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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Bacterial Cellulose Production from the Litchi Extract by Gluconacetobacter Xylinus abc
Xiao-Yan Yang ab
Qing Lin , Xue-Fang Chen a
ab
ab
ab
ab
ab
, Chao Huang , Hai-Jun Guo , Lian Xiong , Jun Luo , Bo Wang , Xiaobc
ab
& Xin-De Chen
Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou, PR China
b
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, PR China c
University of Chinese Academy of Sciences, Beijing, PR China Accepted author version posted online: 02 Sep 2014.
To cite this article: Xiao-Yan Yang, Chao Huang, Hai-Jun Guo, Lian Xiong, Jun Luo, Bo Wang, Xiao-Qing Lin, Xue-Fang Chen & Xin-De Chen (2014): Bacterial Cellulose Production from the Litchi Extract by Gluconacetobacter Xylinus, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.958163 To link to this article: http://dx.doi.org/10.1080/10826068.2014.958163
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Bacterial cellulose production from the litchi extract by Gluconacetobacter xylinus Xiao-Yan Yang1,2,3 , Chao Huang1,2 , Hai-Jun Guo1,2, Lian Xiong1,2 , Jun Luo1,2, Bo Wang1,2 , Xiao-Qing Lin1,2, Xue-Fang Chen2,3 , Xin-De Chen1,2 1
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Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou, PR China, 2Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, PR China, 3 University of Chinese Academy of Sciences, Beijing, PR China
Abstract
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Although litchi has both nutrient and edible value, the extremely short preservation time limited its further market promotion. To explore processed litchi products with longer preservation time, litchi extract was selected as an alternative feedstock for
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production of bacterial cellulose (BC). After two weeks’ static fermentation, 2.53g/L of the BC membrane was obtained. The trace elements including magnesium (Mg) and
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sodium (Na) in the litchi extract were partly absorbed in the BC membrane, but no potassium (K) element was detected in it curiously. Scanning electron microscope (SEM) photographs exhibited an ultra fine network nanostructure for the BC produced in the litchi extract. Analysis of the infrared spectrograms confirmed the pellicles to be a cellulosic material. Interestingly, X-ray diffraction (XRD) results showed the BC
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Corresponding Address to X. D. Chen: No.2 Nengyuan Road, Tianhe District, Guangzhou 510640, PR China. E-mail: [email protected]
membrane obtained from litchi extract had higher crystallinity of 94.0% than that from HS medium. Overall, the work showed the potential of producing high value-added polymer from litchi resources.
KEYWORDS: Cellulose produced by bacteria, litchi processing, fermentation; bio-refinery
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1. INTRODUCTION Bacterial cellulose (BC), synthesized by many bacteria and with high Young’s Modulus, has great potential in the application on many fields such as food, biomedical material,
Generally, the substrate for BC production is usually the low-cost one such as
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cotton-based waste textiles [4], wheat straw [5], elephant grass acid hydrolysate [6], or other residues from agro-forest industries [7]. However, when BC is applied in food industry, it
is better to use substrate for BC production from food resources such as corn steep liquor [8]
, molasses [9], konjac powder hydrolysate [10] and etc. Among various food resources,
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fruit juices was shown as one promising substrate for BC production and the fruit chosen
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was the common one such as orange, apple, pineapple, grape and etc. [11].
Litchi (Litchi chinensis Sonn.), which is a tropical and subtropical fruit of the Sapindaceae family, has been widely planted in South-East Asia, especially in China for many years [12]. Owing to its bright color and delicious taste, litchi has been gradually accepted by consumers and has established great popularity in the international market.
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xylinus is the most common one used for BC production [3].
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papermaking and transducer diaphragms [1-3]. Among various bacteria, Gluconacetobacter
Moreover, many components have been proven to have antioxidant function in the fruit pulp of litchi [13, 14]. Unfortunately, the preservation time of litchi is extremely short (merely 1-2 days without cold storage) and thus limited its market and profit. Exploring processed litchi product could not only increase its preservation time but also expand its market promotion. Therefore, using litchi extract as substrate for BC production seems to
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be a good alternative to obtain BC membrane with nutrient from litchi. In addition, this product could solve the preservation problem of litchi and explore its new application. Thus, it might have potential market competitiveness in future.
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It is known that the composition of the culture medium and fermentation conditions
polysaccharides including the bacterial cellulose [15-17]. These modifications (if any) in
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turn may affect the properties of BC. Therefore, it seems important to investigate the
possible modifications in the physicochemical properties of the BC sheets produced from the litchi extract. To our knowledge, little work has used litchi as raw material for BC production. For the first time, the litchi extract was used directly as the substrate for BC
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production by G. xylinus. In this work, the possibility of BC production from litchi extract was evaluated and the BC yield, structure and composition were also systematically
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measured.
2. MATERIAL AND METHODS
2.1. Preparation and Composition Analysis of Litchi Extract Fresh fruits of litchi at mature stage were purchased from the local market. The bright red
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greatly influence the chemical structure, composition and viscosity of the microbial
fruits without disease symptoms were selected, and then washed thoroughly with water, manually peeled, removed the seeds and extracted the juice with the 99B-4 fruit squeezer (CROWN ELEC., CO. Hong Kong, China). Then the litchi juice was vacuum filtrated twice to remove impurities without crushing by SHZ-Dш circulating water vacuum pump (Gongyi City Yuhua Instrument CO., LTD, China) with vacuum degree close to -0.1 MPa,
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and finally stored at -20 oC until use. The total sugars of litchi extract were measured using anthrone methods [18]. Mineral elements of litchi extract were analyzed by Inductive Coupled Plasma Emission Spectrometer (ICP, PerkinElmer Optima 8000DV ICP system,
2.2. Microorganism, Culture Media and Growth Conditions
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Gluconacetobacter xylinus CH001 (Laboratory of Energy and Biochemical Engineering, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences) was used as the microorganism for BC production in this work.
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The pre-culture was performed on pre-cultivation medium (g/L, mannitol 25, peptone 5, yeast extract 3, initial pH 6.0) at 28 oC and 150 rpm for 24 h. Then, 8 % (v/v) seed culture
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was translated into the litchi extract which was diluted 6 times and autoclaved at 121 oC for 20 min. Then, the static fermentation was carried out at 28 oC for 14 days. To compare the BC product obtained from normal fermentation medium, the fermentation was also carried out on the classical HS medium (g/L, glucose 20, yeast extract 5, peptone 5, disodium hydrogen phosphate 2.7, citric acid 1.15, initial pH 6.0) [19] at the same
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overnight.
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Massachusetts, USA). Before analysis, the sample was digested using nitric acid
fermentation condition.
2.3. Harvest and Weighing of BC After fermentation, the fermentation broth and BC were separated by vacuum filtration. Then, the BC was treated by 1.5% (w/v) NaOH at 80 oC for two hours. After that, the BC
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was repeatedly washed by distilled water until the washing water being neutralization. Finally, the BC was freeze-dried and weighed.
2.4. Structure and Composition Analysis of BC by SEM, FT-IR, and XRD
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The morphologies of freeze-dried BC samples were observed by a Hitachi S-4800 high
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resolution Field Emission Scanning Electron Microscope (FE-SEM, Hitachi, Japan) operated at 2.0 kV and 10 μA.
BC samples were mixed with spectroscopic-grade potassium bromide powder (1% w/w)
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and Fourier Transform Infrared Spectroscopy (FTIR) was carried out in the range of 400 to 4000 cm-1 wavelength with a Perkin-Elmer Spectrum one FT-IR Spectrometer (US).
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The kind of crystallite allomorph was determined with shifting FT-IR bands related to specified groups and bonds [20-22].
Structure, size, and percentage of crystals of BC samples were analyzed by D/max-RA X-ray diffractometer (Rigaku, Japan) with Cu Kα radiation (λ = 0.154 nm) operated at 40
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Mineral elements of BC samples were analyzed according to 2.1.
kV and 100 mA. Samples were scanned from 10° to 50° (2θ range) at a scan speed of 0.008°/step. To determine the sample crystallinity, profile analysis was carried out with a peak fitting program using Gaussian line shapes. The crystal size of cellulose was calculated using Debye-Scherrer’s equation [17]. Crystallinity index (CI) was calculated from the related intensity data by the Segal method (Equation (1)) [23].
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CI
I200 I am / I200
(1)
where I200 is the maximum intensity of crystalline region at 2θ (about 22.8°), I am is the intensity of the amorphous region of the wide-angle X-ray diffraction curves at 2θ (about
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18°).
3.1. Preparation of Litchi Extract
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In this work, the litchi was used that harvested at May, 2013 in Guangzhou. The composition of the litchi extract was analyzed before fermentation. Litchi extract
contained a very high sugar concentration of 183.5 g/L, this undoubtedly made litchi extract an ideal substrate for BC production. In fact, BC static fermentation requires an
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initial sugar concentration less than 25 g/L [3], namely, 1 L litchi extract could be used for
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more than 6 L fermentation substrate for BC production. At this situation, in spite that the cost of litchi is relatively high as fermentation substrate, the high sugar concentration in litchi extract made this bioconversion economically feasible. The trace elements in litchi extract were also measured by ICP. Generally, elements Mg, K, and Na were all found in the litchi extract. Without any further pretreatment, the litchi extract was used as substrate for BC fermentation.
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3. RESULTS AND DISCUSSION
3.2. BC Production From The Litchi Extract In this work, the BC fermentation on the litchi extract was carried out for two weeks and the evolution of BC dry weight was shown in Fig. 1. After one day’s fermentation, the BC membrane could be seen at the surface of static fermentation. From the 2 nd day of
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fermentation, the BC dry weight begun to increase stably, but its increasing rate is relatively slow compared with other researches [24, 25]. This may be due to the large fluctuation of fermentation pH value (mainly going up rapidly in this period). After the 8th day, the BC dry weight increased much faster and it maintained at about 1.4 g/L from
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the 9th day to the 11th day. The highest BC dry weight was got at the 13 th day of static
could be used as carbon source for maintaining the growth of G. xylinus. Compared with
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other BC fermentation, the BC yield on litchi extract was lower. Two reasons might
explain this phenomenon: on the one hand, the viscosity of litchi extract was higher than that of traditional hydrophilic medium, and this might prevent normal sugar metabolism of yeast; on the other hand, the substrate concentration of litchi extract might be too high
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for BC fermentation and thus influence the BC yield.
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It is well known that the element present in the materials could be detected by ICP [26]. To evaluate the element loading of BC samples obtained from litchi extract, the trace elements in both litchi extract and BC samples were both analyzed by ICP. From the ICP results, the litchi extract mainly contain three trace elements, namely Mg, K, and Na, and their concentration were (g/L): 1.838, 30.639, and 5.498, respectively. However, K was
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fermentation. and then, the BC dry weight became a little lower. It is possible that the BC
not found in the BC samples surprisingly. It is possible that K could not be absorbed by BC nano-structure. The concentration of Na and Mg was much lower than that of K in the litchi extract but they were all found in the BC samples (their concentration was 0.0706 and 2.305, respectively), indicating that Na and Mg could be adhered on the BC membrane. Since Na and Mg are the basic elements for human health, the BC membrane
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derived from litchi extract could be an ideal food additive. Overall, the final product BC membrane obtained partly absorbed the nutrient of litchi extract and thus has certain nutrient value. After fermentation, the BC membrane could be separated easily by filtration, and the remaining fermentation broth could be recovered for further
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3.3. Structure Analysis of BC
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In order to show the influence of litchi extract on the BC structure, the analysis of
SEM, FTIR and XRD was carried out for the samples obtained both from litchi extract and classical HS medium.
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The morphologies of BC samples were shown in Fig. 2. As it depicted, both samples showed a reticulated structure consisting of ultrafine fibrils. The gross morphological
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structure seemed to be similar for both specimens. Their transverse dimensions ranged from 10 to 50 nm. However, detailed examination of the micrographs revealed profound morphological differences between the nanofibrils and the reticulated structures of BC produced under different fenmentation media. The fibrils of BC produced from the litchi extract appeared to be more voluble and had more clearly three-dimensional construction
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fermentation or other food application.
than BC fibrils produced using HS medium. But the later showed highly extended and almost uniform in size. The exact width of individual microfibrils in each case, however, is difficult to estimate. The morphological changes in BC sheets affected the microstructures and various properties including the degree of polymerization, crystallinity, content of cellulose Iα and water holding capacity [26, 27]. Besides, the SEM
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figures showed that the nano-structure of BC obtained in this work is much clearer than that got from elephant grass acid hydrolysate [6].
The functional groups of BC were evaluated by FT-IR in the wave number of 4000-400
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cm-1 and the results obtained from litchi extract and HS medium was compared carefully
3352 cm-1 for the stretching vibration of hydroxyl groups (-OH), at 2897 cm-1 for the
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asymmetric stretching vibration of methylene (-CH2 -), at 2856 cm-1 for the symmetric stretching vibration of methyl (-CH3), at 1427 cm-1 for the asymmetric deformation vibration of methyl and methylene, at 1060 cm-1 for C-O-C and C-O-H stretching
vibration of sugar ring and at 897 cm-1 for γ (COC) in plane, symmetric stretching [28].
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Overall, no obvious difference was observed on the BC samples obtained from litchi extract or HS medium, indicating that no influence of using litchi extract as substrate on
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the functional groups of BC. Obviously, the BC obtained has all the characteristic bands of cellulose, indicating its high purity of cellulose when compared with other plant cellulose (besides cellulose, it also contain hemicellulose, and lignin).
XRD patterns obtained from BC samples are shown in Fig. 4. As it depicted, the XRD
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(Fig. 3). The characteristic bands of cellulose synthesized in litchi extract appeared at
figures had a little difference for the BC samples obtained from the litchi extract and HS medium. For the BC sample obtained after fermentation from litchi extract, it showed diffraction profiles characteristic of cellulose I (Table 1), with peaks at 2θ angles of 14.4, 16.7, and 22.5°, corresponding to the (1Ī0), (110), and (200) crystal planes (Fig. 4), respectively. However, the BC sample obtained from HS medium showed obvious
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decrease of peak height for the (110) and (200) crystal planes (Fig. 4) of which the corresponding diffraction angles were much bigger. Furthermore, BC produced in the litchi extract had smaller crystallite size of (110) and (200) crystal planes than BC produced in HS medium (Table 1). Decrease of crystallite size indicates that litchi extract
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prevents the tight aggregation of microbrils to form a ribbon by adhering to the surface of
agreed with the SEM analysis. The increase in intensity of the (110) and (200) crystal
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planes from the litchi extract (Fig. 4) indicates the existence of preferential orientation
growth. As a result, the crystallinity of BC samples produced in litchi extract was bigger than BC produced in HS medium; in contrast, the CI is much smaller. Similar experimental investigation has been reported by Tokoh et al. [30]. Litchi extract is a
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complex natural medium consisting of several ingredients [13, 14]. It might contain some constituent(s) which can serve as the condensed matter to increase the aggregation of
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particles. From these results it can be concluded that the culture medium has a significant effect on crystallite size and crystallinity index. Overall, using litchi extract as substrate for BC fermentation is beneficial to generate more value-added food nano-cellulose.
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nascent microbrils, thus promoting the formation of space grid structure. These results
4. CONCLUSIONS
Without any pretreatment, litchi extract could be used as substrate for BC production
by G. xylinus. After 2 weeks’ fermentation, the final production BC membrane could be generated. Its highest dry weight was 2.53 g/L. It has a more regularly reticulated nanostructure and a higher crystallinity than the BC membrane obtained from classical HS medium. Besides, the BC membrane contained elements Na and Mg. The BC
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membrane generated from litchi extract could be used as one kind of suitable food additive or food sources.
ACKNOWLEDGMENTS
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The authors acknowledge the financial support of National Natural Science Foundation
(S2012040007546), project of Guangzhou Science and Technology (2013J4300031), the
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Support Plan Project of National Science and Technology (2012BAD32B07), and
Foundation of Director of Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences (y407pb1001, y107rf1001).
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Fermentation Crystallinity Size of crystals (Å)
Crystallinity
Crystallite
medium
Index (%)
Structure
(%) (1Ī0)
(110)
(200)
Segal equation
91.00
80.45
254.60 76.22
93.9
Cellulose I
Litchi extract
94.02
83.90
139.83 70.54
89.4
Cellulose I
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HS
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Table 1. Characteristics peaks obtained for BC samples
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Figure 1. Evoluation of BC dry weight throughout the fermentation process. (□) BC dry
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weight; (△) pH.
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Figure 2. FE-SEM pictures of BC samples collected from different fermentation medium.
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(a) product obtained from HS medium; (b) product obtained from litchi extract.
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Figure 3. FT-IR spectra of BC samples collected from different static fermentation
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medium. (a) product obtained from HS medium; (b) product obtained from litchi extract.
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Figure 4. XRD patterns of BC samples collected on different static fermentation medium.
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(a) product obtained from HS medium; (b) product obtained from litchi extract.
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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Bacterial Cellulose Production from the Litchi Extract by Gluconacetobacter Xylinus abc
Xiao-Yan Yang ab
Qing Lin , Xue-Fang Chen a
ab
ab
ab
ab
ab
, Chao Huang , Hai-Jun Guo , Lian Xiong , Jun Luo , Bo Wang , Xiaobc
ab
& Xin-De Chen
Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou, PR China
b
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, PR China c
University of Chinese Academy of Sciences, Beijing, PR China Accepted author version posted online: 02 Sep 2014.
To cite this article: Xiao-Yan Yang, Chao Huang, Hai-Jun Guo, Lian Xiong, Jun Luo, Bo Wang, Xiao-Qing Lin, Xue-Fang Chen & Xin-De Chen (2014): Bacterial Cellulose Production from the Litchi Extract by Gluconacetobacter Xylinus, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.958163 To link to this article: http://dx.doi.org/10.1080/10826068.2014.958163
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Bacterial cellulose production from the litchi extract by Gluconacetobacter xylinus Xiao-Yan Yang1,2,3 , Chao Huang1,2 , Hai-Jun Guo1,2, Lian Xiong1,2 , Jun Luo1,2, Bo Wang1,2 , Xiao-Qing Lin1,2, Xue-Fang Chen2,3 , Xin-De Chen1,2 1
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Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou, PR China, 2Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, PR China, 3 University of Chinese Academy of Sciences, Beijing, PR China
Abstract
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Although litchi has both nutrient and edible value, the extremely short preservation time limited its further market promotion. To explore processed litchi products with longer preservation time, litchi extract was selected as an alternative feedstock for
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production of bacterial cellulose (BC). After two weeks’ static fermentation, 2.53g/L of the BC membrane was obtained. The trace elements including magnesium (Mg) and
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sodium (Na) in the litchi extract were partly absorbed in the BC membrane, but no potassium (K) element was detected in it curiously. Scanning electron microscope (SEM) photographs exhibited an ultra fine network nanostructure for the BC produced in the litchi extract. Analysis of the infrared spectrograms confirmed the pellicles to be a cellulosic material. Interestingly, X-ray diffraction (XRD) results showed the BC
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Corresponding Address to X. D. Chen: No.2 Nengyuan Road, Tianhe District, Guangzhou 510640, PR China. E-mail: [email protected]
membrane obtained from litchi extract had higher crystallinity of 94.0% than that from HS medium. Overall, the work showed the potential of producing high value-added polymer from litchi resources.
KEYWORDS: Cellulose produced by bacteria, litchi processing, fermentation; bio-refinery
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1. INTRODUCTION Bacterial cellulose (BC), synthesized by many bacteria and with high Young’s Modulus, has great potential in the application on many fields such as food, biomedical material,
Generally, the substrate for BC production is usually the low-cost one such as
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cotton-based waste textiles [4], wheat straw [5], elephant grass acid hydrolysate [6], or other residues from agro-forest industries [7]. However, when BC is applied in food industry, it
is better to use substrate for BC production from food resources such as corn steep liquor [8]
, molasses [9], konjac powder hydrolysate [10] and etc. Among various food resources,
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fruit juices was shown as one promising substrate for BC production and the fruit chosen
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was the common one such as orange, apple, pineapple, grape and etc. [11].
Litchi (Litchi chinensis Sonn.), which is a tropical and subtropical fruit of the Sapindaceae family, has been widely planted in South-East Asia, especially in China for many years [12]. Owing to its bright color and delicious taste, litchi has been gradually accepted by consumers and has established great popularity in the international market.
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xylinus is the most common one used for BC production [3].
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papermaking and transducer diaphragms [1-3]. Among various bacteria, Gluconacetobacter
Moreover, many components have been proven to have antioxidant function in the fruit pulp of litchi [13, 14]. Unfortunately, the preservation time of litchi is extremely short (merely 1-2 days without cold storage) and thus limited its market and profit. Exploring processed litchi product could not only increase its preservation time but also expand its market promotion. Therefore, using litchi extract as substrate for BC production seems to
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be a good alternative to obtain BC membrane with nutrient from litchi. In addition, this product could solve the preservation problem of litchi and explore its new application. Thus, it might have potential market competitiveness in future.
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It is known that the composition of the culture medium and fermentation conditions
polysaccharides including the bacterial cellulose [15-17]. These modifications (if any) in
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turn may affect the properties of BC. Therefore, it seems important to investigate the
possible modifications in the physicochemical properties of the BC sheets produced from the litchi extract. To our knowledge, little work has used litchi as raw material for BC production. For the first time, the litchi extract was used directly as the substrate for BC
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production by G. xylinus. In this work, the possibility of BC production from litchi extract was evaluated and the BC yield, structure and composition were also systematically
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measured.
2. MATERIAL AND METHODS
2.1. Preparation and Composition Analysis of Litchi Extract Fresh fruits of litchi at mature stage were purchased from the local market. The bright red
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greatly influence the chemical structure, composition and viscosity of the microbial
fruits without disease symptoms were selected, and then washed thoroughly with water, manually peeled, removed the seeds and extracted the juice with the 99B-4 fruit squeezer (CROWN ELEC., CO. Hong Kong, China). Then the litchi juice was vacuum filtrated twice to remove impurities without crushing by SHZ-Dш circulating water vacuum pump (Gongyi City Yuhua Instrument CO., LTD, China) with vacuum degree close to -0.1 MPa,
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and finally stored at -20 oC until use. The total sugars of litchi extract were measured using anthrone methods [18]. Mineral elements of litchi extract were analyzed by Inductive Coupled Plasma Emission Spectrometer (ICP, PerkinElmer Optima 8000DV ICP system,
2.2. Microorganism, Culture Media and Growth Conditions
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Gluconacetobacter xylinus CH001 (Laboratory of Energy and Biochemical Engineering, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences) was used as the microorganism for BC production in this work.
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The pre-culture was performed on pre-cultivation medium (g/L, mannitol 25, peptone 5, yeast extract 3, initial pH 6.0) at 28 oC and 150 rpm for 24 h. Then, 8 % (v/v) seed culture
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was translated into the litchi extract which was diluted 6 times and autoclaved at 121 oC for 20 min. Then, the static fermentation was carried out at 28 oC for 14 days. To compare the BC product obtained from normal fermentation medium, the fermentation was also carried out on the classical HS medium (g/L, glucose 20, yeast extract 5, peptone 5, disodium hydrogen phosphate 2.7, citric acid 1.15, initial pH 6.0) [19] at the same
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overnight.
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Massachusetts, USA). Before analysis, the sample was digested using nitric acid
fermentation condition.
2.3. Harvest and Weighing of BC After fermentation, the fermentation broth and BC were separated by vacuum filtration. Then, the BC was treated by 1.5% (w/v) NaOH at 80 oC for two hours. After that, the BC
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was repeatedly washed by distilled water until the washing water being neutralization. Finally, the BC was freeze-dried and weighed.
2.4. Structure and Composition Analysis of BC by SEM, FT-IR, and XRD
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The morphologies of freeze-dried BC samples were observed by a Hitachi S-4800 high
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resolution Field Emission Scanning Electron Microscope (FE-SEM, Hitachi, Japan) operated at 2.0 kV and 10 μA.
BC samples were mixed with spectroscopic-grade potassium bromide powder (1% w/w)
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and Fourier Transform Infrared Spectroscopy (FTIR) was carried out in the range of 400 to 4000 cm-1 wavelength with a Perkin-Elmer Spectrum one FT-IR Spectrometer (US).
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The kind of crystallite allomorph was determined with shifting FT-IR bands related to specified groups and bonds [20-22].
Structure, size, and percentage of crystals of BC samples were analyzed by D/max-RA X-ray diffractometer (Rigaku, Japan) with Cu Kα radiation (λ = 0.154 nm) operated at 40
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Mineral elements of BC samples were analyzed according to 2.1.
kV and 100 mA. Samples were scanned from 10° to 50° (2θ range) at a scan speed of 0.008°/step. To determine the sample crystallinity, profile analysis was carried out with a peak fitting program using Gaussian line shapes. The crystal size of cellulose was calculated using Debye-Scherrer’s equation [17]. Crystallinity index (CI) was calculated from the related intensity data by the Segal method (Equation (1)) [23].
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CI
I200 I am / I200
(1)
where I200 is the maximum intensity of crystalline region at 2θ (about 22.8°), I am is the intensity of the amorphous region of the wide-angle X-ray diffraction curves at 2θ (about
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18°).
3.1. Preparation of Litchi Extract
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In this work, the litchi was used that harvested at May, 2013 in Guangzhou. The composition of the litchi extract was analyzed before fermentation. Litchi extract
contained a very high sugar concentration of 183.5 g/L, this undoubtedly made litchi extract an ideal substrate for BC production. In fact, BC static fermentation requires an
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initial sugar concentration less than 25 g/L [3], namely, 1 L litchi extract could be used for
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more than 6 L fermentation substrate for BC production. At this situation, in spite that the cost of litchi is relatively high as fermentation substrate, the high sugar concentration in litchi extract made this bioconversion economically feasible. The trace elements in litchi extract were also measured by ICP. Generally, elements Mg, K, and Na were all found in the litchi extract. Without any further pretreatment, the litchi extract was used as substrate for BC fermentation.
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3. RESULTS AND DISCUSSION
3.2. BC Production From The Litchi Extract In this work, the BC fermentation on the litchi extract was carried out for two weeks and the evolution of BC dry weight was shown in Fig. 1. After one day’s fermentation, the BC membrane could be seen at the surface of static fermentation. From the 2 nd day of
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fermentation, the BC dry weight begun to increase stably, but its increasing rate is relatively slow compared with other researches [24, 25]. This may be due to the large fluctuation of fermentation pH value (mainly going up rapidly in this period). After the 8th day, the BC dry weight increased much faster and it maintained at about 1.4 g/L from
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the 9th day to the 11th day. The highest BC dry weight was got at the 13 th day of static
could be used as carbon source for maintaining the growth of G. xylinus. Compared with
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other BC fermentation, the BC yield on litchi extract was lower. Two reasons might
explain this phenomenon: on the one hand, the viscosity of litchi extract was higher than that of traditional hydrophilic medium, and this might prevent normal sugar metabolism of yeast; on the other hand, the substrate concentration of litchi extract might be too high
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for BC fermentation and thus influence the BC yield.
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It is well known that the element present in the materials could be detected by ICP [26]. To evaluate the element loading of BC samples obtained from litchi extract, the trace elements in both litchi extract and BC samples were both analyzed by ICP. From the ICP results, the litchi extract mainly contain three trace elements, namely Mg, K, and Na, and their concentration were (g/L): 1.838, 30.639, and 5.498, respectively. However, K was
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fermentation. and then, the BC dry weight became a little lower. It is possible that the BC
not found in the BC samples surprisingly. It is possible that K could not be absorbed by BC nano-structure. The concentration of Na and Mg was much lower than that of K in the litchi extract but they were all found in the BC samples (their concentration was 0.0706 and 2.305, respectively), indicating that Na and Mg could be adhered on the BC membrane. Since Na and Mg are the basic elements for human health, the BC membrane
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derived from litchi extract could be an ideal food additive. Overall, the final product BC membrane obtained partly absorbed the nutrient of litchi extract and thus has certain nutrient value. After fermentation, the BC membrane could be separated easily by filtration, and the remaining fermentation broth could be recovered for further
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3.3. Structure Analysis of BC
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In order to show the influence of litchi extract on the BC structure, the analysis of
SEM, FTIR and XRD was carried out for the samples obtained both from litchi extract and classical HS medium.
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The morphologies of BC samples were shown in Fig. 2. As it depicted, both samples showed a reticulated structure consisting of ultrafine fibrils. The gross morphological
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structure seemed to be similar for both specimens. Their transverse dimensions ranged from 10 to 50 nm. However, detailed examination of the micrographs revealed profound morphological differences between the nanofibrils and the reticulated structures of BC produced under different fenmentation media. The fibrils of BC produced from the litchi extract appeared to be more voluble and had more clearly three-dimensional construction
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fermentation or other food application.
than BC fibrils produced using HS medium. But the later showed highly extended and almost uniform in size. The exact width of individual microfibrils in each case, however, is difficult to estimate. The morphological changes in BC sheets affected the microstructures and various properties including the degree of polymerization, crystallinity, content of cellulose Iα and water holding capacity [26, 27]. Besides, the SEM
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figures showed that the nano-structure of BC obtained in this work is much clearer than that got from elephant grass acid hydrolysate [6].
The functional groups of BC were evaluated by FT-IR in the wave number of 4000-400
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cm-1 and the results obtained from litchi extract and HS medium was compared carefully
3352 cm-1 for the stretching vibration of hydroxyl groups (-OH), at 2897 cm-1 for the
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asymmetric stretching vibration of methylene (-CH2 -), at 2856 cm-1 for the symmetric stretching vibration of methyl (-CH3), at 1427 cm-1 for the asymmetric deformation vibration of methyl and methylene, at 1060 cm-1 for C-O-C and C-O-H stretching
vibration of sugar ring and at 897 cm-1 for γ (COC) in plane, symmetric stretching [28].
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Overall, no obvious difference was observed on the BC samples obtained from litchi extract or HS medium, indicating that no influence of using litchi extract as substrate on
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the functional groups of BC. Obviously, the BC obtained has all the characteristic bands of cellulose, indicating its high purity of cellulose when compared with other plant cellulose (besides cellulose, it also contain hemicellulose, and lignin).
XRD patterns obtained from BC samples are shown in Fig. 4. As it depicted, the XRD
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(Fig. 3). The characteristic bands of cellulose synthesized in litchi extract appeared at
figures had a little difference for the BC samples obtained from the litchi extract and HS medium. For the BC sample obtained after fermentation from litchi extract, it showed diffraction profiles characteristic of cellulose I (Table 1), with peaks at 2θ angles of 14.4, 16.7, and 22.5°, corresponding to the (1Ī0), (110), and (200) crystal planes (Fig. 4), respectively. However, the BC sample obtained from HS medium showed obvious
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decrease of peak height for the (110) and (200) crystal planes (Fig. 4) of which the corresponding diffraction angles were much bigger. Furthermore, BC produced in the litchi extract had smaller crystallite size of (110) and (200) crystal planes than BC produced in HS medium (Table 1). Decrease of crystallite size indicates that litchi extract
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prevents the tight aggregation of microbrils to form a ribbon by adhering to the surface of
agreed with the SEM analysis. The increase in intensity of the (110) and (200) crystal
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planes from the litchi extract (Fig. 4) indicates the existence of preferential orientation
growth. As a result, the crystallinity of BC samples produced in litchi extract was bigger than BC produced in HS medium; in contrast, the CI is much smaller. Similar experimental investigation has been reported by Tokoh et al. [30]. Litchi extract is a
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complex natural medium consisting of several ingredients [13, 14]. It might contain some constituent(s) which can serve as the condensed matter to increase the aggregation of
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particles. From these results it can be concluded that the culture medium has a significant effect on crystallite size and crystallinity index. Overall, using litchi extract as substrate for BC fermentation is beneficial to generate more value-added food nano-cellulose.
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nascent microbrils, thus promoting the formation of space grid structure. These results
4. CONCLUSIONS
Without any pretreatment, litchi extract could be used as substrate for BC production
by G. xylinus. After 2 weeks’ fermentation, the final production BC membrane could be generated. Its highest dry weight was 2.53 g/L. It has a more regularly reticulated nanostructure and a higher crystallinity than the BC membrane obtained from classical HS medium. Besides, the BC membrane contained elements Na and Mg. The BC
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membrane generated from litchi extract could be used as one kind of suitable food additive or food sources.
ACKNOWLEDGMENTS
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The authors acknowledge the financial support of National Natural Science Foundation
(S2012040007546), project of Guangzhou Science and Technology (2013J4300031), the
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Support Plan Project of National Science and Technology (2012BAD32B07), and
Foundation of Director of Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences (y407pb1001, y107rf1001).
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Fermentation Crystallinity Size of crystals (Å)
Crystallinity
Crystallite
medium
Index (%)
Structure
(%) (1Ī0)
(110)
(200)
Segal equation
91.00
80.45
254.60 76.22
93.9
Cellulose I
Litchi extract
94.02
83.90
139.83 70.54
89.4
Cellulose I
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HS
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Table 1. Characteristics peaks obtained for BC samples
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Figure 1. Evoluation of BC dry weight throughout the fermentation process. (□) BC dry
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weight; (△) pH.
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Figure 2. FE-SEM pictures of BC samples collected from different fermentation medium.
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(a) product obtained from HS medium; (b) product obtained from litchi extract.
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Figure 3. FT-IR spectra of BC samples collected from different static fermentation
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medium. (a) product obtained from HS medium; (b) product obtained from litchi extract.
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Figure 4. XRD patterns of BC samples collected on different static fermentation medium.
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(a) product obtained from HS medium; (b) product obtained from litchi extract.
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