J. Biomed. Ther. Sci. 2017, 4(1), 7-10 . Article.

Journal of Biomedical &

Therapeutic Sciences

Optimization of extraction and production methods of Hyaluronic Acid (HA)

from Streptococcus zooepidemicus
Surajit Baksi,* Mukandar Khan, Mamta Panchal, Nirav Rao

Hester Bioscience Limited, Ahmedabad-380006, Gujarat, India.

Received on: 08-May-2017, Accepted on: 15-June-2017, Published on: 20-June-2017

ABSTRACT

Hyaluronic acid (HA) is a biopolysacharride present in connective tissues of body. It has high significant economic concern due to its high
use in medical, cosmetic and food industries. It is present in several resources like rooster combs, eye and umbilical cord. Due to its good
chemical and physical properties, it is used in ophthalmic and osteoarthritic surgeries. Presently, it is produced from animal and bacterial
sources. But, the percentage yield is very less at large scale. In present study, effort was done to increase yield of HA from Streptococcus
zooepidemicus, through modification of the fermentation and extraction process. The cell concentration and HA concentration was
measured which showed that HA concentration increased with induced pH stress.
Keywords: Hyaluronic acid, Production, Bacteria, Extraction

in medical, cosmetic and food industries production. Due to its

INTRODUCTION
Hyaluronic acid (HA) is a high-molecular weight
biopolysaccharide, discovered in 1934 by Meyer and Palmer1 in
the vitreous of bovine eyes. It is found in connective tissues and
is concentrated in synovial fluid, the vitreous fluid of the eye,
umbilical cords and chicken combs. HA is a linear biopolymer
with repeating disaccharide units of D-glucuronic acid and N-
acetyl glucosamine, which are linked alternately by glycosidic
linkages.2 HA is an important biological compound and popular
*Corresponding Author: Dr. Surajit Baksi
Hester Bioscience Limited, Ahmedabad-380006, Gujarat, India
Tel: +91 9712838961
Email: drsbaksi.vm@gmail.com
Cite as: J. Biomed. Ther. Sci., 2017, 4(1), 7-10.
©IS Publications JBTS ISSN 2394-2274
http://pubs.iscience.in/journals/jbts
specific properties like high moisture retention, biocompatibility
and viscoelasticity; and use, it has gained high commercial
value in pharmaceutical, biomedical and cosmetic industry
worldwide. It is used in drug delivery, ophthalmic surgery,
osteoarthritis treatment and tissue engineering.3-7 Use of HA is
also mentioned in cancer, wound repair, inflammation, cell
migration and skin healing.8-12 Traditionally, HA is extracted
from rooster combs. In recent years, HA from microbial
fermentation is receiving increased attention for avoidance of
cross-species viral infection and the use of hazardous
solvents.13-15 Currently, large scale production of HA involves
extraction from animal tissues as well as the use of bacterial
expression systems in Streptococci. However, due to concerns
over safety, alternative sources of HA have been pursued which
include the use of endotoxin-free microorganisms such as
Bacilli and Escherichia coli.16-18 Due to high demand of HA in
medical and cosmetic field, rapid and safe production is
strongly desired. Cell free and non-pathogenic bacterial
expressions are required to produce HA, but they have limited

Journal of Biomedical and Therapeutic Sciences J. Biomed. Ther. Sci., 2017, 4 (1), 7-10 7
capacity and hence, in present study, effort was done to increase
the yield by modification of fermentation and extraction
processes.19 Streptococcus zooepidemicus is the bacteria which
is used for large scale production of HA and produces HA with
proper physical and biological properties.20
Here we report the results of high yield production of HA
from Streptococcus zooepidemicus by varying the experimental
conditions.
MATERIALS AND METHODS
Micro-organism and media
Streptococcus zooepidemicus (procured from ATCC) was
used in this study. Fresh slants were cultured at 37 °C for 12 h
and used for inoculation. Seed culture medium consisted of
(g/l): glucose 40, yeast extract (20), MgSO4.7H2O (2.0),
MnSO4.4H2O (0.1), KH2PO4 (2.0), CaCO3 (20) and 1 ml trace
elements solution. The trace element solution consisted of (g/l):
CaCl2 (2.0), ZnCl2 (0.046) and CuSO4.5H2O (0.019). The
fermentation medium contained (g/l): glucose (60), yeast extract
(30), K2SO4 (1.3), MgSO4.7H2O (2.0), Na2HPO4.12H2O (6.2),
FeSO4.7H2O (0.005) and 2.5 ml trace element solution (pH 7.2).
Chemically defined medium was autoclaved at 121 °C for 15
min. Glucose and MgSO4.7H2O solutions were autoclaved
separately. Batch fermentation was done in 5 l fermenter.
In 50 ml seed culture medium, loopful of Streptococcus
zooepidemicus culture was inoculated from a fresh slant and put
on a rotary shaker at 200 rev/min and 37 °C for 12 h. The 10%
seed culture was inoculated into a 5 l fermenter containing 2.5 l
fermentation medium. Agitation was done by 3 6-bladed disk
turbines at the speed of 200 rev/min and pH was automatically
controlled by adding 5 mol/l NaOH solution. Temperature was
maintained at 37 °C and aeration rate was 2 l/min. For inducing
pH stress, pH was set at 7 for first 7 hours. Then it was switched
to higher value (8.0, 8.5 and 9.0) for certain period time (0.5,
1.0 and 1.5 h) and back to 7.0 for 1 h until the end of
fermentation at 18 h (one pH switch cycle every 2 h). Same
fermentation process was carried out using control medium,
where pH was maintained at 7.
Extraction
HA produced in fermentation broth was purified as per
method described in literature21-23 and modifications in its
purification steps were made to improve the recovery and purity
of HA like rpm of centrifugation and reagents concentration.24
Carbazole method was used to estimate crude HA present in
fermentation broth at every 2 hours.25 The fermentation broth26
containing HA was precipitated with 2-propanol (1:1 v/v). The
precipitates were redissolved in 0.15 M NaCl solution to reduce
the viscosity. The nucleic acids and bacteria derived proteins
present in crude samples were removed by lowering the pH of
the broth from 6 to 2 with addition of trichloroacetic acid
(0.2%) and treated with charcoal (2%) for 50 min, followed by
centrifugation at 8000 rpm for 30 min at 4ºC. The solution was
passed from 0.45 μm filters, after removal of cells and charcoal.
Then it was diluted fivefold and further purified by
ultrafiltration in diafiltration mode using a 300 kDa cut off
cassette. Finally, the retentate containing HA sample was
Journal of Biomedical and Therapeutic Sciences
concentrated to original volume. White fibrous aggregates of
pure sodium hyaluronate were precipitated and dried.
Cell concentration was measured from the optical density
(OD) of the broth at every 2 hours at 660 nm with a
spectrophotometer (Speccord 210 plus, Analytikjena,
Germany). The correlation of OD with dry cell weight (DCW,
in g/l) was DCW = 75.44*OD + 1.85.
RESULTS AND DISCUSSION
The dry cell concentration (DCW) was measured every 2
hours interval and obtained as shown in figure 1. Up to 6 h of
fermentation, the DCW was similar in both the media (under
control experimental conditions and under pH stress set up as
experimental conditions were same upto this time duration).
The change in the DCW was observed after changes in the pH
were done at 7 h and successive pH stress. DCW increased to
10.29 g/l in pH stress and 15.31 g/l in control condition. The dry
cell concentration was less under the changed pH stress
conditions compared to the normal experimental conditions. It
was observed that DCW of control was 48.7% higher than pH
stress experimental conditions. Similar observations were found
by Liu et al., 200827, where shift in the alkaline conditions
resulted in less concentration of cells compared to controlled
conditions28
18
pH stress
16
Control
14
12
DCW (g/l)
10
8
6
4
2
0
0 2 4 6 8 10 12 14 16 18
Time (h)
Figure 1: Dry Cell Weight (DCW) at every 2 h interval
HA concentration was measured by carbazole method at
every 2 h interval as shown in figure 2. Two steps were
followed in the method: 1. Hydrolysing HA into uronic acid and
glucosamine with 0.025M sodium tetraborate in sulphuric acid,
2. Coloring uronic acid with carbazole. In initial two hours there
was no increase in HA concentration in both media. The
concentration increased after two hours and it was similar up to
6 hours. Difference in HA concentration was observed when pH
stress was induced in one medium, which led to higher
concentration in pH stress medium up to 18 hours. Maximum of
6.8 g/l concentration of HA was found in pH stress medium,
while it was 5.2 g/l in control medium. There was 30.7% higher
J. Biomed. Ther. Sci., 2017, 4 (1), 7-10 8
production of HA found in pH stress condition and changed in
extraction procedure than control medium. In other researches
also, pH stress induced higher production of HA than in control
condition, where pH of 7 was maintained for 6 hours, while in
present study the duration was 7 hours.14,26-30
8
pH stress
7
HA concentration (g/l)
Control
6 5. G. Kogan, L. Soltes, R. Stern, P. Gemeiner. Hyaluronic acid: a natural
5 applications. Biotechnol. Lett. 2007, 29, 17–25.
4 and hyaluronic acid. Altern. Complement. Ther. 2008, 14, 78–84.
3 information-rich system. Eur. J. Cell. Biol. 2006, 85, 699–715.
2 infections and status of current progress in treatment. Chem. Biol. Lett.
1 9. B. Chhikara. Prospects of Applied Nanomedicine. Appl. Nanomed.
0 10. R. Upadhyay and A. Khed. A comparative study of inhibitive effects
0 2 4 6 8 10 12
Time (h)
Figure 2: Hyaluronic Acid (HA) concentration at every 2 h
interval
There was no Hyaluronic acid produced in first two hours of
experiment in both the controlled and induced pH stress
conditions. The yield was similar in controlled and under pH
stress from 2 h to 6 h of experiment as experimental conditions
were same in both the setups. The pH stress was induced in the
experiment set up for pH stress at 7 h. At 8h, the yield of
Hyaluronic acid became more under pH stress conditions of the
experiment compared to the controlled experimental conditions.
After this, the yield of hyaluronic acids kept on increasing and
was proportionately more under pH stress conditions as
measured at the interval of 2h (upto 18 h). The changed
extraction procedure is useful in extraction of higher amount of
the hyaluronic acid.
CONCLUSION
Conventional methods of production of HA give less yield
and there are concerns of safety in the present methods of
production. Previous research has been done on production of
HA from various sources of animal and bacterial cultures.
Present study was an effort to increase the yield of HA from
bacteria with modification in extraction process. Here with the
change in experimental condition, change in pH stress duration
and extraction gave higher yield of Hyaluronic acid. The pH
stress production of Hyaluronic acid from Streptococcus
zooepidemicus and extraction with modified reported procedure
(as in experimental section) could be applied for higher
production of Hyaluronic acid.
Journal of Biomedical and Therapeutic Sciences
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