International Journal of Modern Agriculture

ISSN: 2305-7246
Volume 10 Issue 3, 2021
Isolation and Molecular Characterization of Exo-Polysaccharide
Producing Weissella Confusa from Buffalo Ruminal Gut
Nowshad Wali 1, Hafiz Talha Qayyum2, Fadia Waheed3, Ghadir Ali4, Muhammad Shehbaz5, Shumaila6, Hafiza
Iqra7, Rida Basharat8, Dr Raheela Bano9, Muhammad Nadeem10, Rahid Khan11, Sadia Noor12
1
Department of Microbiology, Hazara University Mansehra
2
supervisor Laboratory Government THQ Sabzazar Hospital Lahore
3
Institute of Microbiology and Molecular genetics Punjab University, Lahore
4
School of Science, Department of Life Sciences, University of Management and Technology, Lahore, Pakistan
5
Drugs Testing Laboratory, Multan
6
Senior Scientist Nuclear Institute of Agriculture and Biology (Niab)
7
Mphil Biotechnology Bhauddin Zakariya University Multan
8
Gulab Devi Educational Complex, Lahore
9
Assistant Professor Microbiology, Gomal Medical College Dera Ismail Khan
10
Faculty of Life Sciences, University of Central Punjab, Lahore
11
Department of Biotechnology and Genetic Engineering, Hazara University Mansehra
12
Department of Zoology, Abdul Wali Khan University, Mardan.

Corresponding Author; [email protected]

Abstract
Lactobacilli are the most important colonizers of the ruminant’s intestinal tract and are an important
source of probiotics. Among Lactic acid bacteria, Exopolysaccharide producing Weissella confusa
has gained considerable interest due to its importance in the food industry and several health
attributes. The present analysis was carried out to isolate and characterize exopolysaccharide
producing W. confusa from buffalo ruminal gut. The initial characterization of the isolate was done
morphologically and biochemically. Molecular detection of the isolate was done by PCR based
amplification of 16s rRNA gene. Temperature, pH, incubation-time and media conditions were
optimized for the enzyme assay. A total of 35 LAB isolates were cultured for exopolysaccharide
production on MRS agar having ruthenium red dye. The pure isolates were also screened for
proteolytic, amylase, lipolytic, and antibiotic susceptibility testing. Two Catalase and Oxidase
negative strains were selected which shows their probiotic potential.. The two strains identified
as W.confusa (by GenBank accession number MK128505, Mk212944), which was based on 99%
nucleotide homology and phylogenetic analysis. Isolates produced amylase, protease, lipase and also
displayed a high resistance range against selected antibiotics. These isolates also showed
antimicrobial activity against pathogens Listeria monocytogenes as well as against Staphylococcus
aureus. The isolates also produce EPS when grown on ruthenium red MRS agar. The temperature
30°C-37°C and pH 4 were found optimum for enzyme assay. This study unravelled the capability
and safety of EPS producing W. confusa for industrial use, and other potential applications.
Key words: Isolation; Molecular identification; Exopolysaccharides; Weissella confusa

Introduction
Buffalos, whose scientific name is "Bubalus bublis," are the biggest nutritionally and commercially
valuable animals in the Bovidae family. The overall buffalo population in the world is estimated to
be around 150 million. They are employed not just for flesh but also for dairy production due to their

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International Journal of Modern Agriculture
ISSN: 2305-7246
Volume 10 Issue 3, 2021
high mass, huge physique, and ease of growth. Kundi, Nili Ravi and Azakheli buffalo are the most
common buffalo species in Pakistan (1). Franzolin and Wright (2016) suggested that the rumen may
contain 300–400 different species of bacteria, while contemporary techniques such as 16s r RNA
gene sequence analysis and real-time polymerase chain reaction (PCR) revealed that up to 2000
species may exist. While some microorganisms are pathogens that cause infection, other microbes,
known as “Probiotics”, are beneficial to the host and help it fight pathogens (2, 3). Escherichia,
bifidobacterium, Propionibacterium, Enterococcus, Weissella Spp and Bacillus are examples of
bacterial probiotics, however the most frequent and effective probiotics are Lactic acid bacteria
(LAB) (4). Different microorganisms have the ability to produce exopolysaccharides (EPS).
exopolysaccharides is made of long polysaccharides chain (5). They may be papered within the cell
or in secretary form (6). More research is being done on EPS, which have a role in the flavor,
texture, perception and mouth feel of fermented foods (7). Weissella genus has a distinctive
phenotypic trait that allows it to produce dextran. As a result, certain strains of Weissella, such as W.
confusa and W. cibaria, have attracted researchers' interest due to their great potential for producing
dextran, as well as heteropolysaccharides, new non-digestible oligosaccharides, and fractions (8).
These are the exopolysaccharides that are the most significant. It also receives attention because of
its prebiotics potentials, which include the ability to improve metabolism and bowel function,
decrease the risk of illness and diarrhoea, and promote the unique gut microbiota, which includes the
Bifidobacteria (9). Prebiotic oligosaccharides are mostly utilized at the industrial level in the
cosmetics, food, and feed sectors, as well as in the clinical setting and as dietary fibre (10, 11).
Dextran and other similar oligosaccharides produced by W. confusa and W. cibaria are utilised in
therapeutic settings, such as animal therapies, to enhance and activate the gut microbial flora and
decrease illness risk (11, 12).
Galle and Arendt reported in 2014 that EPS may be utilized as a replacement for polysaccharides
derived from plants, which are often employed in the food sector as gelling, stabilizing, thickening
and texturizing agents (5). Food-related functions of EPS are important, although they are also
concerned with general health issues, such as the prevention of cancer and ulcers, activation of the
immune system, and reduction of cholesterol (13). EPS is mostly made by LAB, which provides a
possible method for replacing hydrocolloid additives (14). The present study was piloted to isolate
and characterize exopolysaccharide producing W. confusa from buffalo ruminal gut.

Materials and methods

The present research was carried out at the National Agriculture Research Center (NARC) in
Islamabad, Pakistan. From three distinct sites in ChataBakhtwar, Bharakhao, and Rawalpindi, rumen
samples were obtained from 35 different species of younger and healthier Nili Ravi, Kundi and
Azakheli buffalos. Standard microbiological procedures were followed to transfer the samples using
well-labeled, clean, and sterilized Polythene bags or sterilized flasks. Before processing, the samples
were transported to the NIGAB Laboratory and kept in the refrigerator to avoid external
contaminants (15). The bacteria were isolated using the De Man, Rogosa, and Sharpe agar (MRS)
medium. Gram staining and standard biochemical assays were used to confirm the identification of
bacterial isolates. The molecular characterisation of isolated bacterial strains was conducted out
using 16S rRNA gene sequencing. To do this, the isolates were first cultivated on MRS solid
medium plates and then incubated at 37°C for 24 hours. A new colony was chosen with the aid of a

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International Journal of Modern Agriculture
ISSN: 2305-7246
Volume 10 Issue 3, 2021
sterilised toothpick. Colonies were selected and immersed in a 20L TE buffer solution before being
stirred. In a PCR strip tube, a 0.2(ml) suspension was produced. The PCR tube strips were put in a
water bath at 95°C for 10 minutes to extract the DNA from the isolates. After that, the supernatant
was obtained by centrifugation at 13000 rpm for 2-3 minutes. The DNA of the isolated bacterial
templates was found in the supernatant. The pallet was dumped after centrifugation. The 16s rRNA
gene was amplified using the template DNA that was acquired. The supernatant, which included
1µl of bacterial cell template DNA, was used to achieve the required Gene amplification.
Forward primer: (5’AGAGTTTGATCMTGGCTCAG-3’).
Reverse Primer: (5’ACCTTGTTACGACTT3’).
After that, the template DNA (1µl) and master mix (49 µl) were put in PCR tube and the reaction
volume reached to 50 µl (16). For 1-2 minutes, the sample was centrifuged lightly. The PCR strips
were maintained in the thermocycler for 16s rRNA Gene amplification before the reaction was begun
and condition were set according to previous study (17). The amplified DNA was run on gel
electrophoresis and the bands were observed through the Gel Doc System (17). PCR products from
isolated isolates were sent to Macrogen (Korea) for 16S rRNA gene sequencing. With the use of the
online NCBI BLAST, the acquired sequence was compared to the nucleotide database of 16S rRNA
gene sequences. The top BLAST hits' phylogenetic tree was then chosen to create a phylogenetic
tree. The MEGA7 bioinformatics program was used to measure evolutionary relationships and build
a phylogenetic tree. The EPS production ability of isolated strains was tested similar to previous
study (18). All the data was analyzed statistically by using SPSS version 23. A p value of less than
0.05 was taken as significant.

Results
In this research, 35 buffalo rumen samples were obtained from various areas of Islamabad, namely
Bharakhao, Chatabakhtwar and Rawalpindi. In the microbial biotechnology lab, the specimens were
processed and analyzed. MRS (De Man, Rogosa, and Sharpe agar) agar was used to culture all of the
samples. Pure growth was shown on this media by 24 isolates. For identification, the isolates were
further examined by gram staining and biochemical tests (Table 1)

Table 1: Gram staining and biochemical characteristics of isolates

Gram Morphology Oxidase Catalase

Strain ID
staining test test
Rods
NMCC-M2 Gram positive -ve -ve

Rods
NMCC-M3 Gram positive -ve -ve

Cocci
NMCC-M4 Gram positive -ve -ve

Rods
NMCC-M5 Gram positive -ve -ve

Cocci
NMCC-M7 Gram positive -ve -ve

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International Journal of Modern Agriculture
ISSN: 2305-7246
Volume 10 Issue 3, 2021
Rods
NMCC-M8 Gram positive -ve -ve

Rods
fM-13 Gram positive -ve -ve

Rods
fM-14 Gram positive -ve -ve

Short rods
fM-20 Gram positive -ve -ve

cocci
MF-9 Gram positive -ve -ve

Rods
FI-10 Gram positive -ve -ve

The DNA was extracted using a kit that was readily available (FavorPrep cat No-FAVNK001-2).
Using the primers described in the previous section, the 16s rRNA gene was amplified. Figure 1
shows the Gel Doc system used to analyse the amplified DNA sequence. A band size of 1465-
1522bp was used to confirm 16s RNA.

Figure 1: Agarose gel and 16s rRNA amplified bands of isolated strains:
The image above illustrates the amplified sequence of the 16s rRNA gene from isolated strains on an agarose gel. The
16s rRNA gene length in W. confusa strains with accession number (Mk128505) is 1465 base pairs (bp) in M3, whereas
the 16s rRNA gene length in strains with accession number (Mk212944) is 1470 base pairs (bp). The M line depicts a
DNA ladder with various sizes ranging from 600 to 2100 base pairs. The size of the isolated strains corresponded to
1500bp DNA bands. In the provided image M3 indicate lab ID (NMCC-M3) whereas M8 indicates lab ID (NMCC-M8)
(NMCC-M8).
The data was analysed for phylogeny once it was sequenced and trimmed. Only NMCC-M3 and
NMMC-M8 (BLAST, NCBI) showed 99 percent identity with the available sequence of W. confusa
JCM strain accession number NR113258.1 in a sequence similarity search for all identified strains.
ClustalW of MEGA7 was used to do the phylogenetic analysis, and maximum-likelihood was used
as the statistical metric. The fact that both strains have a bootstrap value more than 50 indicates that

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International Journal of Modern Agriculture
ISSN: 2305-7246
Volume 10 Issue 3, 2021
they are fully accurate and very similar to the W. confusa JCM strain. Figure 2 shows a phylogenetic
study of W. confusa in relation to other LAB using the maximum-likelihood technique.

Figure 2: Phylogenetic tree : Based on examination of the 16S rRNA sequence, a maximum likelihood tree with 50
bootstrap values in MEGA7 shows the phylogenetic relationship of W. confusa. The tree was constructed through
MEGA-7 by using neighbour-joining method (16).

EPS Producing Activity

On the MRS medium with a particular indication, the suspicious LAB isolates were
qualitatively tested (ruthenium red dye). Ruthenium red dye is cationic, meaning it attaches to ionic
sites in the bacterial cell membrane, giving it a red colour. EPS-producing bacteria, on the other
hand, do not interact with ruthenium red dye, which results in a white colony on screening medium.
In the case of both isolates chosen, white colonies of isolates were found, indicating favourable
findings as shown in figure 3.

Figure 0: EPS production by the selected isolates

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International Journal of Modern Agriculture
ISSN: 2305-7246
Volume 10 Issue 3, 2021
Discussion
Because of its probiotic potential and many industrial uses, LAB strains capable of generating EPS
have been intensively researched. These EPS-producing LABS may be found in milk, dairy products,
and the rumen gut of ruminants. The present research isolated and identified W. confusa that
produces EPS from the ruminal gut of buffalo in order to evaluate their potential of
exopolysaccharide production. Thirty five samples of buffalo faeces were collected from various
locations throughout Islamabad, Pakistan. MRS medium was used to isolate lactic acid bacteria
anaerobically. Colony morphology was creamy white and silky. The isolates (NMCC-M3 and
NMCC-M8) were classified as round or punctiform. A previous study reported similar results (19).
Gram staining was used to identify the LAB isolates morphologically. They showed purple rods
under a light microscope following Gram staining, which is a hallmark of Gram-positive bacilli.
Only 19 isolates were recognised as Gram-positive rods in our research. In comparison to our study
same results were reported in another study done by Jose, Bunt (20). The isolates were chosen for
further investigation based on biochemical analyses. Only 14 Gram-positive isolates were catalase-
negative, with only 11 showing oxidase negativity among the 19 Gram-positive isolates. Barakat et
al., for instance, found similar findings (21). Different settings were adjusted for the experiment in
order to ensure that LAB's activity was as efficient as possible. At 30°C and 37°C, the LAB strains
grew the fastest. At 45°C, though, no growth was seen. Same findings were reported by another
previous study done by Lee, Park (22). LAB's probiotic capacity to survive within the
gastrointestinal system is shown by their growth at 37°C. The strains were also unable to generate
bacteriocins, among other things. W. confusa strains had previously been shown to be largely
resistant to high salt concentrations, according to the authors. The strain's unique feature of acid
tolerance and survival at low pH allows it to demonstrate its probiotic potential as well as its capacity
to live in unfavourable circumstances (23). The strains in our research exhibited the best
survivability at pH 2 for 2-3 hours. Montoro, Benomar (24) reported that W. confusa was shown to
have similar survival behaviour at pH 2 without decreasing viability. H+-ATPase activity determines
a strain's capacity to withstand high acid concentrations. Only two isolates were chosen for this study
and were tested for EPS activity. On the MRS medium with ruthenium red dye as an indicator, these
two isolates were then tested qualitatively for EPS production. Osmotic stress, biofilm and hazardous
chemicals are all inhibited by exopolysaccharide. They are also suitable for usage in the textile,
culinary, pharmaceuticals, and chemical industries. Both isolates formed white colonies on the
medium, indicating that the EPS test was positive (25). The identification of LAB species is based on
their physiological and biochemical properties, according to the literature. Furthermore, since 16s
rRNA is the most conserved segment across various LAB strains, it is used for phylogenetic analysis.
Exopolysaccharide-producing LAB was isolated from the ruminal gut of buffalo and genotypically
identified using PCR-based amplification of the 16s rRNA fragment. Sequencing was used to
improve the strain's characterisation. After comparing the sequence to other sequences in the
Genbank database, it was determined that it was 99 percent identical to the W. confusa JCM strain
accession number NR113258.1. Other researchers have also reported similar findings (26).

Conclusion
Our study concludes that isolates of W. confusa from the ruminal gut of buffaloes showed unique
exopolysaccharide production activities.

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 International Journal of Modern Agriculture
ISSN: 2305-7246
Volume 10 Issue 3, 2021
Coming scientific research is attempt on probiotics bacteria to explore novel and particular bacterial
strains for the welfare of the different host such as animals and human. So, the future improvement
and scientific research tendency will be.
 More features of W. confusa will need to be determined before such strains may be used in the
industrial zone. Apart from that, the strains' safety must also be assessed.
 Further processed the isolated strains for whole genome sequencing.

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