American Journal of Food Science and Nutrition

2017; 4(1): 1-6

http://www.aascit.org/journal/ajfsn
ISSN: 2375-3935

Investigating the Conditions for

Nata-de-Coco Production by Newly
Isolated Acetobacter sp.
Huynh Xuan Phong*, Le Thi Lin, Nguyen Ngoc Thanh,
Bui Hoang Dang Long, Ngo Thi Phuong Dung
Biotechnology Research and Development Institute, Can Tho University, Can Tho City, Vietnam

Email address
[email protected] (H. X. Phong)
*
Corresponding author

Citation
Keywords Huynh Xuan Phong, Le Thi Lin, Nguyen Ngoc Thanh, Bui Hoang Dang Long, Ngo Thi Phuong
Acetobacter, Dung. Investigating the Conditions for Nata-de-Coco Production by Newly Isolated Acetobacter
Bacterial Cellulose, sp. American Journal of Food Science and Nutrition. Vol. 4, No. 1, 2017, pp. 1-6.
Cellulose Production,
Nata-de-Coco Abstract
Nata-de-coco, a bacterial cellulose product, which is usually prepared by Acetobacter
xylinum grown in mature coconut water, is a quite popular snack in Vietnam and other
countries. This research was carried out to obtain pure Acetobacter strains that can
Received: March 20, 2017 produce high cellulose and to investigate the effect of fermenting conditions on cellulose
Accepted: March 27, 2017 production. Total aerobic microorganisms in four nata fermenting media, collected from
Published: June 7, 2017 local producers in Ben Tre and Vinh Long provinces, Vietnam, were in the range of 8.79-
9.14 log cells/mL. Eight strains of Acetobacter sp. were isolated and identified by several
biochemical and physiological tests. Acetobacter sp. strain N was selected as the best
cellulose producing bacterium. Mature coconut water that incubated 60 h before
fermentation and initial bacterial density of inoculum at 6 log cells/mL gave the highest
yield (213.87 g cellulose/300 mL fermented mature coconut water). The addition of
glucose into the medium up to 5°Brix was also increased the cellulose production
(233.15 g/300 mL). Furthermore, the addition of both (NH4)2HPO4 and (NH4)2SO4 or
only (NH4)2HPO4 gave higher cellulose yields, 248.23 and 247.70 g cellulose were
obtained from 300 mL mature coconut water, respectively.

1. Introduction
Nata-de-coco is a chewy, jelly-like food, and quite popular snack in Vietnam and other
countries. Nata-de-coco, a bacterial cellulose product, which is usually prepared by
Acetobacter xylinum grown in mature coconut water [1, 2]. This product is not high
nutrient, less energy and high cellulose content. Therefore, it is useful for decreasing
obese people ratio, and stimulating peristaltic intestinal tract that help to regulate
excretion easily. This kind of bacterial cellulose (BC) has introduced into many different
fields such as paper production, bio-membrane used for treatment of burn and skin
trauma and so on. In food, BC was used as membrane for fresh coconut preservation
instead of chemical or sausage preservation besides nata-de-coco manufacturing.
Although there are so many researches on BC now, BC will be still subject that many
scientists are interested in the future because of new their applications [3, 4].
Nowadays, the problem of Vietnam and other countries which cultivate and produce
products from coconut is the elimination of coconut water is into environment, especially
from local producers of coconut’s flesh. This is one of causes of environmental pollution.
Consequently, using of coconut water for nata-de-coco production is necessary because
2 Huynh Xuan Phong et al.: Investigating the Conditions for Nata-de-Coco Production by Newly Isolated Acetobacter sp.

this work not only reduce the coconut water eliminated into to Acetobacter genus were shaking inoculation with the same
environment but also increase the value of coconut. Ben Tre initial density. After 24 h, determined bacterial density of
is known like country of coconut and has many local these strains. Then, these strains were inoculated with the
producers of nata-de-coco in Vietnam. However, almost same bacterial densitiy into culture medium. Culture medium
these local producers produce nata-de-coco at farmer using 300 mL mature coconut water, 24-h pre-incubation,
household scale. Their production process is essential base was adjusted to 5°Brix. The vessel’s size was 17.3 x 13.0 x
on their experience and different bacterial sources even reuse 7.3 cm. All treatments were conducted in triplicate.
bacterium liquid after fermented many times. Hence, quality The data were analyzed by Microsoft Excel (version 2007)
of nata-de-coco is unequal, not stable and low productivity. and Statgraphics Centurion XV (version 15.1.02). Statgraphics
Many recent researches provided that using pure bacterial Centurion XV was used to test for the least significant
starter and ratio of ingredients in producing fermented difference (LSD) with the confidence interval of 95%.
products and naturally in nata-de-coco production kept
important roles to effect on productivity and quality of 2.5. Study on Effect of Incubation Time of
products [5, 6]. Mature Coconut Water
Because of important roles and improving ways in the Mature coconut water was incubated at environmental
future of BC, this research was carried out to isolate and temperature (ca. 28-32°C) for 24 h, followed by adjusted
select good Acetobacter strain with high cellulose production total sugar concentration to 5°Brix, pH 4.5 and inoculated
and to investigate suitable conditions for effective cellulose with the same initial density at 24, 36, 48, 60 and 72 h in
production on selected strain. triplicate. Medium used mature coconut water without pre-
incubation was as control medium.
2. Materials and Methods
2.6. Study on the Influence of Bacterial Cell
2.1 Materials and Chemicals Density

Nata fermenting media were collected from 4 local This experiment was performed to obtain the appropriate
producers in Ben Tre and Vinh Long provinces, Vietnam. bacterial density for highest cellulose production. Initial Brix
Microbiological media and chemicals were purchased from degree of culture medium was regulated to 5°Brix. The
commercial products of Hi-Media (India) and Merck experiment was randomly carried out with 1 factor and 5
(Germany) such as Plate Count Agar (PCA), D-glucose, levels (3, 4, 5, 6 and 7 log cells/mL) in triplicate.
(NH4)2SO4 (SA), (NH4)2HPO4 (DAP), acetic acid, etc.
2.7. Study on the Effect of Sugar
2.2. Determination of Total Aerobic Supplementation
Microorganisms
Experiment was carried out to investigate initial glucose
Total aerobic microorganisms in nata fermenting media concentration for most effective cellulose production. The
were conducted at 3 continuous diluted concentrations on media were supplemented with D-glucose to 5, 6, 7, 8, 9, 10,
PCA medium in triplicate and incubated at 30°C. After 24 h and 11°Brix. Control medium was without glucose added.
and 48 h of incubation, all plates were counted the colonies All treatments were conducted in triplicate.
that were in the range of 25-250.
2.8. Study on the Effect of DAP and SA
2.3. Isolation and Identification of
Acetobacter Strains The purpose of this experiment was to obtain nutrient
sources added for highest cellulose production. Ratio of DAP
Streaked a small amount of nata fermenting medium onto and SA added was 0.2% DAP and 8.0% SA, 0.2% DAP and
mature coconut water medium which consisted of glucose (5 0% SA, 0% DAP and 0.8% SA, 0% DAP and 0% SA [8, 9].
g/L), SA (8 g/L), DAP (2 g/L), agar (40 g/L), and adjusted to
pH 4.5 by acetic acid [7]. Coconut water was incubated at 3. Results and Discussion
30°C in 24 h and sub-cultured several times until get the
purity colonies. Recorded the colony and cell morphology, 3.1. Evaluation of Total Aerobic
Gram reaction, catalase test, acetic acid formation on YPDG Microorganisms
medium (g/L; D-glucose 5, yeast extract 5, glycerol 5, agar
20) supplemented CaCO3 (5 g/L) and ethanol (4% v/v), the Total aerobic microorganisms in 4 samples were in range
color changing of YPGD plus 0.02% bromocresol green. 8.79-9.14 log cells/mL (Table 1). Result of Nguyen and
Pham revealed that A. xylinum density was about 7.51-7.68
2.4. Screening the Cellulose Production of log cells/mL after 6-day culture [9]. These samples were
Isolated Acetobacter Strains achieved higher numbers in because the total aerobic
microorganisms include other aerobic microorganisms beside
Bacterial strains that were preliminarily identified belong of A. xylinum.
 American Journal of Food Science and Nutrition 2017; 4(1): 1-6 3

Table 1. Total aerobic microorganisms in nata fermenting medium.

Samples Local producers Total aerobic microorganisms (cells/mL)* Strains

A Tran Phuoc 8.92 A1, A2
B Mai Thi Phuong 9.14 B1, B2
C Tran Van Chien 9.05 C1, C2, C3
N Hoang Chi Nhan 8.79 N

Note: (*) average values of triplicate.

3.2. Isolation and Identification of strains were rod shaped (Figure 1b, c). Cells shaped of A2,
Acetobacter Strains C2, and N strains were shorter than the others (A1, B1, B2,
C1, and C3). Biochemical test showed that the catalase was
Eight bacterial strains were isolated successfully from 4 positive reaction and Gram was negative reaction. They
nata fermenting medium samples in Ben Tre and Vinh Long formed clear zones on medium containing CaCO3 and
provinces, Vietnam (Table 1). The colonies of all isolated changed the medium color containing bromocresol green
strains were circle, swollen, yellowish (Figure 1a). In from blue to yellow then to green again. Therefore, these
addition, colonies of A1, B2, C1 and C3 strain had mucous strains belong to Acetobacter genus [10-12].
structure. Microscopic examination confirmed that these

Figure 1. Colonies form (a), cells shape (b) and Gram staining (c) of B1 strain.

Cellulose production by these 8 strains after 6 days of

3.3. Screen of Cellulose Production of Newly fermentation was shown in Figure 3. Strains N, A1, B2, C1
Isolated Acetobacter Strains and C3 produced more than 200 g of cellulose per 300 mL of
Eight strains were inoculated at the same initial density of mature coconut water (205.42, 211.78, 204.55, and 211.61 g,
6 log cells/ml into the mature coconut water and incubated in respectively) and their production were not significant
24 h. The bacterial density was reached 8.60-8.89 log difference in total cellulose weights at 95% confidence level.
cells/mL (Figure 2). The density of Acetobacter sp. N was
reached highest number (8.89 log cells/mL) and different
significance with the other strains at 5% of means after 24 h
incubation.

Figure 3. Cellulose yields produced by 8 bacterial strains.

Note: Values on the column was triplicate. Means with different subscripts
are statistically different at the 95% confidence level.

Figure 2. Density of bacteria strains after 24 h.

Cellulose that Acetobacter sp. N produced more white
color and smooth surface than other strains (Figure 4).
Note: Values on the column was triplicate. Means with different subscripts Moreover, statistical analysis of bacterial density at 95%
are statistically different at the 95% confidence level. confidence level after 24 h also indicated that density of
4 Huynh Xuan Phong et al.: Investigating the Conditions for Nata-de-Coco Production by Newly Isolated Acetobacter sp.

Acetobacter sp. N reached highest and difference converted mainly to cellulose [7]. When sucrose was used as
significance with the other strains. This increasing speed of carbon source for cellulose production, the system included
density will limit the contamination of other microorganisms, the hydrolysis of sucrose into glucose and fructose as well as
so cellulose production will be more effective. Therefore, other side reactions besides cellulose forming reaction.
Acetobacter sp. N produced high and nice cellulose was the Therefore, Brix degree decreased more but cellulose
selected strain using in further studies. production was not high. Besides, the presence of sucrose in
the medium in combination with fructose produced lower
amounts of cellulose [3].
The addition of acetic acid to the medium enhanced the
cellulose yield from glucose, but decreased the cellulose
yields on sucrose [13]. Mature coconut water without
incubated medium need more acetic acid to adjust the acidity
to pH 4.5, which is the optimum pH value for cellulose
production. The more incubated time of mature coconut
water was, the more sucrose decreased until only glucose was
detected. The pH value also decreased and acetic acid added
as little, so the yields of cellulose increased. Consequently,
the cellulose yield at incubated time of old coconut water of
24, 36 and 48 h was lower than at 60 h although Brix degree
Figure 4. Cellulose of Acetobacter strain B2 (left), N (center), and C3
(right).
decreased more.
Mature coconut water incubated before fermentation is
3.4. Effect of the Incubation Time of Mature very necessary in production. Materials can be initiative from
Coconut Water many places and acetic acid added into medium was saved.
The coconut water will not become yellow when cooking.
Mature coconut water was incubated at room temperature, Therefore, the production of cellulose will be more effective.
followed by adjusted to 5°Brix, pH 4.5 and inoculated with 7
log cells/mL at 24, 36, 48, 60 and 72 h. Medium used mature 3.5. Effect of Bacterial Cell Density on
coconut water without incubated was as control medium. The Cellulose Production
yield of cellulose was showed in Figure 5 was varied from
125.23 to 211.80 g per 300 mL of fermented mature coconut This experiment was carried out at 3, 4, 5, 6, 7 log
water. cells/mL of bacterial cell densities. The yields of cellulose
increased with an increase in inoculum densities, the
cellulose production was 94.87-213.87 g/300 mL (Figure 6).
The highest of cellulose yields were at initial inocula of 6 log
cells/mL (213.87 g/300 mL) and 7 log cells/mL (202.63
g/300 mL), and there was no significant difference at 95%
confidence level between these two inoculum levels.
However, among the two inoculums levels, 6 log cells/mL
was chosen because of saving inoculum into production as
well as easy to reach the density.

Figure 5. Effect of mature coconut water incubated at times on the

production of cellulose.
Note: Values on the column was triplicate. Means with different subscripts
are statistically different at the 95% confidence level.

Cellulose was produced more effectively with incubated

mature coconut water. The yields of cellulose increased with
an increase in incubated time of mature coconut water and
produced highest at mature coconut water that incubated at
60 h, then yields of cellulose decrease because the nutrient
and glucose concentration decrease in increasing incubated
time of mature coconut water. Figure 6. Effect of bacterial density on cellulose production.
Sucrose was found in freshly coconut water but only
Note: Values on the column was triplicate. Means with different subscripts
glucose was detected after 3-day storage and glucose was are statistically different at the 95% confidence level.
 American Journal of Food Science and Nutrition 2017; 4(1): 1-6 5

3.6. Effect of Sugar Supplementation on initial 4°Brix and 5°Brix, pH value in the medium increased
Cellulose Production slowly. The gluconic acid increased and then decreased
again. pH value, in common with the changing of gluconic
Fermentation media were adjusted to 5, 6, 7, 8, 9, 10, and acid, also increased and then to decreased again because
11°Brix by adding D-glucose. The medium without glucose glucose not used for cellulose synthesis is metabolized via
supplemented was control. The highest of cellulose yields gluconic acid to other substances [13, 14]. Therefore, at
was at 5°Brix, and there was significant difference at 5% initial Brix degree was 4°Brix and 5°Brix which glucose
confidence level with the others (Figure 7). The yield of concentration was not high, accumulation of gluconic acid
cellulose decreased with an increase in initial glucose was metabolized to other substances. Consequently, initial
concentration. The decrease in cellulose yield at high initial glucose concentration supplemented was very important. If
glucose concentration could due to some glucose initial glucose concentration supplemented was low, there
metabolized to gluconic acid, whereas forming cellulose. were not enough substances for production of cellulose. On
When initial glucose concentration increased, the pH value in the contrary, the accumulation of gluconic acid inhibited
the medium after fermentation decreased lower the range 4.0- cellulose production.
6.0 which is the optimum pH range for cellulose production
because of accumulation of gluconic acid [14]. However, at

Values on the column were triplicate. Means with different subscripts are statistically different at the 95% confidence level.
Figure 7. Effect of initial Brix degree on cellulose production.

Besides, mature coconut water will become yellow if it

was boiled in long time. As the results showed in Figure 7,
treatment of 5°Brix was the best choice because of not high
initial glucose concentration will avoid yellow becoming of
mature coconut water and the accumulation of gluconic acid
and save glucose supplemented on production.

3.7. Effect of DAP and SA on Cellulose

Production

DAP and SA added to supply nitrogen source for cellulose

production, whereas glucose was supplied as main carbon
source for cellulose production of Acetobacter. The addition
of nitrogen to the medium before fermentation, cellulose
production was higher than that without nitrogen source
added, 223.87-248.23 g/300 mL and 104.41 g/300 mL, Figure 8. Effect of DAP and SA supplemented on cellulose production.
respectively (Figure 8).
Note: Values on the column were triplicate. Means with different subscripts
are statistically different at the 95% confidence level.
6 Huynh Xuan Phong et al.: Investigating the Conditions for Nata-de-Coco Production by Newly Isolated Acetobacter sp.

These results indicated that this Acetobacter strain need from Acetobacter xylinum TISTR976 and physical properties
more nutrient added besides glucose and nutrient within of BC parchment paper. Journal of Science and Technology,
14: 357-365.
coconut water. The addition of both DAP and SA (248.23
g/300 mL) or only DAP (247.70 g/300 mL) gave highest [3] Embuscado, M. E., J. S. Marks and J. N. BeMiller. (1994).
yields, and there was no significant difference at 95% Factors affecting the production of cellulose by Acetobacter
confidence level between two levels. Thus, the addition of xylinum. Food Hydrocolloids, 8: 407-418.
DAP only was enough nitrogen supplemented. This result [4] Chawla, P. R., I. B. Bajaj, S. A. Survase and R. S. Singhal.
was also agreed with Nguyen Thi Hien [8]. When medium (2008). Microbial Cellulose: Fermentative Production and
was not added DAP and SA, Acetobacter used glucose Applications. Food Technology and Biotechnology, 47 (2):
converted to cellulose, so Brix degree decreased. Moreover, 107-124.
the Brix degree changing also showed that cellulose [5] Ramana, K. V., A. Tomar and L. Singh (2000). Effect of
production was more effective if Acetobacter were supplied various carbon and nitrogen sources on BC synthesis by
both nitrogen and phosphate source. In fact, DAP and SA Acetobacter xylinum. World Journal of Microbiology and
were added to medium with higher ratios of 0,2% DAP and Biotechnology, 16: 245-248.
0,8% SA [9]. In addition, these ratios were different between [6] Melliawati, R. (2008). The evaluation of carrier material for
local producers, although these chemicals must be used incraesing qualities of gel inoculum for nata-de-coco.
carefully because cellulose will be process to food. Hence, Biodiversitas, 9: 255-258.
the results of studies that only addition of DAP, without SA [7] Budhiono, A., B. Rosidi, H. Taher and M. Iguchi (1999).
to medium were very significant in production, so both Kinetic aspects of bacterial cellulose formation in nata-de-
saving to spend on chemicals and more biological safety. coco culture system. Carbohydrate Polymers, 40: 137-143.

[8] Nguyen Thi Hien (2006). Production of sodium glutamate and

4. Conclusions traditional fermentation products. Science and Technology
Publishing House, Hanoi, Vietnam (in Vietnamese).
Eight Acetobacter strains was isolated and identified from
4 nata fermenting medium samples. Colonies of all strains [9] Nguyen Thuy Huong and Pham Thanh Ho (2003). Selection
of Acetobacter xylinum for cellulose production in different
were circle, swollen and yellowish color. Some strains have media at pilot scale. Vietnam Journal of Applied Genetics, 3:
mucous structure. Cells of all strains were short and long rod 49.
shaped. Cellulose production by Acetobacter sp. N was
sufficient from mature coconut water with initial sugar [10] Sudsakda, S., W. Srichareon and W. Pathom-Aree (2007).
Comparision of three enrichment broths for the isolation of
concentration of 5°Brix, culture inoculum of 6 log cells/mL, thermotolerant acetic acid bacteria from flowers and fruits.
and pre-incubated in 60 h (233.15 g/300 mL). The addition of Research Journal of Microbiology, 2: 792-795.
both DAP and SA (248.23 g/300 mL) or only DAP (247.70
g/300 mL) gave higher yields of cellulose production. [11] Kaderel, T. T., T. Miyamoto, R. K. Oniang`o, P. M. Kutima
and S. M. Njoroge (2008). Isolation and identification of the
genera Acetobacter and Gluconobacter in coconut toddy
Acknowledgments (mnazi). African Journal of Biotechnology, 7: 2963-2971.

This research was jointly supported by the Advanced [12] Moryadee, A. and W. Pathom-Aree (2008). Isolation of
thermotolerant acetic acid bacteria from fruit for vinegar
Program in Biotechnology, Can Tho University, Vietnam and production. Research Journal of Microbiology, 3: 209-212.
the New Core-to-Core Program (2014-2019).
[13] Toda, K., T. Asakura, M. Fukaya, E. Entani and Y. Kawamura.
(1997). Cellulose production by acetic acid-resistant
References Acetobacter xylinum. Journal of Fermentation and
Bioengineering, 84: 228-231.
[1] Nugroho, D. A. and P. Aji (2015). Characterization of Nata de
Coco Produced by Fermentation of Immobilized Acetobacter [14] Masaoka, S., T. Ohe and N. Sakota (1992). Production of
xylinum. Agriculture and Agricultural Science Procedia, 3: cellulose from glucose by Acetobacter xylinum. Journal of
278-282. Fermentation and Bioengineering, 75: 18-22.
[2] Suwannapinunt, N., J. Burakorn and S. Thaenthanee (2007).
Effect of culture conditions on bacterial BC (BC) production