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Biochemical characterization and antibacterial properties of fish skin mucus of fresh water fish, Hypophthalmichthys nobilis

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Objective: The present study was undertaken to characterize the biochemical composition and antibacterial activity of skin mucus of fish Hypophthalmichthys nobilis against different human and fish pathogenic bacterial strains viz. Klebsiella pneumonia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus and Aeromonas hydrophilla. Methods: Skin mucus of fish H. nobilis was collected by skin scarping method. Antibacterial activity of mucus extract was carried out by agar well diffusion method and measured in terms of zone of inhibition(ZOI) in mm. Antibacterial activity of mucus extract was then compared with two antibiotic amikacin and chloramphenicol. Minimum inhibitory concentration (MIC) of skin mucus extract was also determined. Results: The biochemical characterization of epidermal mucus extract revealed the presence of proteins as a major component (265±2.64 μg/ml) followed by carbohydrate content (63.66±0.88 μg/ml) and lipid content (0.0077±0.66 g/ml) respectively. Remarkable antimicrobial activity against all the selected microbial strains was observed. Zone of inhibition (ZOI) shown by crude mucus extract against all the bacterial strains was found to be significantly higher than higher than Chloramphenicol. Conclusion: The present study opined that skin mucus of this fish can be used as potential antimicrobial components.
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BIOCHEMICAL CHARACTERIZATION AND ANTIBACTERIAL PROPERTIES OF FISH SKIN
MUCUS OF FRESH WATER FISH, HYPOPHTHALMICHTHYS NOBILIS
Original Article
ANIL K. TYOR, SUNIL KUMARI
Fish and Fisheries Laboratory, Department of Zoology, Kurukshetra University, Kurukshetra 136119
Email: akumar@kuk.ac.in
Received: 05 Feb 2016 Revised and Accepted: 20 Apr 2016
ABSTRACT
Objective: The present study was undertaken to characterize the biochemical composition and antibacterial activity of skin mucus of fish
Hypophthalmichthys nobilis against different human and fish pathogenic bacterial strains viz. Klebsiella pneumonia, Pseudomonas aeruginosa,
Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus and Aeromonas hydrophilla.
Methods: Skin mucus of fish H. nobilis was collected by skin scarping method. Antibacterial activity of mucus extract was carried out by agar well
diffusion method and measured in terms of zone of inhibition(ZOI) in mm. Antibacterial activity of mucus extract was then compared with two
antibiotic amikacin and chloramphenicol. Minimum inhibitory concentration (MIC) of skin mucus extract was also determined.
Results: The biochemical characterization of epidermal mucus extract revealed the presence of proteins as a major component (265±2.64 µg/ml)
followed by carbohydrate content (63.66±0.88 µg/ml) and lipid content (0.0077±0.66 g/ml) respectively. Remarkable antimicrobial activity against
all the selected microbial strains was observed. Zone of inhibition (ZOI) shown by crude mucus extract against all the bacterial strains was found to
be significantly higher than higher than Chloramphenicol.
Conclusion: The present study opined that skin mucus of this fish can be used as potential antimicrobial components.
Keywords: Hypophthalmichthys nobilis, Microorganism, Fish skin mucus, Antibacterial activity, ZOI, MIC
© 2016 The Authors. P ublished by Innovare Academic Scie nces Pvt Ltd. This is an o pen access articl e under the CC BY licens e (http://creativecommons.org/licenses/by/4.0/)
INTRODUCTION
The worldwide emergence of E. coli, K. pneumonia, Haemophilus sp.,
S. aureus and many other ß-lactamase producers have become a
major therapeutic problem. Hospitals worldwide have become
literal breeding grounds for some of the most deadly bacteria. It is
now estimated that half of S. aureus strains found at many medical
institutions are resistant to antibiotics such as Methicillin [1].
Indiscriminate use of antibiotics has become the major factor for the
emergence and dissemination of multi-drug resistant strains of
several groups of microorganisms [2]. Even though pharmacological
industries have produced the number of new antibiotics in the last
three decades, resistance to these drugs by microorganisms has
increased because microorganisms are highly efficient at modifying
or acquiring genes that code for the mechanism of multidrug
resistance [3]. Compounding the problem of multidrug resistance, it
is necessary to search for new antimicrobial agents to combat
infections and overcome the problem of resistance and side effects
of currently available antimicrobial drugs. Several attempts have
been made for exploring new antimicrobial drugs from natural
sources including plant and animal products. In modern society, zoo-
therapy constitutes an important alternative among many other
known therapies practiced worldwide. Zootherapy is the healing of
diseases by use of therapeutics obtained or ultimately derived from
animals [4].
Fishes are a diverse group of animals and comprise almost half the
number of vertebrate species in existence today [5]. Approximately
20 million metric tons of fish by-products are discarded annually
from the world fisheries [6]. Fish by-products are rich in potentially
valuable proteins, minerals, enzymes, pigments or flavors. Fish
mucus, a fish by-product, is a key component of fish innate
immunity. It acts as innate defense barrier of fish skin which
continuously gets replaced and helps to prevent stable colonization
of majority of infectious microbes such as bacteria, fungus into the
fish body [7]. Fish mucus is secreted by epidermal goblet cells and
comprises of mucins and other substances such as inorganic salts,
immunoglobulin, proteins and lipids suspended in water giving it
characteristic lubricating properties [8]. The composition, viscosity,
and rate of mucus secretion vary from species to species and have
been observed to change in response to microbial exposure or to
environmental fluctuation such as hyperosmolarity and pH [9]. Fish
skin mucus has been reported to secrete many antibacterial
peptides [10-11]. Channa striatus is endowed with wound healing,
antinociceptive, platelet aggregation, anti-inflammatory as well as
mild antifungal and antibacterial properties [12]. In addition to
antimicrobial peptides, fish skin mucus also contains C-reactive
protein, lysozymes, lectin, flavoenzyme, immunoglobins etc. which
protects fishes against pathogenic microbes in their surroundings
[13-14]. Antibacterial properties of crude skin mucus from many
fishes have been demonstrated against several human and fish
pathogenic bacteria by many workers [6, 15-16]. H. nobilis is
omnivorous and feeds on larger phytoplankton mostly on algal
blooms [17], thus this species lines with an environment harboring
many infectious microbes. Thus, the present study was focused on
analyzing the biochemical characterization and antimicrobial
activity of skin mucus of H. nobilis.
MATERIALS AND METHODS
Fish collection and acclimatization
Live fish, H. nobilis irrespective of sex, weighing 800-900 grams
were purchased from the nearby fish culture pond and maintained
in F. R. P. tank (1000 L capacity) at Fish and Fisheries Laboratory,
Department of Zoology, Kurukshetra University, Kurukshetra. Half of
the water of the tank was changed on alternate days. Dissolved
oxygen was maintained at a preferable level in the tank with the
help of low-pressure aerators and pumps. The health of fishes was
observed daily, and dead fish or fish with lesions (if any) was
immediately removed. The fish were fed daily at 3% of body weight
with commercial/formulated feed during the acclimatization period.
Fish skin mucus collection
The fish were acclimatized for seven days and kept starved for 24 h
before mucus collection. A collection of mucus was done by 'skin-
scraping' from the body of test subjects. No anesthesia was given
prior to mucus collection. Mucus was taken from 15 fishes dorso
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 8, Issue 6, 2016
Tyor et al.
Int J Pharm Pharm Sci, Vol 8, Issue 6, 132-136
133
laterally by using a sterile plastic spatula. Mucus scraped first was
discarded to avoid any bacterial contamination. Collection of mucus
from ventral region of the fish was avoided to prevent mixing of
urinogenital excreta. Fish skin mucus was placed in vials and kept
frozen at 0 °C until use to avoid bacterial growth and protein
degradation.
Preparation of mucus extracts and biochemical characterization
Two mucus extracts viz. crude mucus extract and aqueous extract
were prepare from the previously preserved mucus. For crude
mucus extract skin mucus preserved from 15 fishes was thawed and
centrifuged at 5000 r. p. m for 5 min. The supernatant was subjected
to qualitative and quantitative assays to estimate the biochemical
constituents. To prepare aqueous mucus extract, collected mucus
was thoroughly mixed with equal quantity of sterilized physiological
saline (0.85% NaCl) and centrifuged at 5000 r. p. m for 5 minutes.
The supernatant was analyzed for biochemical constituents. Protein
analysis was done by Biuret test [18] and Lowry assays [19].
Carbohydrate content was estimated by Anthrone test [20] and
Phenol sulphuric acid reaction [21] and lipid analysis was
performed by free fatty acid test [22] and folch method [23].
Test microorganisms-procurement and maintenance
Antibacterial activity of fish skin mucus extracts was tested for six
human pathogenic bacteria E. coli, K. pneumoniae, P. aeruginosa
(Gram-negative bacterial strains), S. aureus, S. epidermidis and B.
cereus (Gram-positive bacterial strains) and a fish pathogenic
bacteria A. hydrophilla (Gram-negative strain). The bacterial strains
were obtained from Institute of Microbial Technology (IMTECH),
Chandigarh through Department of Biotechnology and Department
of Zoology, Kurukshetra University, Kurukshetra, India. All the
bacterial strains were grown in nutrient broth (0.5% peptone, 0.5%
NaCl, 0.3% beef extract, distilled water, pH adjusted to neutral (6.8)
at 28 °C) under biomedical safety protocols and conditions. 10 ml of
nutrient broth was poured in flask and one loop of target bacteria
was added to the flask and incubated for 24 h at 37 °C in incubator.
Antibacterial assay
In vitro antibacterial evaluation of fish skin, mucus extracts were
assayed by agar well diffusion method [24]. 100 µl culture of
different bacterial strains was spread on different culture plates
containing 15 ml of nutrient agar media [1.5% agar-agar, 0.5%
peptone, 0.5% NaCl, 0.3% beef extract, distilled water, pH adjusted
to neutral (6.8) at 28 °C] by using a sterile cotton swab. Wells were
made with the help of cork borer on the agar nutrient media plates
suitably spaced apart. 100 µl of both mucus extracts, crude and
aqueous were loaded in wells on different plates. The plates were
then incubated at 37 °C for 24 h. The antibacterial activity was assayed
by measuring the diameter (mm) of the inhibition zone formed around
the well [25]. Amikacin and Chloramphenicol drugs were used to
compare the antibacterial effect of fish mucus extracts. NaCl was used
as the negative control along with two antibiotics in the determination
of antimicrobial activity of aqueous mucus extract. Experiments were
conducted in triplicates to determine the reproducibility.
Minimum inhibitory concentration
MIC represents the lowest concentration of an antimicrobial
substance that inhibits the growth of a microorganism. Agar plate
dilution test [26] was performed to determine the MIC of crude skin
mucus extract against all selected microbes. Desired concentrations
of crude mucus extract were prepared by volume/volume dilution
with distilled water and poured in different wells on nutrient agar
plates. Plates were incubated at 37 °C for 24 h.
Statistical analysis
The data so obtained were pooled separately for each parameter and
expressed throughout as means±SE Significant difference in
antimicrobial activity of fish skin mucus of different fishes among
groups was tested by Analysis of variance (ANOVA) Duncan’s
multiple range tests for the experiments. Statistical significance was
settled at a probability value of P<0.05. All statistics were performed
using SPSS Version 11.5 for Windows.
RESULTS
H. nobilis huge secrete amount of mucus which was viscous in
nature. We collected 10-15 ml of mucus/day. In our study, we also
noticed that amount of mucus secretion also vary according to the
season. H. nobilis was reported to secrete more mucus in summer
than in winter.
Biochemical characterization
The presence of proteins in fish skin mucus sample was confirmed
by Biuret test. Change in the colour of skin mucus sample from blue
to purple or violet indicated the presence of proteins. Similarly,
colour change in skin mucus sample from light yellow to blue-green
indicated the presence of carbohydrates in skin mucus of H. nobilis.
Skin mucus sample of H. nobilis gave pink color solution after
addition of dilute alkaline (0.1% NaOH) thus confirming the
presence of free fatty acids in the sample.
The results for quantitative analysis of fish skin mucus have been
presented in table 1.
Table 1: Concentration of biochemical constituents of skin mucus of H. nobilis
Parameters
Value
Protein (µg/ml)
265.00±2.64
Carbohydrate (µg/ml)
63.66±0.88
Lipids (g/ml)
0.0077±0.06
All values are mean±SE of mean, Value of n (No. of experiments) = 6
Table 2: Zone of inhibition (mm) shown by crude mucus extract of H. nobilis against different bacterial strains
Fish
Microbial strains
Crude mucus extract
Amikacin
Chloramphenicol
K. pneumonia
23.58±0.67Bd
33.50±0.458Aab
17.33±0.19Cd
E. coli
32.66±0.56Ba
33.50±0.56Aab
25.66±0.19Ca
P. aeruginosa
27.62±0.62Bc
33.00±0.40Abc
18.33±0.34Cd
S. epidermidis
32.83±0.49Aa
32.00±0.23Ac
21.16±1.29Bbc
B. cereus
29.25±0.57Bb
34.50±0.31Aa
24.43±0.31Ca
S. aureus
26.33±0.96Bc
32.16±0.34Abc
22.16±0.51Cb
A. hydrophilla
25.93±0.71Bc
33.33±0.19Aabc
20.00±0.16Cc
ZOI also include well diameter, All values are mean±SE of mean, Means with different letters in upper case in the same row are significantly
(P<0.05) different., Mean with different letters in lower case in the same column are significantly (P<0.05) different., (Data were analyzed by
Duncan’s Multiple Range test), Value of n (No. of experiments) = 6
Tyor et al.
Int J Pharm Pharm Sci, Vol 8, Issue 6, 132-136
134
Table 3: Zone of inhibition (mm) shown by aqueous mucus extract of H. nobilis against different bacterial strains
Microbial strains
Aqueous mucus
Amikacin
Chloramphenicol
K. pneumonia
13.16±0.49Bbc
26.36±0.93Aa
10.00±00Cb
E. coli
16.55±1.10Ba
25.26±0.62Aab
12.70±0.21Ca
P. aeruginosa
12.73±0.51Bc
25.86±1.38Aa
08.73±0.50Cc
S. epidermidis
16.71±1.04Ba
23.11±1.10Aab
10.10±0.33Cb
B. cereus
15.85±0.94Bab
22.66±0.95Aab
12.13±0.07Ca
S. aureus
11.58±0.50Bc
23.03±1.23Aab
10.06±0.03Bb
A. hydrophilla
16.03±1.16Bab
21.75±1.01Ab
12.76±0.17Ca
ZOI also include well diameter, All values are mean±SE of mean, Means with different letters in upper case in the same row are significantly
(P<0.05) different. Mean with different letters in lower case in the same column are significantly (P<0.05) different, (Data were analyzed by
Duncan’s Multiple Range test), Value of n (No. of experiments) = 6
Table 4: MIC shown by skin mucus extract of H. nobilis against different bacterial strains
Microbial strains
K. pneumonia
E. coli
P. aeruginosa
S. epidermidis
B. cereus
S. aureus
A. hydrophilla
MIC (µl/ml)
25.00
25.00
50.00
50.00
25.00
50.00
50.00
ZOI (mm)
7.00±00
7.00±00
12.73±0.51
16.71±1.04
20.38±6.11
11.58±0.50
16.03±1.16
ZOI also include well diameter, All values of ZOI are mean±SE of mean, Value of n (No. of experiment) = 6 (for both MIC and ZOI)
Antibacterial assay
Effect of crude mucus extract and aqueous mucus extract of H.
nobilis against microbial strains has been presented in table 2 and
table 3 respectively. Both crude and aqueous fish skin mucus
extracts exhibited the ZOI against all tested bacterial strains. Crude
skin mucus extract exhibited maximum ZOI against S. epidermidis
(32.83±0.49 mm) followed by E. coli (32.66±0.56 mm).
In S. epidermidis, ZOI was higher than both the antibiotics, amikacin
(32.00±0.23 mm) and chloramphenicol (21.69±1.29 mm). Crude
mucus extract showed significantly higher ZOI than chloramphenicol
whereas it was insignificant when compared with amikacin (table
2). When the antibacterial activity of aqueous fish mucus extracts
against selected bacterial strains was compared with amikacin and
chloramphenicol, amikacin showed a significantly higher ZOI followed
by fish mucus extract and chloramphenicol. Aqueous fish skin mucus
extract showed maximum ZOI against S. epidermidis (16.71±1.04 mm)
followed by E. coli (16.55±1.10 mm) and A. hydrophilla (16.03±0.16
mm). No ZOI was shown by negative control (NaCl).
In the case of MIC assay, inhibitory concentration of mucus extract
was found to vary for different microbial strains tested. MIC of crude
mucus extract of H. nobilis was found in the range of 25 µl/ml to 50
µl/ml (table 4).
DISCUSSION
Fish skin mucus acts as the first line of defense against microbes [11,
27-28]. Negus (1963) reported that scaleless fishes produce a higher
amount of epidermal mucus than fish with scale [29]. Although
bighead is scaly fish, it also secretes a large amount of mucus. The
quantity and quality of mucus have been reported to differ according
to the season, environmental conditions such as pH, handling stress
and age of fish [30-31] which also supports our findings that amount
of mucus secretion was more in summers as compared to winters.
All these factors play an important role in the susceptibility of a fish
to infection [9, 13].
Crude mucus extract of H. nobilis is constituted of protein as a major
component followed by carbohydrate and lipids. Manivasagan et al.
(2009) investigated that soluble gel of A. maculates was having
12.64 µg/g of protein content,0.08 µg/g of carbohydrate content and
0.005 µg/g of lipid content[32] which also supports our results. Wei
et al. (2010) also reported protein content in both crude and
aqueous mucus extract of Channa straitus [6]. Dhotre et al. (2013)
also characterized the biochemical composition of freshwater fishes
viz. Channa punctatus, Channa gachua, C. carpio and A. dussmieri [33]
and found similar results. Similarly, protein has been reported as a
major component of fish skin mucus of six freshwater fishes viz.
Clarias gariepinus, Channa micropeletes, C. straitus, Oreochromis
niloticus and Hemibagrus nemurus [34]. The presence of protein
content was also investigated in the epidermal mucus of Gaint
snakehead, striped snakehead, Tilapia mossambicus and bagrid
catfish [35]. Our results also go in agreement with the above studies.
Review of the literature reveals that high amount of protein may be
responsible for antibacterial activity shown by fish skin mucus [6,
32, 34-38]. Over the past few years, many antibacterial peptides
have been isolated from different a fish which provides a non-
specific innate immune system to fishes against various pathogen
and help fishes to survive in adverse conditions [36, 38-40].
Crude mucus extract of H. nobilis exhibited strong antibacterial
activity against all selected microbes. The Strong antibacterial
activity of crude fish skin mucus extract has also been observed in
other similar studies [15, 36, 41-42]. Wei et al. (2010) observed that
both crude mucus extract and aqueous mucus extract of C. straitus
showed inhibitory effect against fish pathogenic bacteria A.
hydrophilla (8 mm) and no inhibitory effect against human
pathogenic bacteria E. coli and K. pneumonia [6] whereas crude and
aqueous mucus extract of H. nobilis showed strong antibacterial
activity against both fish and human pathogenic bacteria.
Bragadeeswaran and Thangraj (2011) noticed that crude mucus
extract of eel fish show a strong inhibitory effect against E. coli, P.
aeruginosa and S. aureus and no activity was observed against K.
pneumonia. In the same study, they reported that aqueous mucus
extract was not effective against P. aeruginosa [43]. However, crude
mucus, as well as aqueous mucus extract of H. nobilis, exhibited
antibacterial activity against all the four bacteria tested by
Bragadeeswaran and Thangraj (2011). Loganathan et al. (2013)
reported the inhibitory effect of crude mucus extract of C. straitus
against E. coli, S. aureus and Aermonas sp.[44]. Our findings on crude
mucus extract are in the agreement with above study. Mucus extract
of C. gaucha, C. punctataus, C. carpio and A. dussumieri showed no
ZOI against K. pneumonia [33]. Rao et al. (2015), did not notice the
inhibitory effect of crude and aqueous mucus extract of C.
micropeltes, C. straitus, Chrysichtys nigrodigitatus and T.
mossambicus against E. coli. However, crude and aqueous mucus
extract of H. nobilis exhibited strong antibacterial activity against E.
coli as well as K. pneumonia in contrary [33, 35]. Aqueous mucus
extract of H. nobilis also exhibited strong antibacterial activity
against all pathogenic bacteria taken under study but comparatively
lesser than antibacterial activity shown by crude mucus extract.
Strong inhibitory effect of aqueous mucus extract shown by a variety
of fishes Arius caelatus, A. maculates, C. striatus, Clarias batrachus,
Cynoglossus arel, Hertropneustes fossilis and Mystus gulio [43, 45-48]
support our findings on aqueous mucus extract. Subramanian et al.
(2007) also reported the presence of antimicrobial compounds in
aqueous mucus extract [38]. But in their further studies no
antibacterial activity was observed in aqueous mucus extract of
Tyor et al.
Int J Pharm Pharm Sci, Vol 8, Issue 6, 132-136
135
wider range of fish species including Arctic char (Salvelinus alpinus)
brook trout (Salvelinus fontinalis), Koi carp (C. carpio), striped bass
(Morone saxatalis), haddock fish (Melanogrammus aeglefinus) and
hagfish (Myxine glutinosa)[11]. Strong antibacterial activity
exhibited by aqueous mucus extract of two indigenous fish (Catla
catla and Labeo rohita) and two exotic fishes (Hypophthalmichthys
molitrix and Ctenopharyngodon idella) [49] which also supports our
findings. On comparing the results of Balasubramanian et al. (2012)
with our study, H. molitrix was found to show higher antibacterial
activity than H. Nobilis. Kumari et al. (2011) [16] reported
antibacterial activity of aqueous mucus extract Rita rita and Channa
punctatus against S. arueus (9.75±1.70 mm) but at the same time, no
antibacterial activity was reported against E. coli and P. aeruginosa.
However, our results showed that aqueous mucus extract of H.
nobilis exhibit maximum antibacterial activity against E. coli
(16.55±1.10 mm) followed by P. aeruginosa (12.73±0.51 mm) and
minimum against S. aureus. (2008) [15] also studied the inhibition
effect of aqueous mucus extract of Channa punctatus and Cirrhinus
mrigala against ten pathogenic strains out of which 4 bacterial
strains viz. E. coli, K. pneumonia, P. aureginosa and S. aureus are
common with the present study. Our findings are in agreement with
Kuppulakshmi et al. (2008) [15].
However, contradictory to our result no antibacterial activity was
observed in aqueous mucus extract of 13 fish species [10]. Our
observation on aqueous mucus extract also supports the reports on
the antimicrobial nature of hydrolytic enzymes such as lysoymes,
cathepsin B, trypsin-like proteases in fish mucus [11, 50-52]. Fish
mucus extracts of H. nobilis were found to show strong inhibition
effect against all the microbial strains taken under study. Thus,
suggesting the presence of one or more antibacterial components in
fish skin mucus of H. nobilis. Paradaxin pore forming a peptide, from
Moses fish Pardachius marmoratus [41] and pleurocidin in skin
secretion of winter flounder [36] have been isolated. Ebran et al.
(1999) also reported pore forming properties of protein extracted
from fish epidermal mucus [53]. The action of these antibacterial
peptides is non-specific and rapid; they kill bacteria by a pore
formation in cell membranes followed by disruption and
solubilization [53]. Thus, we may assume that strong antimicrobial
activity of epidermal mucus extracts of H. nobilis against microbial
strains may be due to pore formation ability of their antibacterial
peptides in target cell membrane.
MIC assay was carried out on mucus extracts of some fishes such as
C. statius, Desyatis sephen and Himantura gerradi against many
human and fish pathogenic bacterial strains [6-7]. Rao et al. (2015)
reported the MIC value of Gaint snakehead, striped snakehead,
tilapia and bagrid catfish (C. nigrodigitatus) against different
pathogen ranged from 11.96µg/ml to 31.91 µg/ml. The MIC values
reported in these works was not similar to those obtained in our
study. In our study the minimum concentration of 50 µl/ml of skin
mucus extract of H. nobilis was found to inhibit the growth of human
pathogenic bacteria S. epidermidis, S. areus, P. aeruginosa and fish
pathogen, A. hydrophilla. The minimum concentration of 25 µl/ml
was adequate to inhibit the growth of K. pneumonia, B. cereus and E.
coli. Same fish or different fishes exhibited different antibacterial
activity against different or same bacterial strains. This may be due
to difference in their age, geological and physiological conditions.
Thus, skin mucus extract of H. nobilis needs to be characterized
further, and can be explored as a potent antimicrobial against
infectious bacteria.
CONCLUSION
The present findings suggest that epidermal mucus of H. nobilis is a
good source of antimicrobial compounds. This antimicrobial activity
might be due to antimicrobial proteins present in epidermal mucus
as protein was found to be the major component of mucus. The
epidermal mucus extracts of H. nobilis showed a different zone of
inhibition against different bacterial strains. Thus, indicating
antimicrobial activity of skin mucus of H. nobilis. Further, a detailed
investigation is required for purification and characterization of
specific antimicrobial components of epidermal mucus so that it may
be utilized as potent anti microbe.
ACKNOWLEDGEMENT
The authors are thankful to Department of Zoology, Kurukshetra
University for providing all necessary facility for the study. We are
also grateful to Dr. Sunita Dalal for providing us microbial strains.
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest regarding the
publication of this paper
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... The protein concentration in the bioactive crude extract was found to be 3.9 mg/mL ± 0.16. Several studies carried out on various fishes have also reported the presence of protein content in both crude and aqueous mucus fish extracts which support our results (Wei et al., 2010;Dhotre et al., 2013;Tyor and Kumari, 2016). This total protein content of bioactive crude extract (3.9 mg/mL ± 0.16) was found to be high when compared with earlier studies where the protein content of acidic extract in tilapia was 239.3 ± 7.8 μg/mL, striped snakehead with 53.40 ± 2.00 μg/mL and Hypophthalmichthys nobilis with 265.0 ± 2.64 (Tyor and Kumari, 2016;Rao et al., 2015). ...
... Several studies carried out on various fishes have also reported the presence of protein content in both crude and aqueous mucus fish extracts which support our results (Wei et al., 2010;Dhotre et al., 2013;Tyor and Kumari, 2016). This total protein content of bioactive crude extract (3.9 mg/mL ± 0.16) was found to be high when compared with earlier studies where the protein content of acidic extract in tilapia was 239.3 ± 7.8 μg/mL, striped snakehead with 53.40 ± 2.00 μg/mL and Hypophthalmichthys nobilis with 265.0 ± 2.64 (Tyor and Kumari, 2016;Rao et al., 2015). A protein concentration of 1.2 mg/mL ± 0.027 was found in the crude extracts of epidermal mucus and epidermis of climbing perch Anabas testudineus which is less than the protein concentration of epidermal mucus and epidermis of C. carpio observed in the present study (Al-Raheed et al., 2018). ...
... Similarly, the growth of Klebsiella pneumoniae was found to be inhibited by fish mucus extracts (Hellio et al., 2002). Previous studies have shown a variety of antimicrobial proteins such as paradaxin and pleurocidin from fish mucus that were potentially involved in the protective function against invading pathogens (Cole et al., 1997;Tyor and Kumari, 2016). These results are not surprising because the mucus is the biological interface between fish and their aqueous environment and is composed of biochemically diverse secretions (Pickering, 1974;Ellis, 2001) that play a role in the prevention of colonization by parasites, bacteria and fungi (Ebran et al., 2000;Lemaitre et al., 1996;Caccamese et al., 1980). ...
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Fish epidermis is rich in different pharmacologically active substances, most of which play a crucial role in immunity. In the current study, β-defensin-like protein 1 with high in vitro antimicrobial activity was isolated and characterized from crude epidermal mucus extract of common carp, Cyprinus carpio L. The crude mucus was screened for antimicrobial activity against five bacterial and four fungal pathogens. Crude mucus exhibited varied antimicrobial activity against all the used pathogens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude mucus extract revealed multiple prominent bands with molecular weights corresponding to 6.9 kDa, 14.5 kDa, 24 kDa, 27 kDa and 48 kDa. Using Sephadex G-50 gel filtration liquid chromatography in conjunction with mass spectroscopy (LC/MS), a single peak with a molecular weight of 6908 Da was isolated and characterized. This peptide showed potent antimicrobial activity against all the five bacterial and four fungal strains used. Leclercia adecarboxylata and Enterobacter kobei were most susceptible with minimum inhibition concentration (MIC) value of 0.017 mg/mL, while Aeromonas sobria was least susceptible with an MIC of 2.24 mg/mL. Among the fungal pathogens, Candida glabrata was most susceptible with MIC value of 0.14 mg/mL, while Aspergillus sp. was least susceptible with MIC value of 4.48 mg/mL. The activity was further confirmed by the time kill assay. The antimicrobial peptide sequence determined from mass spectra was “PQSILVLLVLVVLALHCKENEAVSFPWSCASLSGVCRQGVCLPSELYFGPLGCGKGFLCCVSHF”, which consisted of 64 amino acids. Protein Basic Local Alignment Search Tool (BLASTP) of this sequence revealed 100% homology with the β-defensin-like protein 1, which is the first report of this antimicrobial peptide from epidermal mucus of C. carpio from Kashmir waters.
... While Barbel (Barbus callensis), Japanese eel fish (Anguilla japonica) silver grouper fish (Hypophthalmichthys molitrix), grouper fish (Ctenopharyngodon idella), Atlantic inflatable fish (Scomber scombrus), and Atlantic cod fish (Morhua girl) also main selection in producing of antibacterial activity through antibacterial peptides. The second preferred fish in the study of antibacterial activity is the large-headed fish (Hypophthalmichthys nobilis) and shark hound (Mustellus mustellus) [9,15,16]. Thus, other fish species used in antibacterial activity include tuna (Thunnini) [17], barb fish (Barbonymus schwanenfeldiii) [18], anchovies (Engraulis japonicus) [19] and rainbow trout (Oncorhynchus mykiss) [20]. Bioactive peptides can be obtained from various fish parts, including the muscle, skin, viscera, and bone. ...
... Furthermore, the antibacterial activity of hound shark (Mustellus mustellus) from peptides SHVH-E9, SHVH-EE, and SHVH-P were successfully against M. luteus [45]. In addition, the mucous crust of the large-headed grouper (Hypophthalmichthys nobilis) has antibacterial activity against S. epidermidis and E. coli [16]. The Argentine croaker protein antibacterial peptide (Umbrina canosai) showed inhibition of Gram-positive such as L. innocuo, L. monocytogenes and S. aureus, and Gram-negative, followed by Gram-negative A. hydrophilia and Y. enterecolitica. ...
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By-product removal in fish processing is estimated to be between 25 and 70% due to improper fish production handling and significant problems in the fish industry today. Therefore, one of the ways to manage the raw material of by-product is through protein hydrolysis. However, one of the most effective methods for managing this raw material, which includes skin, bones, heads, and viscera, is to convert their protein into peptides via hydrolysis methods, resulting in fish protein hydrolysate (FPH). FPH has been shown to have bioactive properties such as antibacterial, antihypertensive, antioxidative, anticancer, and anticoagulant properties. Bioactivity could be fully utilised in the future in both the nutraceutical and food industries. Numerous studies have been published on the acceptability of FPH in obtaining bioactive properties from various fish, particularly antibacterial activity. For example, the antibacterial peptide was identified as FPIGMGHGSRPA, consisting of 12 amino acids. Its antibacterial activity was tested against B. subtilis using 800 g/mL ampicillin. The inhibition zone increased with peptide concentration. This review discusses functional bioactive peptides derived from fish protein hydrolysate that can be used as antibacterial agents by inhibit Gram-positive and Gram-negative bacterial growth. It also covers fish species, parts, and hydrolysis methods to maximise yields.
... The mucus in the skin of fish naturally prevents invasion of most pathogenic microbes (includingand fungus) into the body of the fishes (VennilA et al. 2011). Mucus of the fish is extensively released by the goblet cells (present in the skin) and it exclusively consists of mucins and other substances like inorganic salts, immunoglobulin, proteins and lipids (Tyor and KuMAri 2016). The mucus of Claris spp. ...
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K e y w o r d s: fish mucus, antibacterial potentials, susceptibility pattern, spot-on-lawn assay and resistance. A b s t r a c t Fish mucus is known for its antimicrobial activities offering protection to fishes from environmental pathogenic attack. This study evaluates the antibacterial potentials of bacteria isolated from fish mucus of Chrysichthys nigrodigitatus and Solea solea on four clinical isolates: Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Aeromonas hydrophila using the spot-on-lawn method and modified disc diffusion method. Susceptibility pattern of fish mucus bacterial isolates was determined using disc diffusion method. Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Citrobacter freundii and Shigella sonnei were the isolates from the fish mucus varieties. Using spot-on-lawn assay, Proteus mirabilis exhibited the highest antibacterial activities against Bacillus subtilis (4.00±1.73 mm) while on the modified disc diffusion assay, Shigella sonnei exhibited highest antibacterial activities of (7.00±0.21 mm) against Aeromonas hydrophila. Among the isolates, only Klebsiella pneumoniae shown resistance to most of the conventional antibiotics used. Hence, bacteria isolated from fish mucus possess some antibacterial properties against clinical bacterial isolates.
... Huge mucus secretion was observed in all experimental fish species. Similar findings were reported by Wei et al. (2010) and Tyor and Kumari (2016) in many fishes such as C. catla, Channa stratius and H. nobilis. All selected fish species exhibited variation in their mucus secretion and Reverter et al. (2018) too observed the same in their work. ...
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The present work aimed at assessing the antifungal properties of fish epidermal mucus of fresh water fish species viz., Catla catla, Cyperinus carpio and Heteropneustes fossilis against seven pathogenic fungal strains : Aspergillus niger, A. clavatus, A. flavus, Candida albicans, C. tropicalis, C. auris and Mucor ramosissimus. Disk diffusion method was used to analyze the antifungal action of acidic and organic mucus extracts of all selected fishes. Zone of inhibition (ZOI) was also recorded to compare the antifungal effect of these fishes and antibiotic fluconazole against the fungal pathogens. Minimum inhibitory concentration (MIC) was calculated for each pathogenic fungal strain. A potent antifungal effect has been noticed in both acidic and organic mucus extracts of all experimental fishes, even higher than fluconazole in some cases. These results support the presence of antifungal components in fish epidermal mucus, which could be used as an alternative to commercial antibiotics in humans as well as animals.
... The aqueous extract of epidermal mucus was prepared according to the process of Tyor & Kumari [8]. Initially, 10 mL of mucus was transferred to each of ten Eppendorf tubes in equivalent quantities and was mixed with an equal amount of sterilized physiological saline solution (0.85% NaCl) and centrifuged at 5000 rpm for 5 minutes. ...
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Purpose: To investigate the occurrence and antimicrobial effects of certain biochemical compounds in the epidermal mucus secretions of fish and to demonstrate their potential for biomedical applications.Methods: Crude, aqueous, and acidic epidermal mucus samples were collected from live ray specimens. Gas chromatography and gas chromatography-mass spectrometry (GC/MS) analyses were performed to identify the biochemical compounds present in the mucus. The spectrophotometric broth microdilution method was used to determine the antibacterial and antifungal properties of the mucus extracts. The bacterial strains, Bacillus subtilis, Escherichia coli, Enterococcus faecalis, and Klebsiella pneumonia, were used for the tests, as well as the fungal strains, Candida parapsilosis and Candida albicans.Results: GC/MS analysis revealed the presence of several hydrocarbon-derived compounds in the epidermal mucus of the two ray species. The acidic extract of G. altavela epidermal mucus produced a high MIC value, indicating the highest inhibitory effect of 8.64 μL against E. coli, while the crude extract of G. altavela epidermal mucus (41.13 μL against B. subtilis) was the least effective. Conclusion: Epidermal mucus extracts, especially when acid-based, displays strong antimicrobial properties against all the tested pathogens. These findings suggest the plants possess some potential for the development of novel antimicrobial components for applications in medicine. Keywords: Fish, Ray species, Epidermal mucus, Antimicrobial properties, Bioactive compounds
... Consequently, a primary concern of researchers has been value-addition to fish and fish products for sustainable fisheries development to curtail fish wastes [8,9]. Emulsions are systems that could change and tilapia have bactericidal properties due to the presence of anti-microbial peptides [52,53]. Hence, ACM could be a potential emulsifier. ...
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Mucus, a waste product produced when African catfish undergoes stress, has lubricating effects and could be a potential emulsifier. Emulsions are thermodynamically unstable; researchers have documented synthetic bio-polymers as emulsifiers, but its sustainability is in question. This research aims to establish some physicochemical properties of African catfish mucus (ACM) and its effect in soya milk emulsions. A Zetasizer and Turbiscan were used to measure stability, morphology was determined with Transmission electron microscopy (TEM), while functional groups in ACM and ACM-stabilized soya milk emulsions were determined using Attenuated Total Reflection Fourier Transform Infra-red spectroscopy. ACM is a stable hydrogel with negatively charged (−36.2 mV) loosely bound electrons with polar and non-polar portions. ACM concentrations of 1, 3, and 5 g w/w stabilized soya milk emulsions after 180 min of storage. The spectra of stabilized emulsion revealed interactions with soya milk droplets. ACM encapsulated the stabilized emulsion and conferred a kind of cohesive interaction and stability. Turbiscan revealed that the mucin formed strong cohesive connections with stabilized emulsions and the mucin exhibited adhesive properties. ACM is an excellent natural emulsifier with mucoadhesive properties as it encapsulates soya milk to enhance stability.
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Background: The skin mucus layer of fish is endowed with biologics including, Antimicrobial peptides (AMPs) that offer a first line of defence against pathogens. Such peptides can either inhibit bacterial growth or completely kill the bacteria and hence are regarded as a viable alternative to traditional antibiotics, in addressing the ever-increasing incidences of antimicrobial resistance. However, one of the major hurdles to AMPs use is their poor haemolytic profile. As a result, a thorough evaluation of prospective AMPs’ bacterial cell membrane disruption and hemolytic potentials in the early phases of drug discovery is critical. The current study presented cell membrane destruction as well as hemo-compatibility of antimicrobial peptides previously isolated from skin mucus of African catfish, Clarias gariepinus. Methods: A previously isolated antimicrobial peptide in the skin mucus of African catfish, C. gariepinus were profiled using 15% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The electrical conductivity and alkaline phosphatase assays were utilised to measure bacterial cell envelope lysis activity as a classical mode of action of the antimicrobial peptides. Afterwards, fresh Rabbit blood cells were then utilised for in vitro hemolytic assay. Results: The peptides were found to be about 5 kDa molecular weight with, ability to damage the bacterial cell envelope causing significant leakage in periplasmic alkaline phosphatase enzyme and cytoplasmic electrolytes. Even at the highest peptide extract concentration of 100 μg/mL, no significant hemolysis was observed on the fresh rabbit blood cells [3.63%;P>.05], signifying their safety on normal mammalian cells. Conclusion: The findings of this study pointed out that antimicrobial peptides in skin mucus of C. gariepinus are potentially safe source of antimicrobial drug leads; however, further studies are still required to search for possibly maximum dose that is safe to host cells but still effective against infecting bacteria.
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This book covers key areas of Microbiology and Biotechnology. The contributions by the authors include CD4 T-lymphocytes, Propionibacterium acnes, biochemical tests, FAME analysis, 16S rRNA gene sequencing, blue growth, circular economy, microalgae, greenhouse gas emission, environmental protection, aquaculture, Japanese encephalitis Virus, metazoonotic disorder, vaccination, nanotechnology, nanoparticles, UV-Spectrophotometry, Silver Nanoparticles, blood stream infections, biotechnology, gene transfer, tissue culture, plant productivity, pathogenic fungi, antibacterial activity, fish immunity, antiretroviral therapy, antimicrobial susceptibility, Nosocomial infection, opportunistic nosocomial pathogen. This book contains various materials suitable for students, researchers and academicians in the field of Microbiology and Biotechnology.
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This research was directed to understand the bactericidal effect of epidermal mucus of two Asian cat fish species viz. Clarias batrachus and Heteropneustes fossilis. Epidermal mucus extracts (raw and diluted) of both cat fish species were tested against several Gram negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, A. hydrophila) and Gram positive bacterial strains (Bacillius cereus, Staphylococcus aureus, S. epidermidis) and antibacterial results were also compared with two standard antibiotics viz. amikacin and chloramphenicol used as positive control. An A. hydrophila challenge experiment was also performed on all selected test fish species to examine the change in the amount of mucus production and its bactericidal impact.. Both epidermal mucus extracts (raw and diluted) of all selected normal and bacterial challenged test objects showed potent bactericidal effect against all pathogenic bacterial strains taken under study. However, former was more effective than later. Also raw epidermal mucus extracts of both normal and bacterial challenged cat fish species exhibited significantly higher ZOI values against all selected microbial strains than diluted mucus extracts and antibiotic chloramphenicol. Hence, these outcomes have clearly revealed that this cost effective natural product acquired from fishes is the key component of their defensive system. Therefore, it could be utilized as a novel ‘antimicrobial’ in human as well as veterinary sector for combating against several bacterial diseases.
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We have made an analytical study of different parameters involved in a method to cuantify easily the antimicrobial activity in samples of natural products.
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A comparative study of the biochemical structure of mucus and skin of nineteen species of fish was done. Complexity and differences were found in protein (simple and complex protein, isoenzymes), carbohydrate, lipid and mineral structure. Intercellular change of biochemical structure depends on Ihe physiological condition of the fish, stage of maturity, hormonal stress and stress. The last can be caused by both endogenous and exogenous irritants. The effect of external factors (season of the year, salinity in an environment, presence of heavy metals, and conditions of aquaculture) causes shifts in biochemical structure of mucus and skin. It has been shown that the skin and the mucus of fishes are the sources of biogenic biologically active signals—kairomones and pheromones. It was further established that the alarm pheromones and the kairomones (for Cyprinidae) and pheromones of stress (for Salmonidae) have a low molecular weight around 1000. It is assumed that the pheromones and kairomones have a peptide nature. Fish response to stress situations caused by alarm chemical signals was studied in particular. Functions of metabolites of skin and mucus and physiological reactions caused by them in fish have been reviewed. Key words: Mucus; Pheromone; Kairomone; Biogenesis
Article
The enzymatic properties of the bacteriolytic activity was examined on the homogenates of skin mucus in yellowtail Seriola quinqueradiata. The activity was determined by the tubidimetric method using both lyophilized cells of Micrococcus lysodeikticus and Pasteurella piscicida as the substrates. The skin mucus showed high bacteriolytic activity against both cells in distilled water or in low molar buffer. The activity against Micrococcus lysodeikticus was maximal at pH 8.0, 30°C and that against Pasteurella piscicida was maximal at pH 8.0, 50°C. Furthermore, the skin mucus after absorption to chitin hardly showed bacteriolytic activity against Micrococcus lysodeikticus but that against Pasteurella piscicida remained. The bacteriolytic activity of the skin mucus against both cells were completely inhibited by ρ-chloromercuribenzoic acid sodium salt (ρ-CMB), and that against Pasteurella piscicida was 40% inhibited by phenylmethylsulfonyl fluoride (PMSF). Ethylenediaminetetraacetic acid (EDTA) increased the activity against Pasteurella piscicida. From the above-mentioned properties, it was suggested that plural bacteriolytic substances existed in the skin mucus of yellowtail.