Production of Exopolysaccharide by Bifidobacteria and Its Viscometric Analysis IJB
Production of Exopolysaccharide by Bifidobacteria and Its Viscometric Analysis IJB
Production of Exopolysaccharide by Bifidobacteria and Its Viscometric Analysis IJB
INNSPUB
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Sham Lal1*, Nisar Ahmed Kanhar1, Pardeep Kumar1, Om Parkash1, Anwar Hussain
Phulpoto1 Majid Ali Maitlo1, Muzafar Hussain Sirohi2, Ameer Ahmed Mirbahar2,
Abdul Majid Ansari3, Safdar Ali Ujjan4, Javed Ahmed Ujjan4, Majeeda Ruk4, Sapna4,
Hamid B. Ghoddusi5
1
Institute of Microbiology, Shah Abdul Latif University, Khairpur, Sindh, 66020, Pakistan
2
Department of Botany, Shah Abdul Latif University, Khairpur, Sindh, 66020, Pakistan
3
Department of Biochemistry, Shah Abdul Latif University, Khairpur, Sindh, 66020, Pakistan
4
Department of Zoology, Shah Abdul Latif University, Khairpur, Sindh, 66020, Pakistan
5
Microbiology Research Unit, School of Human Sciences, London Metropolitan University, London
N7 8DB, UK
Abstract
The exopolysaccharide production by three Bifidobacterium strains was evaluated by optimizing two parameters
(temperature and time). In addition, the role of EPS on viscosity of solutions was observed. Bacterial cultures
were grown in MRS broth supplemented with 0.5 % (w/v) cysteine HCl in anaerobic conditions. Among the
different time (24 h, 48 h and 72 h) and temperature (30ºC, 37ºC and 42ºC) conditions, high EPS production
was observed at 42 ºC after 72 h of incubation. At these conditions maximum amount of EPS was produced by
Bifidobacterium breve 11815 with the yield of 94.64 ± 0.25 ug/ml, followed by B. longum 11818 and B. animalis
ssp. lactis Bb12 with the yield of 90.53 ± 0.34 ug/ml and 58.8 ± 0.25 ug/ml respectively. Viscometric analysis of
EPS performed by viscometer showed highest viscosity of milk (23 ± 1.41 cp) by using EPS produced by B.
animalis ssp. lactis Bb12. This study suggests that the foods in which bifidobacteria are used as starter culture
should be incubated at 42 ºC to obtain maximum probiotic dose and EPS. Finally, EPS produced by B. animalis
ssp. lactis Bb12 can be used for reducing syneresis and improving texture and viscosity of food products.
* Corresponding Author: Sham Lal [email protected]
Effect of temperature and time on growth of 0.13 at 42 ºC after 72 h of incubation (Fig. 1.). For B.
Bifidobacterium strains longum 11818 high cell growth (7.5 ± 0.04 log) was
High cell growth 7.56 ±0.02 log was observed for B. observed at 37 ºC followed by 7.37± 0.07 log at 42 ºC
breve 11815 at 37 ºC temperature followed by 7.51 ± (Fig. 1.). In contrast to above strains for B. animalis
ssp. lactis Bb12 high cell growth (7.4 ± 0.005 log) was determine the initial inoculum added in food product
observed at 42 ºC followed by 7.23 ± 0.005 log at 37 and also to evaluate the viability of bifidobacteria
ºC (Fig. 1.). ANOVA showed that there was significant during storage time period.
difference in growth rate (p ˂ 0.0001) among these
three strains. Effect of temperature and time on EPS production by
Bifidobacterium strains
The influence of temperature and time on the
production of EPS by Bifidobacterium strains was
also determined by incubating the organisms at
different temperature (30 ºC, 37 ºC and 42 ºC) over a
range of time (24 h, 48 h and 72 h). EPS production
was quantified with spectrophotometer by using
glucose as standard. Calibration curve was prepared
(Fig. 2.).
degradative enzymes which hydrolyze the EPS as higher cell count and higher EPS production at 37 °C
reported by Degeest et al., (2001). However, there is for Streptococcus thermophilus and L. casei strains
no any report about EPS degradation of EPS by (Gancel and Novel, 1994; Mozzi et al., 1996). In our
bifidobacteria, therefore this should be researched. study no EPS was produced at 30 ºC by
High production of EPS at high temperature could be bifidobacteria. In contrast in previous studies
due to the fact that some bacteria produce EPS in significant amount of EPS was produced by
stress conditions as reported by Prasanna et al., mesophilic EPS producing lactic acid bacteria (Petry
(2012). In this study high production of EPS was et al., 2000 and Sanchez et al., 2006).
observed at 42 ºC, where as some researchers got the
In this study time and temperature were optimized to EPS and this was confirmed by Grobben et al., (1997)
produce the high amount of EPS by bifidobacteria. and reported that under controlled condition of pH,
However, EPS production could be improved by significantly higher amount of EPS is produced
manipulating medium composition by adding compared to batch fermentations with uncontrolled
different sugar source (Audy et al., 2010), providing pH. In another study, De Vuyst and Degeest, (1999),
different environmental conditions such as oxygen reported that in some cases controlled pH conditions
tension (Gamar-Nourani et al., 1998) and different produced more exopolysaccharide than that of
amount of carbon dioxide (Ninomiya et al., 2009). supplementation with nutrients and molecular
structure and sugar composition of the EPS was also
There are certain limitations of the method by which dependent upon fermentation conditions.
EPS was quantified in this study. Phenol sulphuric
acid method was used to quantify the EPS by using Viscometric analysis of EPS
spectrophotometer; this method is used to detect the Highest viscosity, 23 ± 1.41 cp was observed for EPS
carbohydrates in sample. However there is possibility produced by B. animalis ssp. lactis Bb12, followed by
that EPS produced by bifidobacteria may also contain equal viscocity 19 ± 0 cp by B. breve 11815 and B.
other components such as proteins, peptides, and longum 11818 (Table 1). Viscometric analysis of EPS
phosphate. Therefore research is needed about solutions was recorded at 25 ºC. Due to very low
chemical analysis of EPS. Moreover change in pH of difference in values, ANOVA could not analyse the
the medium could also effect on the production of data. Although viscosity was measured at different
shear rate such as 50, 60 and 100 no change in their study viscosity of the EPS solutions ranged from
viscosity was detected at these shear rates. 15 to 32.1 cP in complex medium and 26.1 to 100 in
minimal medium at pH 7.0, but by decreasing pH of
the solutions, maximum viscosity was recorded with
16600 cP in complex medium and 3000 cP. Other
factors such as temperature, salt concentration of
medium, protein content in the medium, time of
fermentation, EPS conformation or interactions
between EPS and growth media microstructure could
also effect on the viscosity.
Conclusion
Initially, to observe the role of EPS produced by
Bifidobacterium strains in enhancing the viscosity of
medium, EPS was dissolved in distilled water and
viscosity was measured with viscometer (Brookfield,
DV-E, USA). Unfortunately, viscometer could not
detect the viscosity of solution. This could be because
the viscosity of solution was very less than the lowest
detection limit of the viscometer used. To overcome
this problem distilled water was replaced by whole
milk (liquid) but yet there was same problem. Finally
milk was supplemented with 6 % (w/v) skimmed milk
and then viscometer could detect the viscosity of
Fig. 3. Production of EPS by Bifidobacterium strains
solutions and effect of EPSs on viscosity of milk was
at various temperature, A) 37°C, B) 42°C.
measured.
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