The present study was designed to document the antibacterial activity and biochemical composition of the fish skin mucus against different bacterial strains. Nile tilapia was selected for analysis of their mucus sample and the activities of antibacterial against the Gram positive and Gram negative bacteria. Antibacterial potential of fish mucus was evaluated through well diffusion method. Crude mucus extracts showed slightly higher activity of antibacterial activity of Gram negative (Escherichia coli) and Gram positive (Bacillus subtilis) bacteria then the nanoparticle synthesis mucus extract of Nile tilapia. Samples were tested for their hemolytic (0.12 ± 0.01). The fish mucus activities of antioxidants were checked by DPPH (22.64 ± 0.43), reducing power (32.46 ± 0.35), TPC (68.33 ± 1.31), TFC (70.30 ± 2.16) and for biochemical analysis CAT (15.28 ± 0.70), POD (2.19 ± 0.18), SOD (10.05 ± 0.04) and protein estimated (5.09 ± 0.13) was also recorded. Fourier infrared spectroscopy (FTIR) and UV spectra had been used for the characterization of the fish mucus. FTIR results in fish mucus showed the presence of aliphatic primary amines (N-H) and alkenes as a functional group (C=C) at different peaks of spectrum.

Keywords: Fish; Mucus; Nanoparticles; Zinc Oxide

Nile tilapia belongs to the family Cyprinidae [1]. It is native to Pakistan and is establish throughout the streams and in geologically occurring waters, along with fish tanks and fish ponds [2]. Nile tilapia fish is mainly one of the famous fish species. Nevertheless, as compared to other cultured fish species the market value of Nile tilapia is approximately higher than other fishes. This high market value is due to its characteristics attribute such as good flavor, delicious taste, meat texture and the flesh nature. In Bangladesh, this fish species is very famous for its tasty nature in the people of middle to rich. By way of packaging, the Nile tilapia fillet would be high, and it would also be suitable to them [3]. All the fishes live in such surroundings that are rich in microorganisms and are susceptible to attack by opportunistic and pathogenic microbes. The water environment for fish is very competitive due to the presence of large number of microorganisms, so mucus of fish provides protection against these microorganisms and pathogens [4]. The basic natural protected element in fish contains the mucus sheet on the gills, epidermis and digestive tract and also contains the component of blood like phagocytes [5]. Mucus slime makes the fish silky (lubricious). Its slipperiness is due to the presence of immense water constituents and it is also due to the existence of large molecular weight and gel-forming macromolecules [6].

Normally the body of fish is protected by the layer of mucus and this layer is secreted by the many kinds of biological constituents in ectoderm, these are the mucus cells, the sacciform cell, club cells and the epithelial cells [7,8]. The composition of mucus is that it is gel like slimy, viscous and having the diverse mixture of ions, water and enzymes [9-11].The Fish mucus also has some resistance compounds such as immunoglobulin’s, lecithin, interferon, agglutinin, calmodulin, lysozymes, proteolytic enzymes and antimicrobial peptides [12]. The fish mucus having several unwilling defiance parameter which immunoglobulins are, pathogen peptide and harmonize factors that provide both physical and machinal protection [13-19]. For studying progressive begin of human make it to antimicrobial struggle system, skin mucus offers unique opportunities for this purpose, such as antimicrobial peptides in fish (AMPs) has been conserved in the improved vertebrate skin [20,21]. The fish skin mucus is used to solve skin anti-infection defense and to study potential future and clinical applications in dermatology studies.  Because fish skin provides physical, chemical and mechanical barriers to inter-individual communication through metabolism by using visual signals such as pigments, maintain osmotic balance and sensory functions [22]. The main objectives of present study were to do biochemical analysis of mucus and nanoparticle conjugates of ZnO and biological potential of ZnO-Nps to bind with mucus of fish.

Sample Collection

Fish mucus samples of Nile tilapia were collected from the Fish farm of Zoology, Wildlife and Fisheries, University of Agriculture Faisalabad. Different sizes of fish were captured from the pond for the purpose of mucus collection.

Collection of Mucus

The mucus of fish was collected, with the help of spatula directly from the upper side of the fish, not collected from the lower side in order to prevent from the contagion of urine and sperms. For identification, according to the weight of the sampling, the mucus samples were in Eppendorf tubes. These mucus samples were instantly by kept it into box of crushed ice at -200C and shifted to Medicinal Biochemistry Laboratory, Department of Biochemistry, from Department of Zoology Wildlife and Fisheries, University of Agriculture Faisalabad for more studies.

Preparation of Crude Mucus Sample

The extract of crude mucus was prepared from the earlier conserved mucus of fish. Skin mucus saved from the fishes for crude mucus extract and then centrifuge at 1500 rpm for 15 minutes. The supernatant was collected to quantitative qualitative assays for the evaluation of the biochemical components [23].

Preparation of Zinc Oxide Solution

For the silver oxide preparation, ZnO salt of 0.069g was measured by analytical balance and mixed with distill water and make total volume 100ml.

Synthesis of Mucus-Based Zinc Oxide Nanoparticles

Zinc Oxide solution and the purified mucus of Nile tilapia (Oreochromis niloticus) were used for the preparation of mucus based ZnO nanoparticles. All the solutions were prepared in distilled water. Then0.5% (w/v) of homogenous mucus solution was prepared and the concentration of zinc oxide was 1 mm. The mucus-based zinc oxide nanoparticles were synthesized by autoclave the solution [24].
The further details of this process are as below;

Biological Activities of Fish Mucus, and Mucus Based Zinc Oxide Nanoparticles

From the fish skin mucus extract, antibacterial evaluation was determined by agar well diffusion 

Antibacterial Activity
Antibacterial activity was measured Gram positive (Bacillus subtilus) and Gram-negative bacteria (Escherichia coli) through ager well diffusion method [25].

 Antioxidant Activities
Antioxidant activity of fish mucus was determined by using following antioxidant methods.

• Reducing Power

According to the method [26].

• Total Phenolic Content (TPC) Quantification of Fish Mucus

Total phenolic contents (TPC) of fish mucus, mucus bound nanoparticles and ZnO nanoparticles were measured through the process of [27]. In the Gallic acid equivalents (GAE).

• T-C x V / M

Where T – total contents of phenolic compound in mg GAE of mucus extract, C- the concentration of gallic acid calculated from calibration curve in mg/mL, V- the volume of mucus extract in mL. M- The weight of fish mucus extract in grams.

• Total Flavonoid Content (TFC) Quantification of Fish Mucus

The total flavonoid contents (TFC) of epidermal mucus, ZnO nanoparticles and mucus bound nanoparticles was determined through the method [28].

• 2,2-Diphenyl-1-Pierylhydrazyl (DPPH)

For the determination of DPPH, method determined by [26].

Inhibition of Microbial Biofilm

Microbial biofilm inhibition, against Escherichia coli and Bacillus subtitles was performed according to the process of [29].

Hemolytic Activity

The fish mucus hemolytic activity will be measured through the plates of blood agar base. Fish mucus dilutions will be made in (PBS) phosphate buffered saline. Then the plates will be incubated at room temperature [30].

Biochemical Analysis

Biochemical analysis of fish mucus was performed using catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) assays and protein estimation. Catalase activity (CAT), Catalase activity was measure with the method [31]. Superoxide Dismutase (SOD) SOD activity was evaluated with slight changes in accordance with the [32]. Peroxidase activity (POD) The activity of peroxidase (POD) as a hydrogen donator was measured by using guaiacol [33]. Protein estimation [34] method was used to measure the protein.

Characterization

• UV Visible Spectroscopy

Field UV absorbance measurements were done through the process of as same to that determined by [35].

• Fourier Transformed Infrared Spectroscopy (FTIR)

For spectroscopic study of solid part of the fish mucus, nanoparticle based fish mucus. Fourier transformed infrared spectroscopy (FTIR) is used [30].

Statistical Analysis

For statistical analysis, simple mean and standard deviation was applied. Usually, bar graphs were used to express the data to examine the study hypothesis for the characteristics of interest.

Antioxidant Activity of Crude Fish Mucus and Mucus Bound with ZnO Nanoparticles

Antioxidant activity of fish mucus was determined through various assays.

Total Phenolic Contents (TPC)

Plant extract total phenolic contents of are higher than that of fish mucus and mucus based ZnO nanoparticles. Fish mucus has lower phenolic contents and its antioxidant activity is also low. TPC of mucus extract were evaluated by means of Folin-Ciocalten colorimetric procedure, and regression equation of gallic acid calibration curve was used for this purpose. The amount of phenolic per each extract was expressed as gallic acid equivalent. The results obtained from the assay were expressed as mean ± standard deviation of triplicate analyses and are presented [36] [Table 1]. In present research work, total phenolic contents were also measured by Folin-Ciocalten method as this method is fast and simple for quick determination of sample’s phenolic contents. Many earlier reports were found related to the use of this Folin-Ciocalten reagent [37-39].

Total Flavonoids Content (TFC)

In natural compounds, the flavonoids are the vital group, containing vegetables, fruits and cereals. Due to their wide spectrum of biological and chemical activities, including free radical scavenging properties, flavonoids are the most likely important phenolics. Flavonoids are also therapeutic agents against large number of diseases [36,40]. TFC of mucus extract and mucus based ZnO nanoparticles were calculated as catechins equivalents [Table 1].

Reducing Power

The reducing power assay is typically used to determine the capability of an antioxidant to give an electron due to the reducing capacity of a compound. It is the major indicator of antioxidant activity [41]. For the purpose of the sample extract, ability to reduce iron (III) reducing power assay is used. This reducing power assay depends upon the concentration for all samples. Increase in the reducing power of sample or mixture means that there is an increase in absorbance of reaction mixture. The sample which has high reducing power means higher ability to donate electrons and Fe³⁺/ ferric cyanide complex reduced into the ferrous form formation of blue color. The color of test sample changes from yellow to green or blue depends on reducing power of sample. Absorbance was measured at 700nm [Table 1].

Free Radical Scavenging Activity (DPPH)  

DPPH is a well-known free radical which gives strong absorption band at 517nm. The color of DPPH solution is deep violet and its color disappears and changes to yellow when neutralized by antioxidant compound. Free radical scavenging activity of mucus extract was determined by DPPH scavenging assay. The scavenging activities of all samples were concentration dependent. Lower absorbance of the reaction mixture indicated higher DPPH radical scavenging activity [42] (Table 1).

Table 1: Total phenolic contents, Total flavonoid contents and reducing power of mucus and mucus-based ZnO nanoparticles. Given below is the data of triplicates ± SD.

Biofilm Inhibition

Biofilm is a thin layer of mucilage adhering to a solid surface. It comprises group of microorganisms in which cells are attached to the surface. This cell becomes surrounded within a slimy extracellular matrix that is composed of extracellular polymeric substances such as DNA, proteins and polysaccharides [43,44]. In the biofilm form, bacteria are more resistant to various antimicrobial treatments. Bacteria in a biofilm can also survive harsh conditions and withstand the host’s immune system. The purpose of this activity was to find out the potential of fish mucus extract and mucus based ZnO nanoparticles to inhibit biofilm formation. At first, both samples were tested for their antibio film activity and results are given in [Table 2]. The resistance of biofilm is due to the occurrence of some polysaccharides and enzymes that cause the molecules inhibition or receptor inhibition in the pathway of quorum (necessary for formation of biofilm). Lectins are important for colonization and bacterial infection and also play significant role in formation of biofilm which have been inhibited by the polysaccharides [45,46].

Antibacterial Activity

Fish mucus extracts were tested for their antimicrobial activities. For this activity two bacterial cultures were selected. E. coli, B. subtilis, these two bacterial cultures or stains were used in antibacterial activity (Table 2) [47]. Studied the antibacterial activities of extracts from fish epidermis and epidermal mucus [Figure 1].

Table 2: Inhibition of Bacillus subtilis and E. coli biofilm by the mucus extracts and mucus-based nanoparticles of ZnO.

Figure 1: (a) Zone if inhibition of crude mucus and mucus based ZnO nanoparticles against B. subtilis. Zone of inhibition of positive and negative control. (b) Zone if inhibition of crude mucus and mucus based ZnO nanoparticles against E. coli. Zone of inhibition of positive and negative control.

The native fish species like Nile tilapia and Catla catla showed highest antimicrobial activity rather than that of foreign fish species like Ctenopharygodon idella and Hypophthalmicthys molitrix [48]. Antibacterial proteins are secreted by the fish that made the fish able to permeabilize the target cell membrane and in this way perform as a protection obstacle. Antibacterial activity is due to the antibacterial glycoproteins that are found in fish mucus capable to destroy bacteria by formation of huge pores in the membranes of the target cells [49].

Cytotoxic Activity

• Hemolytic Activity

This essay is used to check the hemolysis of different samples. EDTA was used to safe the blooding from clotting. The more hemolytic activity was measured in crude mucus extract of Nile tilapia that is 40%. As a positive control Triton-X was used and its percentage hemolysis was 90%. It is obvious that the hemolytic activity of mucus is less than that of the mucus-based ZnO nanoparticles [Table 3] [50]. Studied the hemolytic assay of snail mucus on human red blood cells. He concluded that Some AMPs were found to exert hemolytic activities. Human red blood cells were used to evaluate the peptide's hemolytic capability. The result showed little hemolytic activity was exerted by mytimacin-AF. At the concentration of 5, 10, 20, 40, 80, 160, and 320 μg/ml.

Table 3: Hemolytic assay of mucus and mucus-based Ag nanoparticles. Given data is the average of three replicates ±S.D.

Biochemical Analysis

Biochemical analysis of fish mucus was done by Catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) assays along with protein estimation [51].

Catalase (CAT)

In this case H2O2 was used as a substrate and the decomposition of H2O2 by the catalase enzyme was observed using UV-vis spectrophotometer. The absorbance measured at 240 nm. The catalase activity was measured in mucus, mucus based ZnO nanoparticles and free nanoparticles. The results indicated that the catalase activity is highest in mucus. Mucus based ZnO nanoparticles also exhibit noticeable catalase activity while free nanoparticles showed less activity when compared with other groups as indicated by graph [Table 4].

• Peroxidase Activity (POD)

The POD activity was assayed using guaiacol as a hydrogen donor by measuring the change at 470 nm [Table 4].

• Protein Estimation (TSP)

In protein estimation assay samples were diluted to obtain protein. Bovin serum albumin used as standard. The standard was prepared containing a range of 200 to 2000 micrograms protein (Bovine serum albumin 2 mg/ml in 1000 ul volumes for setting up the standards). The absorbance (OD) was measured at 595 nm with the help of spectrophotometer [Table 4].

• Superoxide Dismutase (SOD)

The reagents used in SOD assay included phosphate buffer (pH 7.5), riboflavin, nitro blue tetrazolium, Triton-X and methionine. After exposure of 15 min in UV light added riboflavin at the end. The absorbance was measured at 560 nm [Table 4] [52] measured the SOD biochemical analysis.

Table 4: TSP, CAT, POD and SOD values of mucus, mucus-based Ag nanoparticles and free nanoparticles. Given data is the value of triplicates ± SD.

Characterization

• UV Spectra

Normal range of UV-vis spectra used is ranged from 190 nm to 1100 nm through which peaks of different functional groups are find. In these spectra the maximum peak observed at 250 nm and the lowest peak observed at 1100 nm (0.5nm). The observed spectrum peak is highest at between 190 and 300 nm but after 300 nm the peaks begins decline. The maximum absorbance is at 250 nm [Figure 2].
 

Figure 2: Graphical representation of UV spectra, Crude mucus.

• Fourier Infrared Spectroscopy

I.    In the FT-IR spectrum different peaks were observed of mucus, binding material and the nanoparticles. Each spectrum of the sample gave different peaks. The peaks observed for dry mucus contain different functional groups at various wavelengths. FTIR spectrum showed functional groups alkane, alkyl amine and alkyl halides at wavelengths of 1434.16, 1107.92, 876.27 and 618.44 respectively.
II.    The FTIR spectrum of mucus-based ZnO nanoparticles showed four peaks at different wavelengths. The functional groups are secondary amines (N-H), alkanes, alkyl ketones and alkyl amine at wavelength of 3361.89, 1635.29, 1316.09 and 1150.33 respectively [Figures 3 and 4].
 

Figure 3: Graphical representation of FTIR spectra of mucus.

Figure 4: Graphical representation of FTIR spectra of mucus based ZnO nanoparticles.

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Corresponding Author: Dr. Naveed Ahmad M, Department of Zoology, University of Sargodha, Pakistan.

Copyright: © 2022 All copyrights are reserved by Naveed Ahmad M, published by Coalesce Research Group. This This work is licensed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.