International Food Research Journal 19(1): 153-158 (2012)

Microstructure and physical properties of microbial cellulose

produced during fermentation of black tea broth
(Kombucha). II.
Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A. and *Rajeev Bhat

Food Technology Division, School of Industrial

Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia

Abstract: In this study, we evaluated and characterized microbial cellulose produced from Kombucha after
eighth day of fermentation by employing SEM, FTIR, X-ray diffractometry, adsorption isotherm, and by
measuring the swelling properties. Results on SEM revealed microbial cellulose layer to be composed of
a compact cellulose ultrafine network like structure. FTIR spectra showed the presence of a characteristic
region of anomeric carbons (960 – 730 cm-1), wherein a band at 891.59 cm-1 confirmed the presence of β,
1-4 linkages. Results of FTIR spectra also showed microbial cellulose to be free from contaminants such
as lignin or hemicellulose, which are often present in plant cellulose. X-ray diffraction studies exhibited the
overall degree of crystallinity index for MCC to be slightly lower than that of microbial cellulose. Results
on swelling properties indicated microbial cellulose to possess higher fiber liquid retention values (10-160%)
compared to commercial MCC (5-70%). The adsorption isotherm curves showed similarities between microbial
cellulose with that of pure crystalline substance. Overall, results obtained in this study were comparable with the
commercial microcrystalline cellulose, indicating that the process developed by us can be explored industrially
on a pilot scale.

Keywords: Microbial cellulose, SEM, FTIR, X-ray diffractometry, adsorption isotherm, swelling
properties

Introduction pre-requisite to characterize the microbial cellulose

produced from Kombucha for commercial use and
Kombucha is one of the highly popular fermented applications. In the present study, investigations were
traditional beverages consumed for potential health carried out to characterize the microbial cellulose
benefits. Consumption of Kombucha is reported to that was produced during production of Kombucha.
cure some of the common ailments like arthritis, To the best of our knowledge, this is the first time that
indigestion, various types of cancer, hepatotoxicity, this type of characterization of microbial cellulose
etc. (Sreeramulu et al., 2000; Pauline et al., 2001; has been reported from Kombucha.
Hiremath et al., 2002). Kombucha fermentation
is brought about by the symbiosis of yeast species Materials and Methods
and acetic acid bacteria (Teoh et al., 2004) and
the harboring microbes or the starter culture has Materials
been declared to be safe by the US Food and Drug Black tea (Boh, Superior Cameron Highlands
Administration (CDC, 1996), which further paves the Tea, Malaysia) was used as the substrate for the
way for exploring from consumer’s safety prospects. fermentation and starter culture was obtained
Cellulose is one of the most abundant naturally from a local commercial source. Sucrose, used
occurring polysaccharide which forms an integral part as the carbon source was of food-grade (Gula
of most plant materials. However, in several instances, Prai, Malayan Sugar Mfg. Co. Bhd., Malaysia).
commercially available plant based cellulose are Commercial microcrystalline cellulose (MCC,
impure due to the presence of high amounts of lignin Avicel, Philadelphia, USA) was used as comparison
or hemi-cellulose. Compared to the plant cellulose in cellulose characteristic studies.
(PC), bacterial cellulose (BC) can be synthesized
(extracellularly) by bacteria such as Acetobactor Maintenance of tea fungus sample, and preparation
xylinum and Sarcinia. BC is reported to possess of tea broth for fermentation
several advantages which include: high purity, better Maintenance of tea fungus samples and
mechanical strength, crystallinity and hydrophilicity preparation of tea broth for fermentation was carried
with same chemical structure as that of PC (Yoshinaga out as per the procedures detailed earlier in the first
et al., 1997; Phisalaphong and Jatupaiboon, 2008). part of this paper series (Goh et al., 2011).
Considering these facts, it is highly essential and a

*Corresponding author.
Email: [email protected], [email protected] © All Rights Reserved
Tel: 60 4653 5212; Fax: 60 4653 3678
154 Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A. and Rajeev Bhat

Scanning electron microscopy (SEM) studies analogous to water retention value (Tappi Useful
The cellulosic pellicle formed on the eighth day Methods, UM 256, 54/1991). A known amount (1 g)
of fermentation was cut into thin slices and washed of sample was mixed with 40 g of different solutions
with double distilled water. The washed cellulose (water, acetone, dimethyl sulfoxide, and acetic acid).
fragments were centrifuged at 4500 rpm for 2 min. The suspension was allowed to stand for 2 hours,
(Kubota 5100, Tokyo, Japan) and the supernatant and then centrifuged at 3500 rpm for 30 minutes
was discarded. Centrifugation was repeated 4 times (Kubota-5100, Tokyo, Japan). After centrifugation,
to get rid of the adhering mucus. Sample was then the supernatant was drained and the wet sample
freeze-dried before analysis using Scanning Electron precipitate was weighed. The results were expressed
Microscope (Model S-360, Leica, Cambridge, U.K.) as gram of water per gram of sample. The LRV
to study its cellulose network, and distribution experiment for both microbial cellulose and the
of acetic acid bacteria and yeasts. SEM studies commercial MCC was run in duplicate.
were also performed with the commercial MCC to
compare with the microbial cellulose produced from Adsorption isotherms
Kombucha. Sorption isotherms of microbial cellulose and
the commercial MCC were determined at 30°C
Fourier transformed infrared spectroscopy (FTIR) according to the procedure described by Spiess
The cellulose pellicles, harvested on the eighth and Wolf (1983), with minor modifications. Both
day of fermentation were rinsed with distilled water microbial cellulose and MCC samples were pre-dried
(3 times), cut into slices and were freeze-dried. The in vacuum desiccators over P2O5 at room temperature
freeze-dried sample was then ground into powder (30 ± 2°C) for 7 days to obtain “zero water content.
form for crystallinity analysis. The dried samples (200 mg each) were weighed,
The analysis of crystallinity for the microbial to the nearest 0.0001 g, into pre-weighed weighing
cellulose was carried out by FTIR spectrometer bottles. The dried samples, in quadruplicates, were
(Nicolet Avator 360-FT-IR Thermo Electron Inc., equilibrated in air-tight 1-litre Kilner jars containing
San Jose, CA, USA) employing the method of Abbott different saturated salt solutions of known relative
et al. (1988). For data acquisition, software OMNIC vapour pressure (RVP) at 30 °C. The saturated salt
5.1 was used. A total of 200 scans were taken with a solutions used were lithium chloride, potassium
resolution of 4 cm-1 in order to produce the spectra. acetate, magnesium chloride, potassium carbonate,
The commercial MCC powder was also scanned for sodium bromide, strontium chloride, sodium chloride,
comparison purpose. and potassium chloride, with RVP of 0.11, 0.22, 0.32,
0.43, 0.56, 0.69, 0.75 and 0.84, respectively. Samples
X-ray diffractometry were weighed daily. “Equilibrium was assumed to
The freeze-dried samples were ground into powder have achieved when the change in weight did not
for x-ray diffractometry analysis. The diffractograms exceed 0.1% for 3 consecutive weighing. Moisture
were recorded at room temperature (Siemens-5000 content (dry basis) was calculated from the weight
diffractometer, Siemens 5000, Karlsruhe, Germany) gained at this point.
using Ni-filtered Cu Kα radiation (λ = 1.54 A°). The
operating voltage and current were 40 kV and 18 Results and Discussion
mA, respectively. Data were collected at 0.02° 2θ
intervals. The commercial MCC was also sent for Characterization of the microbial cellulose was
x-ray diffraction for the purpose of comparison. conducted and compared with that of the commercial
Degree of crystallinity was calculated from the MCC. The MCC was chosen as the reference in this
diffracted intensity data using empirical method study because MCC is purified native cellulose and
proposed by Segal et al. (1959): it is not a chemical derivative, and there has been
I 002- I am no chemical modification of the cellulose molecule
(Cr.I.) = ------------------------× 100 (Thomas and Pourcelot, 1993).
I 002

where, Cr.I. is the degree of crystallinity, I002 is the Scanning electron microscopy (SEM)
maximum intensity of the (002) lattice diffraction Figure 1a shows the scanning electron micrograph
and Iam is the intensity diffraction at 18-2θ degrees. of microbial cellulose, which is characterized by an
ultrafine network structure and the microbial cellulose
Swelling properties layer, constituted by a compact cellulose network
The liquid retention value (LRV) was determined structure which is on par with the observation of

International Food Research Journal 19(1): 153-158

 Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (Kombucha). II. 155

Klemm et al. (2001). According to Iguchi et al. (2000), Fourier Transformed Infrared Spectroscopy (FTIR)
the cellulosic pellicle comprised of random assembly The position and intensity of absorption bands of
of microfibrils of less than 100 A° in diameter such a substance are extremely specific to that substance.
as seen in the electron micrograph of freeze-dried Like a fingerprint of a person, the infrared spectrum
Kombucha’s cellulosic pellicle. by FTIR is highly characteristic for a substance
(Gunzler and Gremlich, 2002) and can be used to
identify the microbial cellulose by comparing with
the reference commercial MCC.
In this study, the chemical nature of the microbial
cellulose polymer was confirmed by the infrared
spectra of the MCC. Figure 2a shows the FTIR
spectra of the commercial MCC and Figure 2b
shows the FTIR spectra of the microbial cellulose.
In general, both the spectra of MCC and microbial
(a) cellulose depict a similar trend. Absorbance spectra
for microbial cellulose and MCC in the region 4000-
450 cm-1 are reported in Figures 2a and 2b. The
absorbance bands are consistent with that of a glucose
polymer and their assignments were carried out based
on data from literature (Bertocchi et al., 1997).
The characteristic region of anomeric carbons
(960 – 730 cm-1) was identified, where a band at
891.59 cm-1 confirms the presence of β, 1-4 linkages
(Figure 2a and 2b). There is a good correspondence
(b)
between the main bands of the spectra of microbial
Figure 1. (a) Scanning electron micrograph of bacterial
cellulose network. (Magnification: 5,000×); (b)
cellulose and that of MCC. The only difference
Scanning electron micrograph of the commercial MCC. between the FTIR spectra of microbial cellulose
(Magnification: 1,000×) and MCC was restricted to the intensity of some
Figure 1b shows the scanning electron bands. FTIR spectra from Figure 2b also showed that
micrographs of the commercial MCC. Compared to microbial cellulose is free from contaminants such
the produced microbial cellulose, MCC pose a very as lignin or hemicellulose, which is often present in
different structure. The primary particles forming plant cellulose (Bertocchi et al., 1997). Such a high
the MCC aggregates were bigger in size (Figure 2) purity cellulose may find special applications in food
and therefore showed a low degree of coalescence or non food products.
between boundaries (Kothari et al., 2002). In addition, The samples of microbial cellulose are in the
the MCC aggregates showed less smooth surfaces polymorphic form of native cellulose similar to
and were also less densely packed. those obtained by plants as indicated by the position
and intensity of some absorption bands in the
FTIR spectra (Figure 2b). According to Nelson and
O’Connor (1964a), a weak and broad band centered
at 891.59 cm-1, and a strong band centered at 1424.18
cm-1 (CH2 scissoring) were present in the spectra
of the microbial cellulose samples, defining them
as cellulose I. The intensity of the band at 1424.18
(a)
cm-1 has also been correlated with the degree of
crystallinity and was often used as a standard band
for its estimation (Bertocchi et al., 1997).
Measurement of crystallinity of cellulosic materials
was performed by means of infrared crystallinity
ratio (CR), given by the ratio of absorptivities at 1372
cm-1 (C-H bending) to that at 2900 cm-1 (CH2 and
CH stretching) (Nelson and O’Connor, 1964b). The
(b) infrared crystallinity ratio (CR) obtained from this
Figure 2. (a) FTIR spectra of the commercial MCC; (b) FTIR
study is 100 for both the microbial cellulose and the
spectra of the bacterial cellulose.

International Food Research Journal 19(1): 153-158

156 Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A. and Rajeev Bhat

commercial MCC. From these data, it appears that study. On the other hand, the microbial cellulose in
the bacterial cellulose exhibits a very high degree of the present study was only soaked in distilled water
crystallinity, comparable to that of MCC. rather than employing non-aqueous solvents like alkyl
alcohols or ketones. However, findings obtained in
X-ray diffractometry the present study gave a higher degree of crystallinity
According to Mazumdar (1999), X-ray diffraction (Cr.I.) as compared to the cellulose which was
analysis could be used for quantitative determination isolated from hemp, kenaf and sorghum which only
of the crystalline contents in composite samples. The yielded 72, 65 and 63 % of Cr.I., respectively (Focher
X-ray diffraction scans obtained for the commercial et al., 2001). A different content of crystalline and
MCC is shown in Figure 3a. On the other hand, Figure amorphous region in the microbial cellulose and
3b shows the diffractogram of the microbial cellulose. MCC also contributed to the molecular rigidity of the
The main purpose of this study was to compare the bacterial cellulose fibers, which is higher than that of
degree of crystallinity of both the commercial MCC MCC.
and the microbial cellulose. Therefore, estimation of
percentage of crystallinity from these diffractograms Swelling properties
was used to compare the integrated intensities of Cellulose is insoluble in most of the organic
the crystalline peaks along with that of the diffuse solvents due to its crystalline nature. However,
background (Segal et al., 1959). Mantanis et al. (1995) have opined that both
Findings from both the diffractograms show that intracrystalline and inter-crystalline swelling is
the commercial MCC has a higher intensity of the possible in certain solvents. According to Geyer et
crystalline phase compared to the microbial cellulose, al. (1994), Acetobacter xylinum produces cellulose
which depicted a lower intensity of the crystalline in a highly swollen form using glucose as the carbon
phase. In addition, the diffraction angle at 2θ = 19°, source. The water absorption capacity of cellulose
which represents the intensity for amorphous phase has been reported to be varying between 100 and 120
for MCC also showed a higher intensity than the times its dry weight (White and Brown, 1989). Hence,
microbial cellulose. As a result, the overall degree to obtain more detailed information on the state of
of crystallinity (Cr.I.) for MCC is slightly lower swelling properties, comparison was done between
than that of the microbial cellulose. The crystallinity the microbial cellulose and the commercial MCC.
index was 80.9 and 81.3 for MCC and the microbial The liquid retention values (LRV) of the cellulosic
cellulose, respectively. fibers of the microbial cellulose and MCC in four
organic solvents at room temperature are shown in
Figure 4.

(a)

Figure 4. Swelling of bacterial cellulose and the

commercial MCC in organic solvent.
As depicted in Figure 4, the microbial cellulose
showed higher fiber liquid retention values (10-
160%) in all the four organic solvents than that of
MCC (5-70%). In addition, DMSO resulted in the
highest fiber liquid retention values for both the
(b) microbial cellulose and MCC, than that of water.
Figure 3. (a) X-ray diffractogram of the commercial MCC, These results are in agreement with those reported by
(b) X-ray diffractogram of the bacterial cellulose. Mantanis et al. (1995). The very high swelling power
exhibited by DMSO, whose molar volume is quite
Results obtained by Bertocchi et al. (1997) have high, may be reflective of the very high hydrogen
shown a higher degree of crystallinity as compared bonding capability.
to the microbial cellulose produced in the present Since Robertson (1964) suggested that molar

International Food Research Journal 19(1): 153-158

 Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (Kombucha). II. 157

volume is important to swelling, water with low surface of the crystal. Since this is a surface effect,
molar volume but with a high cohesive energy density grinding the bacterial cellulose into smaller particle
(CED) with high hydrogen bonding capability, results increases the moisture content at the low aw due to
in the swelling power of cellulosic fibers in water to the increased surface area per gram allowing more
be lower than DMSO. On the other hand, acetone hyroxyl groups to stick out (Bell and Labuza, 2000).
exhibited the lowest fiber liquid retention values. It is As shown in Figure 5, at low aw, for microbial
likely that the hydrogen bonding capability of acetone cellulose, the interaction of water with cellulose
is the weakest compared to the other three organic molecules is not strong enough to break the interactive
solvents. Apart from this, the cohesive energy density forces between the individual cellulose molecules in
value is also found to be lowest. Therefore, these two the crystal. However, as the aw is increased, the overall
factors might have resulted in the swelling power water-cellulose interactions increase enough to cause
of microbial cellulose and MCC in acetone as the disruption of the cellulose-cellulose interactions, and
lowest. Mantanis et al. (1995) reported that higher this water begins to penetrate into the crystal and
the cohesive energy density of the solvent, the higher exposing new surfaces.
is the cellulosic fibers swelling power. For the commercial MCC, it holds more water
than that of the produced bacterial cellulose, as
Adsorption isotherm shown in Figure 5. This happened because the less
Besides predicting the microbial or physico- crystalline or amorphous noncrystalline material of
chemical stability of foods, knowledge of water MCC can hydrogen-bond water internally, not just on
adsorption isotherms is very important for engineering the surface. However, as the aw and moisture content
purposes related to concentration and dehydration. increases, the cellulose molecules in the amorphous
The sorption isotherms for both the microbial solid become more mobile as the solid becomes
cellulose and the commercial MCC are shown in increasingly rubbery.
Figure 5.
Conclusions

The characterization of microbial cellulose

harvested from the tea broth revealed cellulose
layer to be constituted by a compact cellulose
network structure with the assembly of the ultrafine
microfibrils (by SEM) and exhibited a very high
degree of crystallinity at 81.3, accessed by means
Figure 5. Moisture adsorption isotherms of bacterial of FTIR, which was further confirmed by X-ray
cellulose and the commercial MCC at 30 °C. diffractometry. In addition, the microbial cellulose
It is observed that for the commercial MCC, it also depicted a high swelling power due to its
follows the sigmoidal-shaped curve, which might structure, which easily forms hydrogen bonding as
be caused by the combination of colligative effects, compared to the commercial cellulose. The curve
capillary effects, and surface-water interactions. Two for the produced microbial cellulose represents the
bends are noted on the isotherm for MCC, one around adsorption isotherm for a pure crystalline substance.
a water activity (aw) of 0.2-0.4 and another at 0.65- To conclude, microbial cellulose with high purity
0.75, which results from changes in the magnitude and high degree of crystallinity could be successfully
of the separate physical-chemical effects. The results produced from the fermentation of black tea broth -
obtained in the present study are in accordance with Kombucha.
the results on microcrystalline cellulose reported by
Ardizzone et al. (1999). References
On the other hand, the curve for the microbial
cellulose (BC) represents the adsorption isotherm Abbott, T., Palmer, D., Gordon, S. and Bagby, M. 1988.
for a pure crystalline substance that shows very Solid state analysis of plant polymers by FTIR. Journal
little moisture gain until the aw goes above the point of Wood Chemistry and Technology 8: 1-28.
where water begins to dissolve the crystal surface. Ardizzone, S.,  Dioguardi, F. S., Mussini, P. R., Mussini,
T.,  Rondinini, S., Vercelli, B. and Vertova, A. 1999.
The relative humidity where crystal dissolves is
Batch effects, water content and aqueous/organic
sometimes referred to as the deliquescent point. The solvent reactivity of microcrystalline cellulose samples.
shape of this curve is due to water interacting only International Journal of Biological Macromolecules
via the hydrogen bonds with hydroxyl groups on the 26: 269-277.

International Food Research Journal 19(1): 153-158

158 Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A. and Rajeev Bhat

Bell, L. N. and Labuza, T. P. 2000. Moisture sorption: of Applied Polymer Science 8: 1325-1341.
Practical aspects of isotherm measurement and use, Pauline, T., Dipti, P., Anju, B., Kavimani, S., Sharma, S. K.,
Second ed. American Association of Cereal Chemists, Kain, A. K., Sarada, S.K.S., Sairam, M., Ilavazhagan,
Inc., USA pp. 122. G., Kumar, D. and Selvamurthy, W. 2001. Studies on
Bertocchi, C., Delneri, D., Signore, S., Weng, Z. and toxicity; anti-stress and hepatoprotective properties
Bruschi, C.V. 1997. Characterization of microbial of Kombucha tea. Biomedical and Environmental
cellulose from a high-producing mutagenized Sciences 14: 207–213.
Acetobacter pasteurianus strain. Biochimica et Phisalaphong, M. and Jatupaiboon, N. 2008. Biosynthesis
Biophysica Acta 1336: 211-217. and characterization of bacteria cellulose–chitosan
CDC. 1996. CDC editorial note. The Journal of the film. Carbohydrate Polymers 74:482-488.
American Medical Association 275: 97–98. Robertson, A.A. 1964. Cellulose-liquid interactions. Pulp
Focher, B., Palma, M. T., Canetti, M., Torri, G., Cosentino, Paper Magazine of Canada, 65 : T171-T178.
C. and Gastaldi, G. 2001. Structural differences Segal, L., Creely, J., Martin, Jr. and Conrad, C.M. 1959.
between non-wood plant celluloses: evidence from An empirical method for estimating the degree of
solid state NMR, vibrational spectroscopy and X-ray crystallinity of native cellulose using the X-ray
diffractometry. Industrial Crops and Products 13:193- diffractometer. Journal of Textile Research 29:786-
208. 794.
Geyer, U., Heinze, Th., Stein, A., Klemm, D., Marsch, Spiess, W.E.L. and Wolf, W. 1983. The results of the COST
S. and Schmauder, H.-P. 1994. Formation, 90 project on water activity, in Physical Properties of
derivatization and application of bacterial cellulose Foods, Jowitt, R., Escher, F., Hallstrom, F.B., Meffert,
International Journal of Biological Macromolecules M.E., Spiess, W.E.L. and Vos, G. (eds) (Applied
16: 343-347. Science Publishers, London), pp. 65–91.
Goh, W.N., Rosma, A., Kaur, B., Fazilah, A., Karim, A.A. Sreeramulu, G., Zhu, Y. and Knol, W. 2000. Kombucha
and Bhat, R. 2011. Fermentation of black tea broth fermentation and its antimicrobial activity. Journal of
(Kombucha): I. Effects of sucrose concentration and Agricultural and Food Chemistry 48:2589–2594.
fermentation time on the yield of microbial cellulose Teoh, A. L., Heard, G. and Cox, J. 2004. Yeast ecology of
International Food Research Journal (in press). kombucha fermentation. International Journal of Food
Gunzler, H. and Gremlich, H. U. 2002. IR Spectroscopy, Microbiology 95:119–126.
an Introduction. Wiley VCH, Germany, pp 361. Thomas, C. and Pourcelot, Y. 1993. Preformulation of five
Hiremath, U. S., Vaidehi, M. P. and Mushtari, B. J. 2002. commercial celluloses in drug development: rheological
Effect of fermented tea on the blood sugar levels of and mechanical behavior. Drug Development and
NIDDM subjects. The Indian Practitioner 55: 423- Industrial Pharmacy 19:1947 – 1964.
425. White, D.G. and Brown Jr., R.M. 1989. Prospects for the
Iguchi, M., Yamanaka, S. and Budhiono, A. 2000. commercialization of the biosynthesis of microbial
Bacterial cellulose-a masterpiece of nature arts. cellulose, in Cellulose and Wood- Chemistry and
Journal of Material Science 35: 261–270. Technology, Schuerch C. (ed) (John Wiley and Sons,
Klemm, D., Schumann, D., Udhardt, U. and Marsch, S. Inc. N.Y.), pp. 573-590.
2001. Bacterial synthesized cellulose-artificial blood Yoshinaga, F., Tonouchi, N. and Watanabe, K. 1997.
vessels for microsurgery. Progress in Polymer Science Research progress in production of bacterial cellulose
26: 1561-1603. by aeration and agitation culture and its application as
Kothari, S. H., Kumar, V. and Banker, G. S. 2002. a new industrial material. Bioscience Biotechnology
Comparative evaluation of powder and mechanical and Biochemistry 61:219–224.
properties of low crystallinity celluloses,
microcrystalline celluloses, and powdered celluloses.
International Journal of Pharmaceutics 232: 69-80.
Mantanis, G. I., Young, R. A. and Rowell, R. M. 1995.
Swelling of compressed cellulose fiber webs in organic
liquids. Cellulose 2:1-22.
Mazumdar, S. 1999. A standardless method of quantitative
ceramic analysis using X-ray powder diffraction.
Journal of Applied Crystallography 32:381-386.
Nelson, M.L. and O’Connor, R. T. 1964a. Relation of
certain infrared bands to cellulose crystallinity and
crystal latticed type. Part I. Spectra of lattice types I,
II, III and of amorphous cellulose. Journal of Applied
Polymer Science 8: 1311-1324.
Nelson, M.L. and O’Connor, R.T. 1964b. Relation of
certain infrared bands to cellulose crystallinity and
crystal lattice type. Part II. A new infrared ratio for
estimation of crystallinity in celluloses I and II. Journal

International Food Research Journal 19(1): 153-158