2.1.1. Plant and fish materials.

The ginger (Zingiber officinale), banana (Musa acuminate), and turmeric (Curcuma longa) peels were sourced from a local market in Taif City. The matured catfish were procured from Wadi Mitna. The fish were washed with distilled water, placed in ice-cooled boxes and taken immediately to the laboratory. Also the lime (Citrus aurantiifolia) fruits were purchased from an orchard in Taif City, Saudi Arabia.

2.1.2. Chemical and equipment.

The methanol, Selenite Cystine (SC) broth, Rappaport–Vassiliadis (RV) broth, Xylose Lysine Deoxycholate (XLD) agar, nutrient agar (NA), Baird-Parker agar (BPA), Potato Dextrose Agar (PDA), Mannitol Egg Yolk Polymyxin (MYP) agar, Aspergillus flavus/parasiticus agar (AFPA), Brilliant Green Agar (BGA) plates, and the ICP–OES system (model: iCAP PRO XPS) was produced by Thermo Fisher Scientific Inc. America. The HPLC system, model LC-W100B, was manufactured by Wincom Co. Ltd., China. Phosphate-buffered saline (PBS), and bromocresol green (BCG) were from Acros Organics (Waltham, MA, USA). Sodium carbonate (Na₂CO₃) was obtained from Merck KGaA, Darmstadt, Germany.

2.2.1 Plant extract and oil preparation.

The collected raw materials were carefully sorted to remove any foreign matter, then oven-dried at 60°C. After drying, they were ground using a laboratory grinder and filtered with a 1.00 mm nylon filter. Then, extracts were prepared in compliance with established standards, using food-grade ethanol (98% purity) as the carrier solvent. The plant material powder was totally immersed in the ethanol at 28 ± 5°C for three days, and the mixture was agitated every six hours for ten minutes. After the three days, the mixture was sieved with a Grade 1 filter paper, and the solution obtained was concentrated in a water bath at 40°C, to evaporate the solvent (ethanol) to obtain a thick crude extract [43]. The food grade ethanol (98% purity) was selected over methanol, because of serious health challenges linked to methanol consumption [44].

Additionally, the essential oils were extracted using the steam distillation approach. This is to preserve the oil’s essential healthful qualities, and avoid the occurrence of chemical residues from the extracting solvents. About 3 kg of pulverized plant material was fed into a distillation chamber, and subjected to steam flow to vaporize the oil component, from the parent material. The vaporized oil was condensed, separated from the water with a separating funnel and dried at 45 ± 2^oC for 20 minutes [45].

2.2.2. Experimental treatment plans.

The concentrations of extracts and oils used in this study were chosen according to the range previously utilized by other researchers for essential oils and extracts [39,37]. The lime extract (LE) was integrated into some of the treatments, as a cross-check to establish if the LE can significantly enhanced the antimicrobial effects of the other treatments. Bio-additives with higher acidity level create favorable conditions, for essential oils and extracts to inhibit pathogenic activity, by increasing the acidity of the treatment medium [46]. Tap water was the solvent composition (carrier medium) for each treatment. The experimental design mix ratio is presented below:

Control:100% tap water (untreated borehole water)

T1:2% (w/w) of Lime extract (LE)

T2:2% (w/w) of ginger peel extract (GPE)

T3:2% (w/w) of turmeric peel extract (TPE)

T4:2% (w/w) of banana peel extract (PPE)

T5:1% (w/w) of ginger peel oil (GPO)

T6:1% (w/w) of turmeric peel oil (TPO)

T7:1% (w/w) of banana peel oil (PPO)

T8:2% GPE + 1% GPO + 2% LE

T9:2% TPE + 1% TPO + 2% LE

T10:2% PPE + 1% PPO + 2% LE

T11:1% GPO + 1% TPO + 2% LE

T12:1% GPO + 1% PPO + 2% LE

T13:1% TPO + 1% PPO + 2% LE

T14:2% GPE + 1% TPE

T15:2% TPE + 1% PPE

T16:2%GPE + 1% PPE

Where; LE – lime extract, GPE – ginger peel extract, GPO – ginger peel oil, PPE – banana peel extract, PPO – banana peel oil, TPE – turmeric peel extract, TPO – turmeric peel oil, T – treatment

2.2.3. Fish samples preparation and storage.

The fish was skinned in the laboratory and the flesh recovered, which was cut into the size of about 100 g. The dripping method was used to apply the treatments to the fish samples, at a temperature of 15 ± 3^oC. Each sample was immersed in the designated treatment dose for 4 hours, and then allowed to drip for 20 minutes before being dried in a laboratory oven (model: 637G, Fisher Scientific Inc., USA) at 115°C to attained a moisture level of about 8%.

The 16 fish specimens (both control and the treated) were kept in a container (31 ± 6^oC, 87 ± 6% RH) for eight weeks (the experimental duration. To protect the fish from direct insect and pest exposure while allowing air circulation between the fish samples and the surrounding air, each container was covered with nylon mesh (0.3 µm).

2.2.4. Quality assurance (QA) and quality control (QC).

The blank samples were mixed with the certified reference materials (CRMs) to achieve quality control. Similarly, aseptic and replicate plating methods were adopted, and the tests were performed in three replicates.

2.3.1. Moisture content (MC) evaluation.

The hydration status of the samples was determined through the gravimetric technique, in accordance with the Association of Official Analytical Chemists (AOAC) guidelines [AOAC, 2000]. Individual specimen was oven-dried by using the laboratory oven (model: 637G, Fisher Scientific Inc., USA), at a temperature of 100 ± 2°C until a constant mass was achieved. The moisture level in the fish tissues was computed by using Equation 1.

(1)

Where:

W₁ = Weight of moisture can with the lid (g),

W₂ = Weight of wet fish sample + moisture can with the lid (g), and

W₃ = Weight of dry fish sample + moisture can with the lid (g)

2.3.2. Plant derivative pH determination.

The acidity (pH) level of the extracts and essential oils was measured in accordance with AOAC procedures, by employing a calibrated digital pH meter (model: pHCal, produced by Analab Scientific Instruments Pvt. Ltd., India). Particularly, the device was calibrated with these buffered media at pH 4.0, 7.0, and 9.0, before taking new measurement. After calibration, the electrode was thoroughly cleaned using distilled water, dried, and dipped into the specimen solution. Then, the pH value of the sample was read from the pH meter screen, after the reading stabilized.

2.3.3. Minerals determination.

The calcium (Ca), phosphorus (P), iron (Fe), potassium (K), and zinc (Zn) concentrations in fish muscles (tissues), were determined in accordance with the AOAC 2020 official guidelines, by using the Inductively Coupled Plasma–Optical Emission Spectrometry (ICP–OES) approach. Specifically, these wavelengths were used for mineral determination: Ca – 422.7 nm, P – 213.6 nm, Fe – 259.9 nm, K – 766.5 nm, and Zn – 213.9 nm.

2.3.4. Vitamins determination.

The carotenoids, vitamin A, total B vitamins, and vitamin E profiles in the EOs, extracts and fish samples were determined using an HPLC system. The fat-soluble vitamins (vitamins A and E) were quantified using the following HPLC parameters: using a C18 column with a column temperature of 31°C, a flow rate of 1.0 mL/min, an injection volume of 10 µL, a run time of 20 min, and UV detection at 325 nm for retinol, 450 nm for β-carotene, and 292 nm for α-tocopherol. Likewise, the water soluble vitamin (vitamin B) was measured using the following HPLC parameters: using a column temperature of 30°C, 0.8 mL/min flow rate, 20 µL injection volume, a run time of 25 min, and UV detection which ranged from 260 nm to 360 nm. The correlation coefficient (R²) value for vitamin A was 0.997, β-carotene was 0.998, α-tocopherol was 0.997, and B-vitamins were 0.995. The LOD and LOQ values for vitamin A were 0.02 and 0.20 µg/mL, β-carotene were 0.05 and 0.25 µg/mL, α-tocopherol was 0.02 and 0.10 µg/mL, and B vitamins ranged from 0.01 to 0.20 µg/mL. The relative standard deviation of less than 4%, and CRM recovery ranging from 93% to 105%.

2.3.5. Protein determination.

The protein level in the dried fish specimens, was determined in accordance with AOAC (2000) standard, by employing the Kjeldahl flasks (produced by Fisher Scientific Inc. USA), and following the explanations provided by Al Banna et al. [27].

2.4.1. Total saponin content (TSC).

The TSC values of the extracts, EOs, and water were measured using a UV–Vis spectrophotometer through the spectrophotometric approach. 1 mL of each sample was added to 1 mL of C₈H₈O₃ (8% w/v) and 10 mL of 72% H₂SO₄. The product was placed in a water bath pre-set at 65 ± 1°C for 15 minutes, before the beaker was placed in an icy environment to quench the reaction. Then, a UV-Vis spectrophotometer was used to measure the product optical density at 540 nm, and the TSC was quantified as diosgenin mg DE/g, with diosgenin as the reference standard. These were the spectrophotometric analytical validation parameters: R² of 0.9881, LOQ = 1.82 µg/mL, and LOD = 0.60 µg/mL, calibration range of 0–100 µg/mL, and RSD of <4% [47].

2.4.2. Total curcuminoids content (TCC).

The TCC of the samples was determined through the spectrophotometric approach, with the aid of a UV–Vis spectrophotometer. 1 mL of the sample (water, extract, or EO) was mixed with 25 mL of ethanol, filtered through Grade 1 filter paper, and the optical density was measured at 425 nm, using ethanol as the blank. The spectrophotometric assay validation criteria were: R² of 0.9891, LOD = 0.30 µg/mL, LOQ = 0.91 µg/mL. concentration range = 0–100 µg/mL, and RSD of <4%. The TCC was quantified as mg CE/g, where CE is the curcumin equivalent, using the curcumin solution as the reference standard.

2.4.3. Total alkaloid content (TAC).

The TCC value was determined using a UV–Vis spectrophotometer, through the spectrophotometric method. 1 mL of the sample was dispersed in 10 mL of 2% ethanoic acid, sieved through Whatman No. 1 filter paper into a beaker containing 10 mL of BCG and 10 mL of PBS (pH 4.7), to produce an alkaloid–dye conjugate, which was later separated into the chloroform phase. The optical absorbance was recorded at 480 nm. Specifically, atropine was the standard reference, and these validation parameters were used: R² = 0.9872, LOQ = 1.52 µg/mL, and LOD = 0.50 µg/mL, analytical range of 0–100 µg/mL, and RSD of <4%. The TCC was expressed as mg AE/g, where AE is the atropine equivalent [48].

2.4.4. Total cyanogenic glycosides (TCG).

The TGC was determined through the spectrophotometric technique. 1 mL of the sample was added to 5 mL of PBS (pH 6.5), and the solution was poured into a beaker containing nitrocellulose paper. The beaker (with its contents) was then placed inside a water bath at 34 ± 2°C for 10 hours to ensure complete reaction. The absorbance of the resulting solution was measured at 530 nm, using KCN as the reference. These validation parameters were used: R² = 0.9915, LOQ = 0.91 µg/mL, LOD = 0.30 µg/mL, and RSD less than 4%. The TGC was expressed as mg KCE/g, where AE is the atropine equivalent.

2.4.5 Total stilbenoid level (TSL).

The TSL of the samples was measured using the spectrophotometric technique. 11 mL of the solution were prepared by dissolving 1 mL of the sample in 10 mL of methanol, which was closely followed by filtration. Then, the optical density of the filtrate was measured at 310 nm using a UV–Vis spectrophotometer. Trans-resveratrol was the reference, while these were the spectrophotometric analytical validation parameters: R² was 0.9895, LOD was 0.25 µg/mL, LOQ was 0.80 µg/mL, and RSD less than 4%. The result unit was mg RE/g, where RE is resveratrol equivalent.

2.4.6. Total phenolic acids (TPC).

The spectrophotometric technique was employed to measure the total phenolic content (TPC) of the samples. The sample was poured into a volumetric cylinder, and the volume was adjusted to 11 mL with methanol. The solution was sifted, and a 1 mL portion of it was added to 5 mL of 10% phenol reagent and 2 mL of 7.5% Na₂CO₃ solution. Thereafter, the product was kept at 25 ± 2^oC for 1 hour, before the absorbance was taken at 750 nm, and the unit of the result was mg GAE/g [49]. These were the spectrophotometric analytical validation parameters: R² of 0.9931, LOQ = 1.36 µg/mL, and LOD = 0.45 µg/mL, calibration range of 0–100 µg/mL, and RSD less than 3%

2.5.1. Total viable Bacterial Count (TVBC).

The Plate Count Agar (PCA) was used to determine the total viable bacteria load of the fish samples.

2.5.2. Bacterial strains determination.

Staphylococcus spp.

Exactly 10 g of ground fish muscle was mixed into 90 mL of sterile buffered peptone water to produce a 1:10 dilution, which was later diluted to 10⁻⁶ using sterile diluent. 0.1 mL of the aliquot was dispersed onto the BPA plate, which was then cultured at 37°C for 24 h. The Staphylococcus spp. clusters were detected by their black to gray, shiny coloring, and their counts were recorded. The Staphylococcus spp. count was then calculated using Equation 1, and the results given as CFU/g.

Salmonella spp.

The aliquot was injected into SC (Selenite Cystine) broth and incubated at 40°C for 24 hours. 0.1 mL of the enriched broth was inoculated on an Xylose Lysine Deoxycholate Agar plate, and cultured at 40°C for 24 hours, and the Salmonella spp. colonies were recognized by their red coloration and counted.

Bacillus spp.

Exactly 0.1 mL of the aliquot was spread onto the Mannitol Egg Yolk Polymyxin Agar plates, which were incubated at 34°C for 24 hours. The Bacillus spp. was identified and enumerated based on its pink coloration, and the population was calculated using Equation 1.

Listeria spp.

The aliquot (0.1 mL) was spread over an Oxford agar plate, and maintained at 35°C for 48 hours. The clusters produced were identified by their grey-green coloration, and the population was counted and calculated using Equation 1.

2.5.3. Fungal count.

The fish muscle (10 g) was mixed with sterile Buffered Peptone Water (90 mL) to produce a 1:10 dilution medium, which was later diluted to 10⁻⁴ using sterilized buffer. To determine the fungal count on the fish body, dilutions of the homogenized sample were plated on Potato Dextrose Agar (PDA) and incubated at 30°C for 4 days. Then, the colonies formed were counted using a digital colony counter (model J-3, manufactured in China), and the results were expressed as CFU/g.

2.5.4. Fungal strains determination.

Aspergillus spp. (black mold).

The aliquot (0.1 mL) was streaked on PDA plate, and incubated at 28°C for 4 days, Then, the black mold was identified based on its globose conidial heads and the appearance of biseriate phialides.

Aspergillus spp. (flavus group).

Exactly 0.1 mL of the portion was spread on an AFPA plate and incubated at 27°C for 6 days, and the Aspergillus spp. (flavus group), growth was recognized by its green-to-olive coloration with rough conidial heads. The Aspergillus spp. (flavus group), fungal burden was computed by using the expression presented in Equation 1.

Rhizopus spp.

Precisely, 0.1 mL of the prepared sample was spread on a PDA plate and incubated at 28°C for 5 days. The Rhizopus spp. was then identified based on its cottony white mycelia. The Rhizopus spp. colonization levelwas calculated using Equation 1.

Penicillum spp.

The portion (0.1 mL) was surface-plated onto PDA plates and incubated in an oxygen-rich environment at 25°C for 8 days. The Penicillium spp. was then identified based on its powdery texture with a pale reverse.

2.5.5. Aflatoxins determination.

The HPLC technique was used to detect and measure the population of the AFs in the dried fish samples in accordance with procedures described by Coskun [50], Water and 0.1% formic acid was the mobile phase A, while methanol and acetonitrile mixed at a ratio of 6:4 was the mobile phase B. Particularly, 28^oC column temperature, 20 µL injection volume, 20 minutes run time, and 1.0 mL/min flow rate were some of the basic HPLC operating parameters, used to measure the AFB₁ and AFB₂ content in the fish tissues. Essential parameters used for the validation of the mycotoxin analysis are presented in Table 1, and the All AFB₁ and AFB₂ concentrations were expressed as µg/kg.

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Table 1. Validation data.

https://doi.org/10.1371/journal.pone.0347254.t001

3.1.1. Bioactive compounds composition.

Table 2 presents the bioactive compounds of the tap water (borehole water), plant oils and extracts used in fish preservation. These essential phytochemicals provide insight into the antimicrobial and preservative effects of the pant derivatives. It was observed that turmeric extract, contain the highest concentrations of curcuminoids, whereas banana peel extract and oil have the highest levels of saponins. Additionally, ginger extract showed the greatest amounts of alkaloids. Interestingly, cyanogenic glycoside was absent in both turmeric extract and oil, although trace amounts were found in the extracts and oils of banana and ginger. Notably, the tap water recorded no detectable phytochemical compounds.

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Table 2. Results of tap water and essential oils and extract phytochemical screening.

https://doi.org/10.1371/journal.pone.0347254.t002

Curcuminoids possess strong antioxidant and antimicrobial properties, exhibiting broad-spectrum antimicrobial activity against numerous varieties of bacteria, fungi, and viruses, by causing disruption to the microbial cell membranes [51]. Curcuminoids have the capability of causing microbial membrane fluidity, resulting in the seepage of proteins and nucleic acids from the cells, thereby exposing the cells to redox imbalance, destabilization, and eventually death of the pathogen [52]. In addition, saponins and alkaloids, have shown potent antimicrobial efficacy against broad spectra of pathogens [53,54]. Saponins tend to inhibit biofilm formation, thereby depriving bacteria of vital nutrients and anchorage sources. Saponins have a hydrophilic chain that can attack the proteins and lipids constituents of the biofilm, thus increasing cell permeability and reducing the cohesiveness of the biofilm. Alkaloids are capable of obstructing enzymatic actions and receptor molecules, as well as depriving microorganisms of essential amino acids. This hinders the cells’ survival [55]. These mechanistic actions will collectively result in significant decline microbial activities in the preserved fish samples, during the prolonged storage duration. This analysis of these chemical composition profiles indicated that, turmeric products exhibit the strongest antimicrobial properties, while having the least anti-nutritional factors.

3.1.2. Vitamins and pH composition.

Table 3 displays the compositions of pH, carotenoids, vitamins B and E, in the plant oils and extracts used for fish preservation. It was observed that TPE and PPE generally have the higheer carotenoids concentrations; PPE products recorded the maximum B vitamin level, while GPO had the highest vitamin E concentration. These vitamins contribute not only to the nutritive enhancement of the preserved fish but also offer additional preservation benefits due to their antioxidant properties. Vitamins B complex and E support microbial growth inhibition, indirectly contributing to preservation by minimizing spoilage [56]. Higher concentrations of B vitamins retard the survival of spoilage microorganisms, although this effect can be extremely reliant on the overall composition of the food matrix and the microbial community that is present [57]. Though vitamins A and E have the ability of enhancing microbial metabolism, these vitamins safeguard against oxidative degradation, thus prolonging the fish shelf life and maintaining their quality [58]. Particularly, these vitamins will enrich the vitamin and antioxidant content of the processed fish samples, through diffusion into fish tissues, surface binding with the fish muscles, and synergistic stabilization of the fish bioactive compounds. These actions will help to avert the fish fat rancidity, hence, preserving the fish integrity during storage [3,59].

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Table 3. Essential oils and extract vitamins profile.

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3.4.1. Total viable bacteria.

The bacterial populations in the fish samples results are presented in Table 6. The findings revealed that the treatments played significant roles, in inhibiting bacteria loads in the fish tissues during storage (p ≤ 0.05). It was T8, T9, and T10 samples, consistently maintained the minimum viable bacterial populations during the storage (excellent antimicrobial effect). This affirms the antimicrobial potency of these treatments used in this research, as their viable bacterial populations were lower than 300 CFU/g at week 8. Also, the control unit recorded the maximum total viable bacterial count (TVBC), attaining 1094 CFU/g at the eight week. T1 and T7 enriched fish samples portrayed weaker anti-bacterial action. The elevated bacterial count in the control unit, affirms the vulnerability of unpreserved fish tissues to bacterial intrusion during storage, supporting previously published reports by Tayel et al. [24]. Unprocessed fish tissues are susceptible to rapid microbial enzymatic reactions, leading to tissue decay, thereby reducing shelf life and the nutritional quality of the fish product [81].

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Table 6. The total viable bacteria count (x10² CFU/g).

https://doi.org/10.1371/journal.pone.0347254.t006

This study’s results reflect that the three-ways hybridization of LE with the other plants extracts or EOs ((T8 to T13), enhanced these treatments antibacterial potency. This is because the lime extract’s acidity level provides a conducive environment, which enhances the anti-bacterial performance of the phytochemical compounds in the other bio-additives. Lime extract has a lower pH value due to its high citric acid and ascorbic acid content, and some bacterial strains, such as Staphylococcus spp., Salmonella spp., Bacillus spp., and Listeria spp., are very sensitive to acidic media. The acidic environment tends to weaken the bacteria’s cell walls, disrupts the osmotic pressure, thereby impairing their function [82,83,84]. According to Nieto Marín et al. [85], acidic environment tends to increase microorganisms’ cell membrane permeability, hence enhancing the penetration (infiltration) rate of the essential bioactive compounds of the treatments, leading to improved antimicrobial effect and storability.

Remarkably, the smaller bacterial counts recorded in T3, T6, T9, T11, T13, T14, and T15, in fish samples at the eight week, can be linked to the significant concentrations of vitamins, phenolic acids, saponins, curcuminoids, and stilbenoids in the additives products used (Tables 2, 3). Curcuminoids are potent antibacterial compounds, with the ability to disrupt the bacterial membrane’s impermeability, thereby causing leakage of intracellular constituents, and resulting in cellular metabolic dysfunction and death of the bacterial cell [86]. Additionally, curcuminoids have a strong ability to chelate heavy metals, thereby distorting the microbial respiratory system, and reducing the availability of non-essential nutrients to the microorganism. Saponins generate free radicals (H₂0₂, OH.), which causes oxidative stress to the cells. Phenolic acids greatly impair amino acid and ATP production in bacterial cells, leading to a massive shortfall in energy and genomic metabolism, thereby inhibiting bacterial growth and activity [87]. Phenolic acids have strong ability to destroy bacterial cell membranes, thereby increasing membrane permeability and leakage of essential nutrients. Also, phenolic acids’ metal chelation and enzyme suppression, can affect the nutrients’ availability to the bacterial cells [88]. Saponins can cause impairment the cell’s DNA and protein structures, resulting in nucleotides degradation and lipid peroxidation, which can lead to cell rupture [89,86]. Active phytochemical compounds possess strong antimicrobial properties, hence retarding bacterial growth and enhancing food storability [90,53]. This creates protective barriers around the fish tissues, resulting in antimicrobial activity and preventing nutrient degradation [29,68,40,91]. Vitamins are antioxidants with the ability to mitigate oxidative stress, thereby enhancing fish nutritional integrity and creating a bacteriostatic environment. These mechanistic actions enhance high bacterial mortality rate, and this can be linked to the lower bacterial counts in the treated fish samples [92,75].

Generally, the microbial counts recorded in this study were lower than those reported by Birie et al. [93], Al Banna et al. [27], and Rana et al. [94] for dried fish samples; however, this study’s findings were similar to the observations stated by Majumdar et al. [95] and Jensen et al. [59]. The variations in the microbial load reported by the various authors can be attributed to storage conditions, type of treatment(s) applied, experimental errors, and the methodology used [96,97].

3.4.2. Isolated bacterial strains.

Table 7 presents the results of bacteria isolated from the fish tissue, both prior to and following the storage period. It was noted that the treatments had a significant impact on the pathogenic growth (p ≤ 0.05). The use of plant extracts and oils as preservatives effectively inhibited the development of disease-causing agents, thereby enhancing the safety and quality of the preserved fish. The results revealed that the preserved fish samples, showed microbial populations within the following ranges: Staphylococcus spp. at 41–91 CFU/g, Salmonella spp at 14–29 CFU/g, Bacillus spp. at 3–14 CFU/g, and Listeria spp. at 5–19 CFU/g. These bacterial counts demonstrated that Staphylococcus spp. had the highest population, whereas Bacillus spp. had the lowest in the enriched fish samples. Consistent with the TVBC readings, the Control and T1 exhibited higher Staphylococcus spp., Salmonella spp., Bacillus spp., and Listeria spp. populations during storage. This can be linked to the lower amounts of bioactive compounds in T1, and the absence of photochemical compounds in the borehole water (Table 2) used in the Control unit. Remarkably, the results of this study aligned with the documented findings of Aberoumand and Baesi [98], who noted that saltwater treatment disrupts microbial growth in preserved Kotr fish.

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Table 7. Isolated bacteria in the stored fish (x10² CFU/g).

https://doi.org/10.1371/journal.pone.0347254.t007

The optimized antibacterial performance observed mainly in the T8–T13 samples, can be attributed to the collaborative antibacterial mechanistic actions, between the hybridized bio-additives bioactive compounds. Curcuminoids compound is highly effective against Gram-positive bacteria, as it retards cellular partitioning, mitosis, and quorum sensing of Staphylococcus spp. Curcuminoids also destroy the Staphylococcus spp. Murein layer, resulting in the massive seepage of the cell nutrients, and thereby leading to cell death [86]. Also, Curcuminoids have a broad spectrum of action against Gram-negative bacteria, and they can inhibit the production of ATP and motility genes in Salmonella spp. These actions cause severe free radical damage to the cells, which can result in a high mortality rate of Salmonella spp. bacteria [99]. Additionally, phenolic acids have strong antimicrobial action against Gram-positive bacteria such as Bacillus spp., as they severely inhibit protein production within the cell, interrupt spore production, and disrupt enzymatic reactions. This causes a deficiency of spores, and a lower rate of Bacillus spp. reproduction [100].

Apart from the low pH of LE, which corrodes the bacterial cell membrane, leading to increase permeability and excessive seepage of intracellular constituents, lemon extract contains substantial amounts of monoterpenes and phenolics, which can interfere with cellular energy metabolism and enzymatic actions. Monoterpenes have the potential to disrupt lipid, amino acid, and protein production, and also creating respiratory disorders, hence causing serious damage to bacterial cells [101]. Therefore, the hybridization of these mechanistic activities (enzymes suppression, metabolic blockade and cell wall destabilization), can enhance the antimicrobial effectiveness of other plant-derived antibacterial treatments [102].

Also, the lower nutrient levels recorded in the Control and T1 samples can partially contribute to the high invasion of Staphylococcus spp., Salmonella spp., Bacillus spp., and Listeria spp. in the fish tissues. For instance, some nutrients such as vitamins have antibacterial effects, which act as defense mechanisms against pathogenic invasion [41]. Interestingly, this laboratory investigation results buttressed the previous findings of Kunová et al. [29], on the utilization of bio-additives in preventing fish spoilage. Notably, T5, T6, T8, and T9 exhibited greater potency for inhibiting Salmonella spp. and Listeria spp. growth to negligible levels; T6 demonstrated greater effectiveness against Bacillus spp. and Listeria spp., while T4 displayed a strong anti- Staphylococcus spp. activity. This affirmed the synergistic antimicrobial effects of the plant extracts and plant oils, and potentiation action of the lime extract. This study has revealed that plant derivatives can be used as a potential substitute for synthetic antibacterial preservatives, leading to the enhancement of food safety and public health, as previously documented by Christaki et al. [38] and Petcu et al. [92].

3.4.3. Fungal contamination.

The outcomes of the total fungal count are given in Table 8 and Fig 2, and the results indicated that the different treatment options significantly impacted fungal survival within the fish tissue. During the eight-week storage period, the fish samples in the control group exhibited the highest total fungal population, reaching a total viable fungal count (TFC) of 1176 CFU/g. Similarly, fish muscles fortified with bio-extracts and EOs (T1 to T16), demonstrated suppressed fungal reproduction; though each treatment exhibited different anti-fungal effectiveness. Furthermore, the dendrogram plot (Fig 2) validates the various treatments’ antifungal efficacy. T8, T9, T11, and T12 verified strong efficacy; T13, T14, T15, and T16 showed moderate efficacy; while T1, T4, and T7 exhibited the lowest antifungal efficacy. Above all, the control group displayed the lowest antifungal efficacy. The high fungal proliferation, associated with control experimental group, highlights the vulnerability of untreated fish muscles to fungal infection (2017). Similarly, Zhang et al. [103]

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Table 8. The total fungal count (x10² CFU/g).

https://doi.org/10.1371/journal.pone.0347254.t008

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Fig 2. The dendrogram of the treatments and total fungal count.

https://doi.org/10.1371/journal.pone.0347254.g002

Fundamentally, the results documented in T1 to T16 revealed that, bioactive compounds have varying capability of compromising fungus membrane impermeability, spore propagation inhibition, which will results in depreciation of the fungus performance [104]. Curcuminoids’ is known for its ability to compromise of fungal cell stability. This results in severe outflow of the essential constituents of the cell, therefore exposing the cell to advance antifungal activities [105]. Additionally, saponins, alkaloids, and phenolic acids, which are present in most of the treatments, are highly effective in disrupting the enzymatic activities of fungi. These mechanisms will inhibit the assimilation of essential nutrients; obstruct the performance of the fungus, leading to a reduction in reproduction [103,106,107]. Saponins interrupt the structural integrity of fungal cells, cellular respiration, and the electron transport chain, resulting in membrane destabilization, metabolic crisis, cytolysis, and ultimately, fungal cell [108]. Furthermore, phenolic acids, due to their acidity nature, tend to lower the pH of the fungal cells intracellular constituents, as well as metals chelation. This can result in protein inactivation, ROS damage, and metabolic quiescence, which can significantly affect fungal cells activities [87]. Generally, alkaloids retard fungal cell homeostasis and survivability by disrupting cell membrane fluidity, DNA unwinding enzymes actions, and fat hydroperoxides [109]. Generally, data obtained from this investigation have confirmed that most plant derivatives tend to play an indispensable role in food safety and preservation.

3.4.4. Isolated Fungal strains.

The results of the isolated fungal species from the fish specimens are presented in Table 9. Notably, the treatment plans significantly affected the survival and growth of individual fungal species in the fish tissues during the storage period (p ≤ 0.05). Though the identities of the fungal strains were not established by the molecular technique, the utilization of AFPA and PDA coupled with structural features during the clinical analysis usually assisted, in the tentative identification of Aspergillus flavus and Aspergillus niger isolates. Notably, the population of fungal strains was highest in the control unit compared to the enriched fish samples. At the eighth week of storage, the control group showed Aspergillus spp. (black mold), Penicillium spp., Aspergillus spp. (flavus group), and Rhizopus spp. counts of 84, 51, 99, and 85 CFU/g, respectively. Similarly, at the eighth week of storage, treated fish samples recorded A. niger ranges from 11 to 63 CFU/g, Penicillium spp. count that varied from 8 to 41 CFU/g, A. flavus population which was between 33 and 81 CFU/g, and Rhizopus spp. count which was between 11 and 72 CFU/g. Additionally, the dominance of A. flavus and Rhizopus spp. in most of the specimens depict the susceptibility the fish muscles to these fungal strains.

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Table 9. The isolated fungi in the fish (x10² CFU/g).

https://doi.org/10.1371/journal.pone.0347254.t009

Specifically, T8, T9, T11, and T12 exhibited the most potent antifungal activities, and this can be attributed to the mechanistic actions of the hybridized bio-additives, which were enhanced by the lemon extract acidity. The low pH lemon extract interferes with the fungal cell wall integrity, leading to greater osmotic pressure, and weakens the fungal resistance pathways [110]. This exposes the cell to secondary antifungal actions, by potent antifungal phytochemical compounds present in the other materials used for the hybridized treatments formulation. At the eight week, Aspergillus spp. (black mold) and Rhizopus spp. counts in the T8, T9, T11 and T12 experimental units, were smaller than 15 CFU/g and 20 CFU/g, respectively. Equally, T13, T14, T15, and T16 shown moderate antifungal activity; however, T1, T4, and T7 displayed very low antifungal effects. The antifungal effectiveness observed in this research, was similar to documented reports of these scholars [12,13,59], which stated that plant derivatives are potential antifungal agents in the fish industry.

The antifungal actions of the most potent treatments (T8, T9, T11, and T12), can be linked to the mechanistic actions of their basic phytochemical compounds. Gingerols, shogaols, zingerone, curcuminoids, turmerones, and phenolic acids, are abundant in ginger and turmeric rhizomes. These compounds have the ability of disrupting fungal cell mitochondrial physiology, as well as interfering with ATP production, hence resulting in the poor fungal growth rate and high mortality rate [111]. Gingerols belong to the lipophilic family, which has the ability to increase fungal permeability, and inhibit spore and ATP formation, eventually resulting in fungal cell lysis [112]. Zingerone hinders fungal proteins biosynthesis, leading to disruption of mycelial growth. This causes drastic reduction of fungal activities, in the fish muscles during storage. Furthermore, the antioxidant actions of the treatments help reduce oxidative stress, which enhances their antimicrobial properties, and consequently inhibits the fungal growth that can cause food deterioration [28,91].

Saponins have the ability to retard the binding of the fungal cell to ergosterol, an essential compound required by the microbes for active growth. This results in nutrient antagonism and poor spore formation, particularly in Aspergillus spp., Penicillium spp., and Rhizopus spp. [113]. Also, saponins cause the seepage of cytoplasm from A. niger, thereby retarding the mycelial growth [114]. It has been documented that saponins compromise the Penicillium spp. cell membrane performance and spore germination, which leads to impaired spore production and a high death rate [115]. Basically, phenolic acids interfere with Aspergillus spp. growth, by inhibiting the production of mycotoxin genes and disrupting the precursor pool [116]. Alkaloids have strong anti-Rhizopus spp. activity by weakening the hyphal membranes of the cells, causing excessive osmotic pressure around the cells and leading to cell failure [117].

3.4.5. Aflatoxins profile of the fish samples.

The results of the aflatoxin levels in the fish samples are presented in Table 10. It was observed that at week 0, all treatment units and control groups recorded no detectable aflatoxins. However, at week eight, there was a substantial variation in aflatoxin concentrations among treatments, indicating the differential effectiveness of the various treatments. This study’s findings have depicted that, the enriched fish samples AFB₁ and AFB₂ concentrations varied from 0.00 to 4.86 µg/kg, and 0.00 to 3.71 µg/kg, respectively. Then in the Control group, the AFB₁ and AFB₂ concentrations were 7.20 µg/kg and 5.69 µg/kg, respectively. Notably, T8 to T11 unveiled the most resilient anti-toxigenic effects, recording LOD value for AFB₁ and AFB₂ at the eight week. Also, T12 and T13 displayed good anti-aflatoxin activity, with the AFB₂ concentration smaller than 0.95 µg/kg. On the contrary, T1, T4, and T7 exhibited relatively moderate anti-aflatoxins action. This ranking confirms that, the treatments contain numerous phytochemicals, having different proficiency of inhibiting aflatoxin production in dried fish muscles [118]. It is well documented that phenolic acids have an effective anti-aflatoxin production effect, by inhibiting the activities of aflatoxigenic fungal strains, and their aflatoxin binding affinity. This occurs through mechanisms such as destroying the cell membrane, mineral chelation, and inhibiting ROS and the aflatoxin gene cluster [116]. Alkaloids disrupt the ability of Aspergillus flavus and Aspergillus parasiticus to produce aflatoxins, by constraining their cells’ oxidative equilibrium, protein and ATP production, as well as free radical scavenging mechanisms of the fugal cells. Furthermore, curcuminoids are known for their high potency in interfering with aflatoxigenic fungal activity, by constraining mitochondrial respiration, ATP production. This will eventually trigger free radical damage, causing excessive cell degeneration, and will reduce the formation of aflatoxins by the fungal cells [119,104].

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Table 10. The Aflatoxin levels in the fish samples (µg/kg).

https://doi.org/10.1371/journal.pone.0347254.t010

Remarkably, the AFB₁ and AFB₂ levels documented this study, aligned with the conclusions reported by [120] and Tian et al. [121]. Tian et al. [121] further elaborated that the active bioactive compounds found in plant oils and extracts have strong anti-aflatoxin formation potential. Fascinatingly, despite the presence of AFB₁ and AFB₂ in most of the treated fish samples, their concentrations were within the 4 µg/kg. However, some of the treated outcomes (T1 and T7) had AFB₁ levels that exceeded 4 µg/kg, but were lower than 10 µg/kg. In summary, the antimicrobial effectiveness of these bio-additives, mainly the active constituents of ginger and turmeric, projected greater efficacy in preventing the formation of aflatoxins in dried fish samples. This enhances their prospects as natural preservatives and nutrient enhancers, leading to higher food safety and improved nutrient quality of stored fish products.