10529) was purchased from China Center of Industrial Culture Collection (CICC) (Beijing, China). Strain Gluconacetobacter xylinus BCA263 (ATCC 53263) was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Strain Gluconacetobacter xylinus P1 was isolated and stored in the Food and Microbial Technology laboratory, School of Life Science, East China Normal University at China.
The BNC samples produced by different strains under static and agitated conditions were freeze-dried on a plastic dish to form thin films. The infrared spectra of the films were measured using Fourier transform infrared spectrometer Spectrum 2000 (PE company, America). All spectra were recorded with a resolution of 2 cm−1 in the range from 4000 to 500 cm−1. Baselines for each sample spectrum were normalized using the spectrum software (Santos et al., 2015).
2.3. Culture media
2.7. X-ray diffraction (XRD)
The seed medium for BCC529 and P1 strains were Hestrin and Schramm (HS) medium with some modification (%, w/v) (Schramm & Hestrin, 1954): glucose, 2.0; peptone, 0.5; yeast extract, 0.5; Na2HPO4, 0.27; critic acid, 0.115; corn steep liquor, 1.0. The initial pH was adjusted to 6.0. The fermentation medium for BCC529 and P1 strains
The freeze-dried BNC films were placed in the X-ray holder (D/max2500, Japan). Scans were performed over the 5-40°, speed of 10°/min, and 2Ɵ range using steps of either 0.02° in width. The crystallinity of BNC was calculated according to the formula: CrlXRD=(I200-Iam)/ I200×100% (Santos et al., 2015). 2
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Fig. 1. The photo images of BNC produced by three strains under static (A) and agitated (B) culture conditions.
evaluated. The formula of stabilizers was composed of BNC 18.3%, xanthan gum 6.2%, carboxymethyl cellulose 12.1%, maltodextrin 6.4% (g/L). As a control assay, a suspension containing 1.5% coffee, 0.08% baking soda, 3% non-dairy creamer, 2.5% whole milk powder, 6% sugar, 0.05% sucrose ester and 0.15% monoglyceride in distilled water was prepared by heating at 60 °C for 20 min. Then a stabilizer containing different BNCs samples were added into a control suspension at concentration of 0.35% and the mix was sheared at a high speed of 10000 r/min for 10 min, the coffee milk beverage was filtered through a 180-mesh sieve and the solution was homogenized twice at 65–75 ºC and 30 MPa. Finally, the solution was packed in bottles and sterilized at 115 °C for 20 min. These coffee milk beverages were allowed to stand at room temperature for 24 h. During the time, the statuses of the beverages were observed, and photographs were taken. Determination of Precipitation rate: the coffee milk beverage was weighed (denoted as m), and then was centrifuged at 3000 r/min for 15 min. The precipitate was collected and weighed (denoted as m1). The precipitation rate was calculated according the formula: precipitation rate (%) = m1/m × 100%. Determination of suspension stability: the coffee milk beverage was centrifuged at 4200×g for 15 min. The supernatant was collected and measured its absorbance at 660 nm. Determination of Zeta potential: the coffee milk beverage was diluted, and then the ultrasonic treatment was conducted. After that, the zeta potential of the solution was measured by Dynamic Light Scattering Instrument DynaPro NanoStar (Wyatt, America).
2.8. Scanning electron microscope (SEM) The freeze-dried BNC films were sprayed with gold and observed by scanning electron microscopy EVO (Zeiss, Germany) operating at 10 or 15 kV to observe its microstructure (Cakar, Ozer, Aytekin, & Sahin, 2014). The diameter of BNC was calculated by software Image J to obtain the approximate nanometer range of the fiber filament. 2.9. Mechanical properties A texture analyzer (TA-XT, Stable Micro System, UK) was used to measure BNC mechanical properties according to ASTM D882 (Concepts, 2014). The purified BNC membranes obtained from static culture of three strains were cut into strips (2 × 7 cm). The initial grip separation and crosshead speed used were set at 40 mm and 1 mm/s, respectively. In each test, Tensile strength (TS) and elongation (E%) were calculated using Eqs. (1) and (2), respectively (Antoniou, Liu, Majeed, & Zhong, 2015). TS = F/(L × X)
(1)
E% = 100%×(l-l1)/l1
(2)
Where F is the tensile force (N), L the width of the film (mm), X the thickness (mm), l1 is the initial length of the film and l is the length of the film at breaking point. The slope of the curve in the linear elastic region was calculated to obtain Young’s modulus. 2.10. Thermogravimetric analysis (TGA)
3. Results and discussion
The TGA of the freeze-dried BNC films were carried out by Thermal Gravimetric Analyzer TG209 (Naichi, Germany) to test its heat resistance. The experimental conditions were 25–600 °C, 10 K/min, and N2 was fed at a flow rate of 30.0 mL/min (Cheng et al., 2009).
3.1. Morphology and yield of BNC In this study, the morphology and yield of BNC produced by three strains under static and agitated culture conditions were compared. It has been reported that the morphology of BNC are greatly affected by different cultivation conditions, such as static and agitated cultivation (Hestrin & Schramm, 1954; Toyosaki et al., 2014). All three strains under static culture conditions produced relatively uniform BNC
2.11. Capability to stabilize coffee milk The capability of BNC to stabilize a coffee milk beverage was 3
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the absorption peak near 1059 cm−1 was generated by COCee stretching vibration; 617 cm−1 may be the peak of some inorganic impurities (Fig. 3). These groups were basically consistent with the organic compound groups contained in the molecular formula of cellulose produced by other aerobic bacteria (Vazquez, Foresti, Cerrutti, & Galvagno, 2012). BNCs produced by agitated culture had a larger hydrogen-bonding vibration, which was the reason why it had stronger water retention. 3.3. XRD analysis of BNCs XRD analysis was used to compare the microstructural changes in BNC samples from different culture conditions and especially to estimate if the shaking causes any disturbance in the crystallization process. The XRD pattern had two main diffraction peaks at 14.5° and 22.7° for both BNC samples from static and agitated culture conditions, which was a characteristic peak of cellulose I (Fig. 4). The result indicated that the BNCs produced by three strains under different culture conditions all belonged to cellulose I, which was consistent with previous reports (Gea et al., 2011; Oh et al., 2005). The crystallinity was calculated, and the results were shown in supplement Table 1. The crystallinity of BNCs produced by three strains under static culture were significantly higher than those under agitated culture. Since the shearing force under agitated culture affected the polymerization of BNC (Singhsa et al., 2018), the degree of polymerization during the formation of BNC was reduced and the crystallinity was low under agitated culture condition.
Fig. 2. Yield of BNC produced by three strains under static and agitated culture conditions.
membranes which were consistent with previous report (Verschuren, Cardona, Nout, De Gooijer, & Van den Heuvel, 2000). Among them, the membranes of BCA263-S and BCC529-S were thinner and more transparent than P1-S (Fig. 1A). While BNCs produced by three strains under agitated culture condition showed forms of fibrous suspensions and pellets (Fig. 1B). BNCs produced under agitated culture condition appeared in forms of spherical shape and irregular granules that were well dispersed in culture medium, only a few BNCs were attached to impeller. The yield of BNC produced from BCA263, BCC529 and P1 under static condition was 3.9744 g/L, 2.4768 g/L and 1.4016 g/L respectively. While the yield of BNC produced from BCA263, BCC529 and P1 under agitated condition were 1.6992 g/L and 1.6608 g/L 1.7184 g/L respectively. The results demonstrated that the yield of BNC from BCA263 and BCC529 under static condition was higher than those under agitated condition, while oppositely from P1 (Fig. 2). Singhsa, Narain, and Manuspiya (2018) have also reported that the yield of BNC was mainly affected by types of BNC-producing strains and methods of fermentation. From the results, it can be concluded that strain BCA263 and BCC529 were more suitable for static culture. However, strain P1 was more suitable for agitated culture. Strain P1 might be a potential BNC-producing strain to apply to large-scale, agitated and aerated fermentation systems.
3.4. SEM imaging of BNCs In addition, the BNC produced by three strains under static and agitated conditions was observed using SEM. The results showed that BNCs obtained under static culture formed a denser network structure, while the network structure of BNC obtained by agitated culture was loose and porous (Fig. 5). It was consistent with the higher crystallinity of the BNC under static culture observed by XRD. Among them, BNC produced by BCC529 and BCA263 strains under agitated culture condition had larger pores than P1 strain associated with low crystallinity. The results indicated that the network structure of BNC was closely related to its crystallinity. The BNC produced under different culture conditions had a diameter of 20–40 nm via calculation, which belonged to the nanometer scale (Table 1).
3.2. FT-IR analysis of BNCs To further analysis of the effect of static and agitated culture conditions on the properties of BNC, BNCs produced by three strains were compared by FT-IR, X-ray diffraction, SEM, tensile test and TGA, respectively. FT-IR showed that the strong absorption peak of 3345 cm−1 was the stretching vibration absorption peak of eOH; 2896 cm−1 and 1400–1200 cm−1 were the stretching vibration absorption peaks of CeH; the absorption peak near 1658 cm-1 was the expansion of C]O; 1428 cm−1 was the deformation vibration peak of methylene (eCH2);
3.5. Mechanical properties of BNC membranes The Tensile strength (TS), elongation (E%) and young’s modulus were used to estimate mechanical properties of BNC membrane produced by three strains under static culture conditions. As shown in Table 2, there are little difference in tension strength among the three BNC membranes. The percentage elongations of the membranes of P1-S
Fig. 3. FI-IR spectrum of BNC produced by (A) BCC529, (B) P1, and (C) BCA263 strains under static (black curves) and agitated (red curves) culture conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 4
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Fig. 4. X-ray diffraction of BNC produced by (A) BCC529, (B) P1, and (C) BCA263 strains under static (black curves) and agitated (red curves) culture conditions. Table 1 Cross-section diameter of BNC produced by three strains under static and agitated culture conditions. Strain
BCC529
BCA263
Culture system diameter/nm
Static 29.13 ± 6.53
Agitated 29.51 ± 8.03
P1
Static 32.85 ± 9.27
Agitated 29.49 ± 5.86
Static 40.99 ± 9.63
Agitated 35.64 ± 8.10
Fig. 5. SEM images of BNC produced by three strains under static and agitated culture conditions. (A and B) BNC produced by BCC59 strain, (C and D) BNC produced by P1 strain, and (E and F) BNC produced by BCA263 strain, respectively.
3.6. TGA of BNCs
Table 2 Tensile test of BNC produced by three strains under static culture condition. Sample
Tension strength/MPa
E%/ %
Young’s modulus/ MPa
BCC529-S P1-S BCA263-S
0.235 ± 0.064 0.234 ± 0.044 0.264 ± 0.037
33.564 ± 6.266 12.118 ± 1.348 28.914 ± 2.082
0.722 ± 0.252 1.968 ± 0.527 0.92 ± 0.172
Using a thermogravimetric analyzer to detect the thermal stability of BNC, as shown in Fig. 6, with the increase of temperature, the quality of BNC was decreasing. At the beginning, free water was evaporated. BNC began to decompose after 200 °C, and the decomposition rate reached the maximum at around 300 °C, and the decomposition rate became stable after 350 °C (Fig. 6). The result indicated that BNCs produced by three strains under static culture condition were more resistant to high temperatures and had good flame retardancy, which may be due to its dense network structure (Supplement Table 2).
was the smallest, and the young’s modulus of the membranes of P1-S was the highest, which further proved that P1 strain was more suitable for agitated culture. BNC membranes produced under static culture condition by three strains had great stretchability, and they were preferably used as a supporting material in biomedical field such as wound healing, artificial blood vessels, and tissue engineering.
3.7. Capability to stabilize coffee milk Finally, BNC produced by different strains under different culture conditions was applied to coffee milk beverages to determine and 5
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Fig. 6. Thermogravimetric analysis of BNC produced by (A) BCC529, (B) P1, and (C) BCA263 strains under static (black curves) and agitated (red curves) culture conditions.
Fig. 7. (A)The state of the coffee milk beverage with BNC as a stabilizer was allowed to stand for 24 h (Control means no BNC added); (B) Determination of suspension stability of coffee milk beverages with different BNC; (C) Determination of precipitation rate of coffee milk beverages with different BNC.
suspension stability showed that the OD660 of P1-A, BCC529-A and BCA263-A were higher than P1-S, BCC529-S and BCA263-S respectively (Fig. 7B), indicated that BNC produced under agitated culture condition more suitable as a stabilizer using in coffee milk beverages. P1-S and P1-A have higher OD660 than others, indicating BNCs from P1 strain has high capacity of improving the stability of coffee milk beverages. The results of precipitation rate were opposite to suspension stability, but the conclusion was consistent. It is worth noting that the value of the control group was higher than all experimental groups, and P1-A was the lowest one (Fig. 7C). This results demonstrated that BNC produce by strain P1 under agitated culture condition was the best one as a
compare the capacity of BNC as a key component of stabilizer. The control group showed obvious stratification, and the addition of BCA263-S and BCC529-S in the experimental group also appeared stratification after standing for 24 h. However, the addition of P1-S, P1A, BCA263-A and BCC529-A in the experimental groups were not stratified (Fig. 7A). Since BNC produced under agitated culture condition showed larger pores and smaller crystallinity, it was no surprise that BNC produced under agitated culture condition more suitable as a stabilizer using in coffee milk beverages. The properties of coffee milk beverages with different BNCs were further analyzed, such as suspension stability, precipitation rate and Zeta potential. The results of 6
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online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115323.
Table 3 Determination of zeta potential of beverages with different BNCs. Types of BNC
Zeta potential/mV
CK BCC529-S BCC529-A P1-S P1-A BCA263-S BCA263-A
−42.6 −44.1 −46.5 −48.0 −52.8 −43.8 −44.8
± ± ± ± ± ± ±
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stabilizer using in coffee milk beverages among three strains. The results of precipitation rate and Zeta potential of P1-A confirmed our conclusion (Table 3).Taken together, it can be concluded that BNCs produced under agitated culture was more suitable as a stabilizer in beverages than those under static culture, and P1 strain in this experiment showed more advantages than the other two strains. 4. Conclusion In summary, BNCs from Komagataeibacter xylinus BCC529, Gluconacetobacter xylinus P1 and Acetobacter xylinum BCA263 under static and agitated culture conditions were compared. BNCs produced by three strains under static culture condition were uniform in film shape, while under agitated culture condition BNCs formed small clumps or fragments. BCA263 strain and BCC529 strain were more suitable for static culture, while P1 strain was more suitable for agitated culture. In comparison of properties, The FT-IR results showed that these groups of eOH, CH, CH2, COC, COeeee], were basically consistent with the organic compound groups contained in the molecular structure formula of cellulose produced by other aerobic bacteria, and the hydrogen bond generated by the agitated culture had a strong hydrogen-stretching vibration, indicating strong water retention of BNCs under agitated culture condition ; The XRD results showed that the crystallinity of BNC produced by the static culture of the three strains was higher than by agitated culture; the mechanical properties results showed that the percentage elongations of the membranes of P1S was smaller and the young’s modulus of the membranes of P1-S was higher than that of BNCs produced by BCC529 strain and BCA 263 strain; the image of SEM showed the network structure of the BNCs under static culture condition were denser than those under agitated culture condition. While both of them had diameters in the range of 20–40 nm belonged to nanostructure; TGA showed that NBCs produced by static culture condition were more resistant to high temperature and good flame retardancy. The addition of BNC can increase the stability of the coffee milk beverage, and BNCs produced by agitated culture was more suitable as a stabilizer in beverages. Taken together, BNC produced by P1 strain under agitated culture condition was promising for food industrial applications. Acknowledgements This work was supported by the National Natural Science Foundation of China (31670138) and Taixing Dongsheng Biotechnology Co. Ltd., China. Among many colleagues from the lab, we are especially grateful to Associate Prof. Yang, Xuexia and Professor Zhang, Qiang for their advice during this study. We thank Material science and Engineering School, Donghua University for the test of FTIR and X-ray diffraction. We thank Xie, Xiujuan from Taixing Dongsheng Biotechnology Co. Ltd for the beverage technical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the 7
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