1 Introduction

Fish is not only a popular delicacy but also a vital source of protein in the Bangladeshi diet, providing consumers with around 60% of their total animal protein intake. The average per capita consumption of fish in Bangladesh is between 20 to 25 kg, which is significantly higher than the world average of 13 kg. In 2008, freshwater and marine fish contributed approximately 80% and 20% of this per capita fish consumption, according to the Department of Fisheries [1]. These figures indicate that fish accounts for about 12.4% of food expenditure in general households and 8.84% in poor households. A wide variety of freshwater and marine fish species are available in different product forms, catering to the buying power of the consumer, and playing a significant role in the Bangladeshi diet [2]. However, the fish market faces a significant challenge in the form of fish spoilage, primarily resulting from enzymatic autolysis, oxidation, and microbial growth. Studies have shown that autolytic changes and lipid degradation via auto-oxidation are responsible for the initial quality loss and chemical spoilage during fish storage [3, 4]. Additionally, the growth of microorganisms from the water in which the fish live exacerbates the situation.

Chitosan possesses remarkable preservative qualities that can combat spoilage in vegetables, fruits, and fish [4]. This is achieved through its ability to eliminate or impede bacterial growth [5]. The efficiency of chitosan as a preservative is attributed to its solubility, degree of deacetylation (DD), and viscosity-average molecular weight, Mη [6]. Normal chitosan, a large and highly viscous polycationic polymer with over 5000 glucosamine monomers, undergoes breakdown and reduction in acetyl groups when exposed to gamma radiation, resulting in a less viscous solution [7]. Given that the preservative activity of chitosan is contingent on the degree of deacetylation, it is anticipated that irradiated chitosan will demonstrate superior preservative activity compared to non-irradiated chitosan. By extracting chitosan from waste prawn shells, we can explore its potential as a preservative agent, addressing the environmental concern of prawn shell waste in processing areas.

Chitosan’s abundance, non-toxic nature, and biodegradability make it an exceptional choice for various applications [8]. Its cost-effectiveness compared to synthetic polymers, along with its neutral taste, further enhance its appeal. Furthermore, chitosan exhibits valuable biological properties such as analgesic, antitumorigenic, hemostatic, hypocholesterolemic, and antioxidant properties, making it an attractive natural food additive and preservative for pharmaceutical, biomedical, and industrial uses [9]. The regulatory approval of chitosan as a functional food ingredient in Asian countries like Korea, coupled with its inclusion in the European Pharmacopoeia, underscores its significance [10]. Additionally, the production of chitosan-enriched dietary products in Japan and the approval for its inclusion in food by the Codex Alimentarius Commission further accentuate its potential in various industries [11].

This study aims to demonstrate the potent antimicrobial effects of both irradiated and non-irradiated chitosan against 16 reported bacteria responsible for fish spoilage. Labeo rohita, a fish of significant economic importance in Bangladesh, was selected as the representative sample for this research. In addition, this study includes an evaluation of the effectiveness of 5% formalin as a preservative, an agent with a long history of use. Furthermore, the study explores the assessment of total viable count (TVC), total volatile basic nitrogen (TVB-N), and pH changes as key indicators of fish freshness.

2 Materials and methods

2.1 Extraction of chitosan from waste prawn shell

Chitosan was skillfully extracted from underutilized prawn shells through a meticulous process (details in supplement). This entailed demineralizing the prawn shells with a diluted mineral acid, followed by dilute alkali and heat treatment to produce pure chitin through deproteinization. The chitin was then meticulously hydrolyzed into high-quality chitosan using concentrated alkali and consecutive heat treatment [12].

2.2 Gamma irradiation to isolated chitosan

Firstly, chitosan was dried in an oven for one hour at 105 °C and then carefully packed into seven polyethylene bags in duplicate and one control (total 15), each containing 10 g. Subsequently, the bags were securely sealed and subjected to varying strengths of gamma radiation (2 kGy, 5 kGy, 20 kGy, 30 kGy, 40 kGy, 50 kGy, and 100 kGy) using a Co-60 machine (details in supplement) at a rate of 1000 krad/hr. Following the radiation process, the samples were diligently stored at room temperature in a dark environment until further used.

2.3 Determination of viscosity and molecular weight of chitosan

In this study, both irradiated and non-irradiated chitosan (as a control) were dissolved in a carefully formulated solution of 0.1 M acetic acid and 0.2 M NaCl, at a balanced 1:1 ratio (v/v), resulting in the creation of chitosan solutions ranging from 0.2% to 0.8% (w/v). After elimination of insoluble materials through filtration using Whatman #4, the passage time of the solutions was precisely measured three times employing an Ostwald viscometer and a capillary immersed in a water bath set at 25 °C. Subsequently, the running times of the solution and solvent were skillfully utilized to ascertain the intrinsic viscosity [η] and molecular weight of the chitosan.

$$ \eta_{{{\text{rel}}}} \left( {\text{Relative viscosity}} \right) = {\text{t2 }}\left( {\text{efflux time of solution}} \right) \, /{\text{ t1 }}({\text{efflux time of solvent}}) $$
$$ \eta_{{{\text{sp}}}} \left( {\text{Specific viscosity}} \right) = \eta_{{{\text{rel}}}}^{{ - {1}}} $$
$$ \eta_{{{\text{inh}}}} \left( {\text{Inherent viscosity}} \right) = {\text{ln }}\eta_{{{\text{rel}}}} /{\text{c}} $$
$$ \eta_{{{\text{red}}}} \left( {\text{Reduced viscosity}} \right) = \eta_{{{\text{sp}}}} /{\text{c}} $$
$$ \left[ \eta \right] = {\text{lim }}\eta_{{{\text{sp}}}} \, /{\text{ c}} = {\text{lim ln }}\eta_{{{\text{rel}}}} /{\text{c}} $$
$$ {\text{c}} \to 0{\text{ c}} \to 0 $$

where, c: Concentration of chitosan solution (g/mL, %). η red was plotted on a graph to calculate the intercept of the plots on the ordinate at c = 0 which gives intrinsic viscosity [η] (mL/g).

Moreover, the viscosity-average molecular weight of chitosan solutions was calculated using the Mark Houwink equation that uses the relationship between intrinsic viscosity and molecular weight.

$$ \left[ \eta \right] = {\text{K}}\left( {{\text{Mw}}} \right)^{{\text{a}}} ({\text{Mark Houwink equation}}) $$

where ‘K’ and ‘a’ are constants for a given solute–solvent system and temperature. Values of ‘K’ and ‘a’ were 1.81 × 10–3 and 0.93, respectively [13].

2.4 Determining the degree of chitosan acetylation using FTIR spectroscopy method

The FTIR spectrum of the chitosan-KBr pellet was analyzed to provide both qualitative and quantitative insights. The KBr disks were prepared following the method with minor adjustments [14]. In summary, approximately 2 mg of dried chitosan powder and 200 mg of KBr were blended and triturated with an agate mortar. Subsequently, an IR hydraulic press with a pressure of 6 tons for 60 s was utilized to compact the mixture. The spectra of chitosan samples (in the form of KBr disks) were comprehensively obtained using an advanced FTIR Spectrometer (Supplier: Perkin Elmer Model: T60U) with a wave number range of 4000–400 cm−1. The degree of deacetylation (DD) of the chitosan samples was accurately calculated using the baseline as per Baxter et al. (1992). The computation equation for the baseline is given below:

$$ {\text{DD }} = { 1}00 \, - \, \left[ {\left( {{\text{A1655 }}/{\text{ A345}}0} \right) \times {115}} \right] $$

where A1655 and A3450 are the absorbances at 1655 cm−1 of the amide-I band as a measure of the N-acetyl group content and 3450 cm−1of the hydroxyl band as an internal standard to correct for differences in chitosan.

2.5 Fish sample collection and processing

A total of 25 premium aqua-cultured Rohu fishes, Labeo rohita (Hamilton), each weighing around 800 ± 200 g, were selected from five reputable firms located in the prime Savar and Gazipur areas of Bangladesh (5 fishes from each firm). Every effort was made to ensure the fish were handled with care, immediately stunned upon capture, and cleaned with potable water. The fish were expertly cut into uniform pieces weighing about 50 g each. Following the careful harvest, all the fish samples were promptly washed, packed on ice, and transported to the laboratory within 2 h. Recognizing the importance of preserving freshness, the fish pieces were ingeniously dipped into various strengths of chitosan (Non-irradiated and 2 kGy, 5 kGy, 20 kGy, 30 kGy, 40 kGy, 50 kGy, and 100 kGy-irradiated) and different concentrations of 50 kGy irradiated chitosan solution (3 g/L, 5 g/L, and 10 g/L) as well as into blank that is 2% acetic acid for 10 min. After this preservation process, the fish samples were carefully air-dried for 15 min in a laminar hood and packed with transparent polyethylene bags for storage at a precisely controlled temperature of 4 °C (± 1) for 16 days. The Research Ethics Committee reviewed and approved the detailed study protocol.

2.6 Identification of bacteria in fresh and stored Labeo rohita (Hamilton) fish samples

10 g of both fresh (samples immediately after capture, not stored) and stored-fish muscles were blended with 90 mL of 0.1% peptone and 0.85% NaCl salt (w/v) to prepare a perfectly homogeneous slurry. The resulting mixture was then serially diluted tenfold and evenly spread on selective plates containing PCA (Plate Count Agar), MacConkey, Cetrimide, MSA (Mannitol salt agar), and XLD (Xylose lysine deoxycholate) Agar media. Following an overnight incubation at 37 °C, counting of colony-forming units (CFU) and recording of colony morphology were done. After careful selection of different morphological colonies, restreaking was performed on PCA plates three times to obtain pure bacterial cultures. To further compare, pure cultures of 16 fish-spoilage bacteria were collected from the Centre for Advanced Research in Sciences (CARS) as well as the Microbiology department at the University of Dhaka (details in supplement and Supplementary Table 1) [15] to provide a comparative analysis of their properties with the isolated bacterial strains.

2.7 Radiation dose fixation against 16 bacteria at a fix concentration

The antibacterial activity of chitosan was determined using Kirby-Bauer disc diffusion. 5 g/L (w/v, in 2% (v/v) acetic acid) concentrated chitosan solution was prepared for each 20 kGy, 30 kGy, 40 kGy and 50 kGy radiation dose. 16 fish-spoilage causing bacteria were grown in nutrient broth at 37 °C for 18–24 h and then diluted to 1:200 in nutrient broth. The inoculum, thus, was expected to contain 105 to 106 CFU/mL. A sterile cotton bud soak with bacterial inoculum was spread uniformly all over a nutrient agar plate. Sterile filter paper (Whatman number 4) discs (6.0 mm diameter) were dipped in 20 kGy, 30 kGy, 40 kGy, and 50 kGy radiated chitosan solutions as well as non-irradiated chitosan solution (as a control) and then placed on the inoculated nutrient agar plate. Approximately 10 µl solution was applied to each disc. As a blank (no chitosan), sterile 2% acetic acid was used. After 24 h plate incubation at 37 °C, differences between the diameter of the paper disc (6 mm) and that of the growth inhibition zone (mm) were recorded.

2.8 Concentration fixation against 16 bacteria at a fix radiation

The same method was used to fix the radiation dose. Therefore, 6 mm filter paper discs were dipped in various concentrations of 50 kGy radiated chitosan solution (1 g/L, 3 g/L, 5 g/L, and 10 g/L) as well as 2% acetic acid blank. The discs were then positioned over the inoculated nutrient agar plate. Following an overnight incubation at 37 °C, the outcomes were recorded carefully.

2.9 Determination of freshness quality

2.9.1 Sensory assessment of fish samples

Fish samples were labeled and assessed by a panel of 6 experts using established methods to evaluate consumer acceptance based on odor, flavor, and texture [16]. Additionally, a 10-point scale was used to determine the total sensory score, with samples scoring 4 or above considered acceptable. Consent form explaining study objectives, participants’ right to withdraw, and study guidelines were signed by the participants to affirm their voluntary participation and their right to withdraw from the study if they so desired. The questionnaire of each respondent was coded to ensure anonymity. The Research Ethics Committee reviewed and approved the detailed study protocol.

2.9.2 Determination of pH of fish samples

The pH of the Labeo rohita was measured after complete blending of its flesh. 10 g of fish flesh was homogenized in 90 mL of distilled water and the pH was measured using a pH meter (HI 110 series).

2.9.3 Determination of total viable count of fish samples

A homogeneous blend was prepared by aseptically weighing 10 g of fish muscles and combining them with 90 mL of 0.1% peptone water and 0.85% NaCl salt (w/v) in a sterilized blender. Utilize ten-fold serial dilution to spread 0.1 mL of this blend onto nutrient agar plates (using PCA media, OXOIDTM). After a 24-h incubation at 37 °C, the colony-forming units (CFU) on each nutrient agar plate were counted to establish the total number of viable bacterial cells (TVC) capable of forming colonies.

2.9.4 Determination of TVB-N of fish samples

The measurement of total volatile basic nitrogen (TVB-N) in minced fish species is crucial for assessing fish spoilage. Utilizing a steam distillation technique, a homogenized blend was prepared: combining 200 mL of 7.5% aqueous trichloroacetic acid solution with 100 g of fish muscle. The resulting mixture was then subjected to filtration and condensation using a Kjeldahl-type distillatory. This method involves collecting 40 mL of the distillate in a conical flask containing 10 mL of 4% aqueous boric acid solution, along with 0.04 mL of methyl red and bromocresol green indicator for titrating ammonia. The precision of this approach was further demonstrated by the use of a 0.05 mL graduated burette containing 0.025 N H2SO4 for titration. This method ensured the complete neutralization of the green boric acid solution, signifying accurate measurement of TVB-N and enabling thorough assessment of fish quality.

$$ {\text{Calculation}}: \, \left( {{\text{mgN}}/{1}00{\text{g}}} \right): \, \left( {{\text{14mg}}/{\text{mol}}*{\text{a}}*{\text{b}}*{3}00} \right)/{ 25} $$

a: mL of sulphuric acid. b: normality of sulphuric acid.

2.10 Statistical analysis

From each batch of chitosan, two independent samples of chitosan were prepared for irradiation and further experiments. After that, experiments were conducted in triplicate (n = 2 × 3). Results were presented as the mean values ± standard deviation (n = 2 × 3). ANOVA were performed wherever appropriate. p value < 0.05 was considered as statistically significant.

3 Results

3.1 Extraction of chitosan from unused prawn shell

7.5 g chitosan was produced from 50 g primary processed dried prawn shell. So, the yield was 15%. Prawn shells contain 18–20% chitin, which after deacetylation can produce chitosan. Previous articles reported that 12–18% chitosan can be extracted from the prawn shell.

3.2 Effects of irradiation on viscosity

The viscosity of non-irradiated chitosan was 144.8 mL/g, indicating a highly viscous solution. Viscosity significantly decreases with an increase in radiation dose, as illustrated in Table 1. Upon irradiation, there was a rapid and significant decrease in viscosity up to 100 kGy, after which the decrease in viscosity slowed down. Notably, at 50 kGy, the viscosity of irradiated chitosan measured 64.5 mL/g, which is less than half of the viscosity of non-irradiated chitosan (144.8 mL/g).

Table 1 Effect of different radiation doses on the physicochemical properties of chitosan
Full size table

3.3 Effect of radiation on the molecular weight

The molecular weight of non-irradiated chitosan was 187,128.43 Daltons. Intriguingly, high levels of ionizing gamma radiation caused the glycosidic bonds of the chitosan to break. As shown in Table 1, there was a sharp and significant decrease in molecular weight between 2 and 20 kGy radiation. Increasing the radiation intensity further decreased the molecular weight, albeit with less impact. A 50 kGy dose of gamma radiation was significantly reduce the molecular weight of chitosan compared to the non-irradiated form.

3.4 Effect of radiation on the degree of deacetylation (DD)

The degree of deacetylation of non-irradiated chitosan was 73.80%. Following exposure to radiation at levels of 2 kGy, 5 kGy, 20 kGy, 30 kGy, 50 kGy, and 100 kGy, the degree of deacetylation significantly increased to 74.33%, 77.76%, 78.32%, 78.88%, 79.50%, and 79.99%, respectively. The effects of radiation on the degree of deacetylation of the chitosan are shown in Table 1. Notably, a rapid increase in deacetylation was observed between the radiation doses of 2 kGy and 5 kGy, indicating the exciting potential for enhancing chitosan properties through controlled radiation exposure.

3.5 Radiation dose and concentration fixation against fish-spoilage bacteria

3.5.1 Bacterial status of fresh and spoil Labeo rohita

The following bacteria were isolated from Labeo rohita using selective media: Pseudomonas sp, Salmonella sp, Shigella sp, Staphylococcus sp, total gram-negative, and positive bacteria. When fresh Labeo rohita was examined, it was discovered that 65% of the bacteria were gram-negative and 35% were gram-positive. After 6 days of preservation at 4◦C without chitosan treatment, the percentage of gram-negative bacteria increased to 81%, while gram-positive bacteria increased to 19%. Notably, in fresh Labeo rohita, 20% of the gram-negative bacteria were identified as Pseudomonas sp, whereas in spoiled (stored without preservation) Labeo rohita, this number increased to 55%. These findings are consistent with previous research by Gram et al. (1996), highlighting the prevalence of Pseudomonas spp. during the aerobic storage of fish in ice [15]. It is evident that the bacterial composition in fish undergoes significant changes during storage (Fig. 1).

Fig. 1
figure 1

Percentage of gram positive and gram-negative bacteria in fresh and spoiled (stored without preservation) Labeo rohita (n = 25, weight = 800 ± 200 g) after 6 days storage at 4 (± 1) ◦C

Full size image

3.5.2 Antimicrobial activity of non-irradiated and radiated chitosan

The study investigated the effectiveness of chitosan, both non-irradiated and irradiated at different levels (20 kGy, 30 kGy, 40 kGy, and 50 kGy), against 16 bacterial species known to cause fish spoilage (Table 2). The results show that irradiated chitosan has significantly stronger antimicrobial properties compared to non-irradiated chitosan, with antimicrobial activity increasing as the radiation dose increases. As shown in Table 1, the chitosan irradiated at 50 kGy exhibited the highest antimicrobial activity (13.96 mm) among all the chitosan samples tested. Therefore, it can be concluded that chitosan irradiated at 50 kGy is a promising option for preserving Labeo rohita and extending its shelf life.

Table 2 Antimicrobial activity (determined by Kirby-Bauer disc diffusion method) of non-irradiated and 20 kGy, 30 kGy, 40 kGy and 50 kGy irradiated chitosan at a stable concentration (5 g/L)
Full size table

3.5.3 Antimicrobial activity of differently concentrated chitosan solution

Various concentrations of 50 kGy-irradiated chitosan (1 g/L, 3 g/L, 5 g/L, and 10 g/L) were prepared and tested against 16 reported bacterial strains responsible for fish spoilage (Table 3 and Supplementary Table 2) to determine their antimicrobial activity. A 2% acetic acid solution was used as a blank, as chitosan was dissolved in it. Some bacteria, such as Alcaligenes spp. and Serratia spp., exhibited resistance to non-irradiated and low concentrations of irradiated chitosan but were susceptible to irradiated chitosan at higher concentrations. Conversely, other bacteria were susceptible to chitosan even at low concentrations. Based on the findings from Table 3, it is evident that a higher concentration of chitosan solution proves to be a superior preservative compared to a lower concentration. Specifically, a 10 g/L (1% or 10,000 ppm) chitosan solution demonstrated a significantly larger average zone of inhibition (15.05 mm) against 16 reported spoilage-causing bacteria of fish. This highlights the potential of chitosan as an effective preservative agent in different applications of food industry.

Table 3 Antimicrobial activity (determined by Kirby-Bauer disc diffusion method) of 50 kGy-irradiated chitosan at different concentration (3 g/L, 5 g/L, 10 g/L)
Full size table

3.6 Determination of freshness quality of stored fish samples

It has been determined that chitosan treated with 50 kGy of radiation showcases superior antimicrobial activity when compared to other variations. Furthermore, research reports have highlighted a direct correlation between the concentration of chitosan and its antimicrobial effectiveness [5]. Therefore, we have chosen to focus our study further on chitosan that has undergone a 50 kGy radiation treatment.

3.6.1 Sensory assessment

The sensory attribute plays a crucial role in determining the acceptability of stored fish products. According to Amerine (2013), human preferences lean towards fish samples with a sensory score exceeding 4.0 [17]. To investigate this, the sensory scores of Labeorohita samples were assessed and plotted against the storage time (Fig. 2).

Fig. 2
figure 2

Sensory score of blank (without chitosan treated), 5% formalin and 3 g/L, 5 g/L, and 10 g/L irradiated chitosan (50 kGy) treated Labeo rohita samples during storage at 4 (± 1) °C. Symbols represent mean values of six measurements ± S.D. (n = 2 × 3). *indicates a statistically significant difference in comparison to blank

Full size image

Initial findings revealed no significant differences in sensory scores between the blank and chitosan-treated samples for the first 2 days of storage. However, a noticeable decline in freshness score was observed in the control samples post this period, rendering them unacceptable by day 6. In contrast, samples treated with 10 g/L concentrated chitosan and 50 kGy irradiation experienced a much slower decrease in freshness score, remaining acceptable until day 16. Interestingly, it was also found that the sensory score of 5% formalin-treated (positive control) Labeo rohita control samples decreased even more slowly than that of the 10 g/L concentrated chitosan solution.

3.6.2 pH changes

The pH of fresh fish muscle is typically neutral, but it becomes acidic after fishing due to the formation of lactic acid from glycogen. Over time, the pH of stored fish muscle gradually and then rapidly increases due to the accumulation of basic end-products from bacterial spoilage [18]. Both chitosan treated and untreated samples had similar initial pH values of approximately 6.21, but the pH values of both the blank and chitosan-treated fish samples fluctuated similarly during storage (Fig. 3).

Fig. 3
figure 3

pH of blank (without chitosan treated), 5% formalin and 3 g/L, 5 g/L, and 10 g/L irradiated chitosan (50 kGy) treated Labeo rohita samples during storage at 4 (± 1) °C. Symbols represent mean values of six measurements ± S.D. (n = 2 × 3). *indicates a statistically significant difference in comparison to blank

Full size image

They decreased over the first 2–4 days and then increased, likely due to the production of volatile basic components by fish-spoilage bacteria, such as ammonia and trimethylamine [19]. In contrast, the pH of 5% formalin-treated samples remained consistently stable, most likely because it effectively eliminated basic compound-producing bacteria at the beginning of the storage process.

3.6.3 Total viable plate count (TVC)

The number of bacteria present fluctuated according to the chitosan concentration. The initial total viable plate count was (3.20 ± 0.2) × 104 CFU g−1, a figure consistent with previous studies (102–106 CFU g−1) [20, 21]. Over the 16-day storage period, the bacterial count in blank samples continually increased, exceeding the acceptable level (107 CFU g−1) on day 6, with no lag phase observed (Fig. 4).

Fig. 4
figure 4

Total Viable Count (TVC) of blank (without chitosan treated) and 3 g/L, 5 g/L and 10 g/L irradiated chitosan (50 kGy) treated Labeo rohita samples during storage at 4 (± 1) °C. Symbols represent mean values of six measurements ± S.D. (n = 2 × 3). *indicates a statistically significant difference in comparison to blank

Full size image

The chitosan treatment initially demonstrated no bactericidal effect. However, after 2 days of storage, chitosan-treated samples exhibited a reduction in bacterial plate count. The sustained inhibitory effect of chitosan was evident with a 3-day lag phase for 3 g/L chitosan, a 6-day lag phase for 5 g/L chitosan, and an 8-day lag phase for 10 g/L chitosan solution. Throughout the storage period, the plate count of Labeo rohita treated with 10 g/L chitosan solution remained consistently lower than that of the blank samples and surpassed the maximum acceptable limit on day 16. In fish treated with 5% formalin, no visible bacterial colonies were detected over the 16-day period. This demonstrates the efficacy of formalin in eradicating bacteria, with the added benefit of preventing further contamination or growth due to the use of an airtight polyethene bag.

3.6.4 Total volatile base nitrogen (TVB-N)

The initial TVB-N levels were consistent in both the blank and irradiated chitosan-treated Labeo rohita samples, measuring at 18.06 mg (100 g)−1. The control samples exhibited a 2-day lag phase, while the 5 g/L chitosan-treated samples showed an 8-day lag phase, and the 10 g/L chitosan-treated samples had a 12-day lag phase (Fig. 5).

Fig. 5
figure 5

Total Volatile Basic Nitrogen (TVB-N) of blank (without chitosan treated), 5% formalin, and 3 g/L, 5 g/L, and 10 g/L irradiated chitosan (50 kGy) treated Labeo rohita samples during storage at 4 (± 1) °C. Symbols represent mean values of six measurements ± S.D. (n = 2 × 3). *indicates a statistically significant difference in comparison to blank

Full size image

Subsequently, there was a rapid increase in TVB-N levels until the end of the storage period, indicating bacterial proliferation. Bacteria possess a notable capacity for the oxidative deamination of non-protein nitrogenous compounds [22]. A TVB-N level of 35–40 mg per 100 g of fish muscle is commonly recognized as an indication of spoilage [23]. However, various studies have reported varying acceptable TVB-N levels for different fish species, specific treatments, and processing conditions, for example, 35–40 mg per 100 g by Connell (1990) and 25–35 mg per 100 g by Ababouch et al. (1996) [24, 25]. From Fig. 5, it has been noted that 5% formalin produces a notably lower amount of TVB-N than 10 g/L chitosan, potentially due to formalin's ability to eliminate all bacteria responsible for oxidative deamination.

4 Discussion

The discovery of chitosan by Rouget in 1859 has vastly increased the significance and versatility of this biopolymer [26]. Chitosan, classified as chitin with a degree of deacetylation of 70% or above, holds potential for a wide range of applications. Using chitin-rich prawn shells as a raw material, around 40% chitosan can be produced, with a molecular weight typically ranging from 100,000 to 1,200,000 daltons. Numerous reports suggest that the antimicrobial activities of chitosan are closely linked to its molecular weight [6]. Furthermore, viscosity, a key indicator related to molecular weight, plays a vital role in determining the commercial viability of chitosan. High molecular weight chitosan exhibits greater viscosity and has demonstrated increased antimicrobial activity against E. coli and Bacillus spp. Moreover, research has indicated that physical and chemical treatments can significantly impact the viscosity of chitosan. The deacetylation reaction is a pivotal factor in influencing the physicochemical properties of chitosan, such as solubility, biodegradability, antimicrobial activity, and wound healing properties [8, 9]. This renders chitosan as a highly promising biopolymer with diverse potential applications.

The antimicrobial actions of chitosan are determined by its unique chemical and structural properties, making it effective against both gram-positive and gram-negative bacteria [6]. Chitosan, a cationic polymer with a positively charged amino group, can penetrate and disrupt the cell walls of gram-positive bacteria, leading to leakage of essential intracellular components and inhibiting their growth. However, its effectiveness against gram-negative bacteria is limited due to its inability to pass the outer membrane Aside from direct cell wall disruption, chitosan also binds to DNA, inhibiting mRNA synthesis and potentially impairing fungal pathogens Furthermore, it can chelate metals, trace elements, and essential nutrients, contributing to its comprehensive antimicrobial efficacy. It is important to note that the concentration of the solvent, acetic acid, also exerts a substantial influence on the antimicrobial activity of chitosan and its derivatives [27].

Mastering the molecular weight and viscosity of chitosan unlocks its potential for diverse applications. This study has harnessed the power of radiation to create low molecular weight chitosan and explore its impact. This innovative approach leverages high-energy ionizing rays to induce beneficial chemical and biological changes in chitosan without the need for harmful additives. Notably, the use of gamma-ray radiation has been found to significantly enhance the antimicrobial properties of chitosan through cross-linking and degradation, opening new possibilities for its practical applications. Furthermore, the distribution of molecular weight in irradiated chitosan plays a crucial role in influencing its antimicrobial activity. Particularly, the 3–5 × 104 molecular weight fraction has shown remarkable potential in suppressing microbial growth.

It is crucial for chitosan to be biocompatible, ensuring that it does not produce any adverse effects on the host. Through testing, chitosan has demonstrated excellent tolerance by various tissues, including the skin, ocular membranes, and nasal epithelium, making it suitable for diverse biomedical applications [8, 9]. Research indicates that chitosan has low or no toxicity based on in vivo studies, sparking significant interest in its potential for food applications. Notably, the oral LD50 (median lethal dose) of chitosan in mice exceeds 16 g/kg of body weight per day, surpassing that of sucrose [9]. However, it is essential to acknowledge that impurities in chitosan, if present, may impact its toxicological profile. Further studies are needed to understand chitosan’s effects on additional spoilage pathogens and other fish species.

5 Conclusion

The utilization of chitosan, a renewable, non-toxic, and biodegradable resource, has been receiving increasing attention. Nonetheless, there has been limited research on the antimicrobial properties of chitosan against fish-spoilage microorganisms. This study sought to investigate the impact of un-radiated and irradiated chitosan on the refrigerated-storage life of Labeo rohita through microbiological, chemical, and sensory analyses. The findings revealed that untreated Labeo rohita had a refrigerated-storage life of 6 days, while samples treated with 10 g/L chitosan exhibited a refrigerated-storage life of 16 days, based on various spoilage parameters. Furthermore, the study showcased the potential of 50 kGy gamma-irradiated chitosan as a natural preservative for prolonging the refrigerated-storage life of Labeo rohita. In comparison, the commonly used preservative formalin has significant adverse effects on human consumption and is not biodegradable. In contrast, chitosan is biodegradable and safe for human consumption.