Effect of Artificial Formaldehyde Treatment on Textural Quality of Fish Muscles and Methods employed for Formaldehyde Reduction from Fish Muscles
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DOI: 10.1016/j.focha.2023.100328
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Effect of Artificial Formaldehyde Treatment on Textural Quality of Fish Muscles and Methods employed for Formaldehyde Reduction from Fish Muscles
Naresh Kumar Mehta , Durba Pal , Ranendra K. Majumdar , M. Bhargavi Priyadarshini , Rupali Das , Gangotri Debbarma , Pratap Chandra Acharya
PII: S2772-753X(23)00150-8
DOI: https://doi.org/10.1016/j.focha.2023.100328
Reference: FOCHA 100328
To appear in: Food Chemistry Advances Received date: 21 November 2022
Revised date: 24 May 2023 Accepted date: 26 May 2023
Please cite this article as: Naresh Kumar Mehta , Durba Pal , Ranendra K. Majumdar , M. Bhargavi Priyadarshini , Rupali Das , Gangotri Debbarma , Pratap Chandra Acharya , Effect of Artificial Formaldehyde Treatment on Textural Quality of Fish Muscles and Methods em- ployed for Formaldehyde Reduction from Fish Muscles, Food Chemistry Advances (2023), doi:
https://doi.org/10.1016/j.focha.2023.100328
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Highlights
Artificial formaldehyde treatment manipulates the textural parameters of fish muscles
Treated fish hardness deceives the consumer as if it is a fresh fish
The chewiness of treated muscles increased by 16–121% compared to untreated fish
Lukewarm water washing is the most effective process in reducing formaldehyde from fish muscles.
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Effect of Artificial Formaldehyde Treatment on Textural Quality of Fish Muscles and Methods employed for Formaldehyde Reduction from Fish
Muscles
Running Headline- formaldehyde contamination and its remedial measures in fish muscles
Naresh Kumar Mehta1*, Durba Pal1, Ranendra K. Majumdar1, M. Bhargavi Priyadarshini1, Rupali Das2, Gangotri Debbarma3 and Pratap Chandra Acharya3
1Department of Fish Processing Technology and Engineering
„College of Fisheries‟, Central Agricultural University, Lembucherra, Tripura, India
2 Post Harvest Technology, ICAR- Central Institute of Fisheries Education, Mumbai, India
3Department of Pharmacy, Tripura University (A Central University), Tripura India.
* Corresponding author- Naresh Kumar Mehta, PhD Assistant Professor
Department of Fish Processing Technology and Engineering
College of Fisheries, Central Agricultural University, Agartala, India-799210 Email- nareshfishco@gmail.com, mehta.fpte.cof@cau.ac.in
https://orcid.org/0000-0002-0688-886X Mobile No.- +91 9967088756
Keyword- Fish, formaldehyde, Contaminants, Quality, Water washing, Food safety
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Abstract
The influence of treatment of three different concentrations of formaldehyde on fish muscles textural quality was determined using spectrophotometric and reverse phase high- performance liquid chromatography (RP-HPLC) methods. The experimental fish treated with 1, 5 & 10 % formaldehyde for 5 min and as a result, the residual content was enhanced to almost 7, 11 and 15 times respectively. The hardness and chewiness of fish muscles increased with increase in formaldehyde concentration and the value increased by 80 and 121
% respectively in the fish treated with 10 % formaldehyde. In order to reduce formaldehyde content in fish, formaldehyde-Treated fish was washed in running tap water, saline water and tepid water separately for a period up to 30 minutes and thereby the content got reduced to the acceptable label in 20, 20, and 10 min respectively. Similarly, treated fish was fried for five minutes and content was reduced to acceptable limits. Overall, this study could establish that formaldehyde treatment not only reduced bacterial load but also improved the textural properties of the fish muscles, creating the impression that the fish is fresh. Tepid water washing and frying process were effective in reducing formaldehyde content in fish muscles.
1. Introduction
The maintenance of top quality of fresh fish is a challenging task, as it is a highly spoilable food item. Though, there are several methods of fish preservation namely chilling, freezing, canning and smoking however, each of these methods have got its own merits and demerits.
Hence, traders keep on searching some short cuts to enhance the shelf life of the fish without spending much money and labor, so that, they can get maximum benefits. Driven by this aim, they started using some uncalled chemicals such as formalin, which is regarded as Class I carcinogen by the “International Agency for Research on Cancer”. Hoque et al. (2016)
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proposed that bio-chemical interactions between added formalin and fish muscles may produce noxious products and exert residual ill effects to the consumer. Yeasmin et al. (2010) studied the effect of formalin treatment to rohu fish during ice storage, and revealed an enhanced denaturation of fish protein in treated fish that resulted in lower solubility and gelling ability of formalin treated fish.
It is a well established fact that formalin exerts a carcinogenic effect and it is well highlighted, but its effect on muscles quality has not been studied in detail. Hoque et al.
(2018) treated the fish with varied concentration of formalin and revealed that the content gets increased as many as thirty times with increasing concentrations (0.5 to 4% @ 5 min each). In another study, formalin treated ice stored fish were found to have reduced protein solubility, gel-forming ability (Yeasmin et al., 2010). The same fish detected with low non protein nitrogen content in the muscle compared to untreated fish due to low bacterial load ((Jinadasa et al. 2022; Yeasmin et al. 2010). Further, once the formalin is added to fish artificially, it is very difficult to remove completely. However, Jinadasa et al. (2022) suggested that washing fish with the tap water and cooking to 70 °C (core temperature ) are the best methods to reduce health risks from the formalin adulteration. Kundu et al. (2020) detected high formalin content in rohu (19.66 mg/kg) and catla fish (23.3 mg/kg) that got reduced gradually due to boiling for five minutes. Further, it is proven that frying also found to be effective in order to reduce formalin in rohu fish. A synonymous study was conducted on five fish species in Bangladesh, wherein they studied the effects of pre-treatment (such as dipping in water for 1h, dipped in 1h 5% brine) and cooking effects (boiling, frying etc.) on formaldehyde content in fresh fishes (9.4–32.6 mg/kg), and reveled a significant reduction in the formaldehyde content due to all the pretreatments and cooking methods (Bhowmik et al., 2020). Similarly, a study conducted in Malaysia also established a marginal reduction in formalin due to boiling and frying in fish (Aminah et al., 2013). However, contrasting to
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abovementioned studies, a study on the squid reported to have increased the formalin content due to boiling, especially at “80 °C and 100 °C”. However, they could not explain any reason behind this increase in the content.
Considering the seriousness of this health hazard in fishes, slowly many food agencies around the world are coming up with the permissible limits for formalin in fish. For instances, Indian food regulator, Food Safety and Standard Authority of India (FSSAI) has established upper limit of 2mg/kg for freshwater fish and 100mg/kg for brackish water/marine origin fishes (FSSAI, 2019). Natural levels of formaldehyde in fish are between 6.5–
293 mg kg−1 depending on different species and tolerable levels of formaldehyde for humans are 100 mg kg−1 (European Food Safety Authority, 2014). In addition to above, Malaysian Food Regulation, 1985 established a maximum limit for formaldehyde in fish and fish products of 5 mg/kg. Other than this, it is important to mention that formaldehyde does form naturally in many food items including fish. Hence, it is sometimes difficult to differentiate whether it is added or formed naturally. Though, authors personally feels that formaldehyde cannot be removed 100% from the food as because it also forms naturally in various foods including fish in limited quantity, hence people should be discouraged to use of such chemical.
In literature search, there was no proper study which could establish the effects of added formaldehyde on muscle texture, functionality of fish muscles proteins and possible methods for formaldehyde reduction to acceptable level. So, this study was performed to understand the effects of added formaldehyde on the textural and functional properties of fresh fish along with a aim of developing simple and safer technique for removable of the same. Though, in our previous study (Mehta et al., 2021), we have studied the effect of 10 % formalin on the muscle quality. Therefore, in this study, effect of varying concentrations of artificial formalin
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treatment on catla fish Catla catla) muscle textural quality parameters and different formalin mitigation techniques was investigated.
2. MATERIAL AND METHODS
2.1 Fish collection and formaldehyde treatment
The live Catla fish (Catla catla) with an average length and weight of 30.56 ± 3.2 cm and 1.2
± 0.2 kg respectively were purchased from the market and were transported to the laboratory of the Department of Fish Processing Technology and Engineering, College of Fisheries, Central Agricultural University, Lembucherra, India. After sacrificing the fishes were thoroughly cleaned and whole fish subjected to different formalin treatments by dipping in 1, 5 & 10 % formalin solution for 5 min. We decided to use 1 to 10 % formalin basically because in most cases the formalin concentrations were used in the range of 1 to 10%
(Hoque, et al., 2018; Barokah, et al., 2022, June). In addition to literature search we also had a non-formal survey with fish vendors; it was found that a varying degree of concentration (up to 10 %) of formaldehyde concentration is mostly used to preserve the fish.
The different concentration formaldehyde solutions were prepared using commercial 37-41 % formaldehyde (HiMedia; CAS No: 50-00-0). The whole treated fish were stored at refrigerated temperature for 24 h by wrapping them in polythene packaging material and the analysis was performed.
2.2 Analysis of formaldehyde content 2.2.1 The spectrophotometric method
The spectrophotometric method with some modification (Benjakul et al., 2003) using Nash‟s reagent was applied to determine the formaldehyde content (mg/kg) in fish. Nash‟s
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reagent (Nash, 1953) was prepared by mixing ammonium acetate (15 g), acetyl acetone (0.3 mL), acetic acid (0.2 mL) and the volume were brought to 100 mL by adding water. Nash‟s reagent is a light sensitive and was kept in a dark-glass bottle covered with aluminum foil. A 0.1 N NaOH and 0.1 N HCl were used to adjust the pH of the distillate to be nearly 7.0.
The fish samples were cut into small pieces. A finely chopped fish (30 g) was added to 60 mL of 6% TCA solution and homogenized uniformly using a mortar and pestle. The mixture was filtered through a Whatman No. 1 filter paper, the filtrate (5 mL) was collected and pH of the filtrate was adjusted to around 7.0 with NaOH or HCl. The extract was then stored in a deep freezer for 30 min. The Nash‟s reagent (2 mL) was added to the extract and heated in a water bath at 60°C for 30 min. The absorbance of the extract was measured at 415 nm immediately using a UV-Vis spectrophotometer. The standard curve was prepared using a 10-ppm standard formaldehyde stock solution.
2.2.2 Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method
2.2.2.1 Sample preparation
About 5 g fish sample (fresh and formaldehyde treated separately) was taken and homogenized with distilled water at room temperature (25–30 °C), shaken for 30 min in a shaker incubator at 4 °C and 150 rpm. Thereafter, the sample was centrifuged for 10 min 5000 rpm at 4 °C, filtered through a syringe micro filter (0.45 mm) and filtrate was used for the RP-HPLC analysis.
2.2.2.2 Analytical condition of RP-HPLC
A 20 µL sample solution was analyzed with a poroshell 120 EC- C18 column(4.6 x 150mm, 4µm) using an Agilent 1260 infinity ll model equipped with a PDA. Several trials (more than 10) were run to detect formaldehyde with an appropriate retention time (RT) using various compositions of water and methanol as solvents in a RP-HPLC system due to no or low
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absorptivity of these solvents even at a lower wavelength. Other organic solvents were avoided as they may interfere with formaldehyde. After several trials, the formaldehyde peak was observed at RT of 2.5 min using a solvent system of water:methanol (85:15) as the mobile phase at wavelength of 220 nm. The flow rate was fixed to 0.5 ml min-1 and the operating time of 10 min. Some minor impurities were also observed as the method was developed using a lab-grade formaldehyde.
2.2.2.3 formaldehyde quantification
The fish samples prepared were analyzed in RP-HPLC and compared to the standard formaldehyde retention time for qualification. The peak area of the sample solution was substituted in the calibration equation of the standard curve to calculate the formaldehyde concentration (Figure 3 in supplementary data).
2.3 pH
The pH of fish sample was measured according to the method described by Mehta and Shamasundar (2016). A 5 g quantity of fish meat was thoroughly mixed in 45 mL of distilled water, and the pH of the resulting slurry was determined with a pH meter (Labman LMMP- 30). The pH meter was calibrated with a standard buffer solution of pH 4.2 and 9.2 preceding measuring the pH of the sample.
2.4 Protein solubility
The solubility of protein extracted from fresh and formaldehyde treated fish was estimated using phosphate buffer (“15.6 mmolL-1” Na2HPO4, 3.5 mmolL-1 KH2PO4, containing 1.1 M Potassium chloride, pH 7.5). The fish muscles were homogenized in phosphate buffer at 9000 rpm for 2 min using laboratory homogenizer and then centrifugation was performed (9000 × g) at 4 ºC @ 15 min. The pellet homogenate before
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centrifugation and the supernatant obtained after the centrifugation were analysed for the amount of soluble protein (Robinson & Hodse, 1940). The calculation was performed using following formulae,
Protein solubility = (amount of soluble protein before centrifugation/ amount of soluble protein after centrifugation) × 100
2.5 Water holding capacity (WHC)
The WHC for fish samples were measured according to the method of Jauregui et al.
(1981) with slight modification. Meat sample of 5 g was weighed and put between two layers of filter paper (Whatman No. 1). Sample was placed at the bottom of 50 mL centrifuge tubes and centrifuged at 5000 × g for 10 min at 4 °C. Immediately after centrifugation, the sample was removed and reweighed. WHC was calculated as equation 1.
WHC (%) = (W1-W2)/W1× 100………..Equation 1.
Where, “W1”
= weight before centrifugation (g) and “W2 “
= the weight after centrifugation. The reported WHC value was arrived after average of three replicates.
2.6 “Texture Profile Analysis” (TPA)
The TPA was performed for fresh and formaldehyde-treated fish fillets using Texture Analyser (TA. XT2i Stable Micro System, Surrey, England) with a 75 mm compression plate as a probe with 50 kg load cell. The dimensions of fish fillets used for test was 2×2×2 cm.
The hardness, cohesiveness, springiness, gumminess and chewiness were recorded (Mehta et al., 2021).
2.7 ‘Total plate count’ (TPC)
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TPC was determined for fresh and formaldehyde treated samples by pour plate technique. Exactly 10 g of fish meat was taken and macerated in 90 mL 0.9% saline and the serial dilutions were prepared. 1mL of sample from each dilution was mixed carefully to molten media (TPC agar) and poured onto sterilized Petri dishes. After solidification, the plates were incubated at 37± 1ºC for 24 h. Colonies obtained were enumerated manually (Udochukwu et al., 2016).
2.8 Different water washing schemes and frying of fish
The fish treated with 10 % formaldehyde solution was used for this experiment. The fish was washed in the running tap water with the flow rate of 2 L/min. The sample was drawn after 5, 10, 15, 20, 25, 30 and 35 minutes of washing in running tap water and formaldehyde content was determined. Similar scheme of washing was followed using stagnant lukewarm water (Temperature 50-55 °C) and 5 % saline water. For frying, 10 % formalin treated fish samples was cut into fillets of standard dimensions (3.5 cm length × 2.5 cm width) and the same were fried at 180 °C for 5 minutes in soybean refined oil in a automatic fryer. Thereafter, formaldehyde content was determined in the fried fish fillets.
2.9 „Statistical analysis‟
The data obtained were analyzed by one way analysis of variance (ANOVA) using SPSS 16.0 software. At least, three replicates were performed for each experiment.
3.0 RESULTS & DISCUSSION
3.1 Effect of formaldehyde treatments on residual formalin content of fish
In the preset experiment, the initial formaldehyde content in the untreated (fresh) Catla fish was 1.66 & 2.31 mg/kg (Fig 1) using spectrophometric method and RP-HPLC method respectively. More or less similar amount of formalin contents (1.9-2.11 mg/kg) were
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reported in two freshwater cat fishes (Kundu et al., 2020). In general, the quantity of naturally formed formaldehyde during post mortem is much lower in freshwater fish compared to marine fish (Jaman, 2013) due to higher amount of TMAO in marine fish. In present study, after treatment of the experimental fish with 1, 5 & 10 % formaldehyde solutions for 5 min, the residual content enhanced to 12.75 &14.59, 19.49 & 44.56 and 26.24 & 61.86 mg/kg fish (Fig. 1) analyzed with spectrophotometric method and RP-HPLC method respectivly. This indicated that the sensivity of formaldehyde detection of RP-HPLC method was significantly (p<0.05) higher than that of the spectrophotometric method. Significant (p<0.05) difference in formaldehyde contents were observed among different treatments. Similar increment in the residual formaldehyde was documented when the fish was treated with 5% formalin (Sanyal et al., 2016). Further, Hoque et al. (2018), reported that regardless of the fish species or analytical methodologies utilized when fish is treated with increasing concentration of formalin and exposure time indicated higher absorption of formaldehyde in fish muscles.
This increment in the residual formalin in the fish muscles due to formaldehyde combines with unsaturated lipids along with double bonds and forms a complex compound containing free carbonyl group which probably originates from formaldehyde (Castell & Smit, 1972). In addition to this, aldehyde compound may be easily bonded to the amino acids in a small amount (Rahmadhani et al., 2017).
3.2 Effect of formaldehyde treatment on pH of fish muscle
The pH of the fresh fish was observed to be 6.48, which was within the suggested limit of 5.5 to 6.5 depending on the different fish species (Kim, 2022) indicating raw material is of good quality. On treating with increasing concentration of formaldehyde, the value decreased accordingly (Table 1). A non-significant (p>0.05) reduction was observed in the samples treated with 1 & 5 % formaldehyde concentrations. However, in the case of fish treated with 10 % formaldehyde, the pH value (6.17) reduced significantly (p<0.05) (Table
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1). This decrement in the pH of the fish muscle might be due to the acidic pH of the formaldehyde (Burke, 1933) and the formation of post mortem lactic acid. The findings of the experiment corroborated with Sanyal et al. (2016) who reported that the pH of fresh fish (control) was 6.72and that reduced to 6.59 when treated with 5% formalin. Similarly, Yeasmin et al. (2016) observed a lower pH value in 5 % treated rohu fish in comparison with the control, where the high pH is due to the production of alkaline bacterial metabolites coinciding with high aerobic plate counts (APC).
3.3 Effect of formaldehyde treatment on water holding capacity (WHC)
In the present study, the WHC of the fresh fish muscle observed was 69.67 % and treating the fish with 1, 5 and 10 % formaldehyde reduced the WHC significantly (p<0.05) by 3.78 %, 5.02% and 15.26% respectively (Table 1). This reduction in water holding capacity may be due to the strong reactivity of the formaldehyde with myofibrillar protein resulted in the formation of crosslinks and displacing the water causing the toughening of the flesh and thereby, a reduction in water holding capacity (Haard & Simpson, 2000) gave rise to lower acceptability and functionality (Li et al., 2007). This may be confirmed with the increment in the concentration of the formalin might be resulting in strong water displacement causing subsequent decrement in the WHC.
3.4 Effect of formaldehyde treatment on protein solubility of fish muscle
The protein solubility of untreated fish in the present study was found to be 87.34 % that reduced significantly (<0.05) to 82.54%, 75.93 % and 69.33% in fish samples treated with increasing formaldehyde concentration (Table 2). The reduction in the protein solubility can be due to the protein denaturation causing the aggregation as effected by the formalin treatment (Morisasa et al., 2020). The marked reduction in the pH value also found to be concurrent with the protein solubility and this might be due to shifting of isoelectric pH of the muscle protein. A similar reduction in protein solubility was reported for the protein extracted
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from rohu fish treated with 5 % formalin solution as compared to fresh rohu (Yeasmin et al., 2010). Initial myofibrillar protein solubility of formalin treated fish was 58% which was significantly lower in compared to fresh fish (86.70%) (Yeasmin et al., 2010). Yeasmin et al.
2010 suggested that the formalin contamination results a marked denaturation of the muscle protein causes lower protein solubility.
3.5 Effect of formaldehyde treatment on muscle texture profile
In the rigor or pre-rigor stage, fish remains stiffened leading to flesh hardness, which is one of the criteria to assess the freshness of the fish. Furthermore, the flesh gets softer shortly after the rigor mortis is resolved. Therefore, to understand the influence of formaldehyde treatment on fish flesh hardness compression test was performed using a texture analyzer. In the present study, the fresh fillets had a hardness of 193.63 N. Hardness values of the fillets increased by 6.07 %, 46.86 %, and 65.11 % in fish treated with 1%, 5%, and 10% formalin, respectively in compared to untreated fish fillets (Table 1). The results indicated that an increase in formaldehyde concentration resulted in a considerable (P<0.05) increase in hardness value, residual formalin content and decrease in the WHC (Fig. 1, Table 1). The findings of the investigation were corroborated with Yeasmin et al. (2010), who reported that after dipping rohu Fish (Labeo rohita) in 5% formaldehyde for 5 minutes, the muscle texture became slightly firmer as compared to untreated fish. Similar results were observed by Morisasa et al. (2020) when the fish sample was soaked in 1000 ppm formaldehyde (FA). The reason for the increase in the hardness could be due to reduced water holding capacity and dehydration of muscles. In addition, the protein aggregation stimulated by formalin treatment is also one of the important factors for muscle stiffening (Morisasa et al., 2020). According to Sotelo et al. (1995), formaldehyde accumulates during poor storage conditions of fish and reacts with protein, causing protein denaturing and muscle toughness.
In the current investigation, the value of WHC and solubility was found to have reduced
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significantly as a result, the hardness value might have increased. Consumers are deluded by the increased hardness of formalin-treated fish muscles since fresh fish or fish in rigor have a firm texture, which is an indication of prime quality. As a result consumers are unable to make the difference between the contaminated and fresh fish.
Likewise, the chewiness value of untreated fish was 20.65 N, but after treatment with 1, 5, and 10% formaldehyde, it increased by 16, 74, and 121 %, respectively, as compared to the chewiness value of fresh fish. Since chewiness is the consequence of gumminess and springiness, the values of these parameters were found to increase as the formaldehyde concentration increased. The increase in the chewiness indicated that fish muscle would be firm as good as or better than fresh fish. With an increase in formaldehyde concentration, the values of springiness, cohesiveness, and gumminess increased as well, however, adhesiveness declined. Fish containing formaldehyde between 10-20 mg/kg may not be considered palatable as a human food (Yasuhara & Shibamoto, 1995).
Further, the value of the chewiness of untreated fish was 20.65 N, after treatment with 1, 5 & 10 % formaldehyde that increased by 16, 74 and 121 % compared to fresh fish chewiness value. Chewiness is the product of gumminess and springiness, therefore values of these parameters also found to be increased with increase in the formalin concentration. The increase in the chewiness indicated that fish muscle would be firm as good as or better than fresh fish. The values of springiness, cohesiveness & gumminess also increased, whereas adhesiveness recorded a decreasing trend with increase in formaldehyde concentration.
3.6 Effect of formaldehyde on the total plate count (TPC)
In the present investigation, the initial microbial load on fish samples was 5.27 log10
CFU/g (Table 1) indicating the good quality of the fish. The microbial load for the good quality fish was reported to be in the range of 2 to 6 log10 CFU/g (Huss, 1995). This load varies from species to species depending on the place of harvest, condition, and temperature
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of post-harvest operations (Perigreen et al., 1987). Further, in the present study, when fish samples were treated with 1, 5 and 10 % formalin, the TPC value significantly (<0.05) decreased to 4.98, 4.7 & 4.6 log10 CFU/g, respectively. The result indicated that with increasing formalin concentration, bacterial load decreased significantly (p<0.05) which corroborated with the results of Sanyal et al. (2016) in mrigal and Yeasmin et al. (2013)in rohu fish when treated with 5 % formalin. This decrease in the bacterial load on fish might be related to the bacteriostatic/bactericidal property of formalin itself. Mezbah et al. (2014) established that formaldehyde acts as an antibacterial and antifungal agent; therefore, it may inhibit microbial infestation on the fish skin or in the fish fillet, and delay the spoilage of formalin treated fish samples. Neeley (1963) reported that bacterial cell division was inhibited in the presence of 20 to 50 μg / mL (equal to 2–5 %) of formaldehyde.
3.7 Effect of different washing methods and frying on residual formaldehyde in fish muscle
The fish treated with 10 % formaldehyde (dipping time: 5 min) were employed to develop an appropriate and inexpensive remedial approach for the removal of the added formaldehyde from fish muscle. The residual formaldehyde content of fish samples dipped in 10% formaldehyde was 26.24±0.08 mg/kg. For various lengths of time, the aforementioned treated fish samples were washed in running tap water, tepid water, and saline water (Table 2). From the results represented in Table 2 and Fig.2, it was observed the residual formaldehyde content reduced significantly in running water, tepid water and saline water as the exposure time increased. Frying the treated fish muscle for 5 min lowered the residual content to 4.04 mg/kg. The scientific panel of food regulators i.e. The Food Safety and Standard Authority of India (FSSAI) fixed an Adhoc maximum limit for formaldehyde content with 4 mg/kg for freshwater fish and 100mg/kg for brackish and marine water fish (FSSAI, 2019). Similarly, many countries have set the maximum limit of
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formaldehyde/formalin for fish and fishery products such as the United States Environmental protection agency (2 mg/kg) (Xuang et al. 2009), Malaysian food regulation act (1885) (5 mg/kg formaldehyde), the Ministry of Agriculture of China and the Italian ministry of health (10 mg/kg) whereas Yasuhara and Shibamoto (1995) has fixed the maximum limit of 10-20 mg/kg.
Considering Indian regulation (4mg/kg), washing fish muscles in running water (20 min), tepid water (15 min) and saline water (20 min) lowered the values to 2.12, 0.78 and 3.51 mg/kg respectively. As a result, it was apparent that tepid water washing, especially compared to saline water or running tap water is more successful in removing residual formaldehyde from fish muscles. It's worth noting that formalin is water-soluble and volatile, which indicates it may be washed away or/evaporated from the fish's body with tap water, warm water, or saline water. The higher reduction in warm water is possibly due to the warm water heat causing enhanced evaporation of formaldehyde from the muscles due to its volatile nature. Hoque et al. (2016) observed a drastic decrease in the formaldehyde in cooked fish sample (0.98 to 5.93 mg/kg) compared to fresh fish (5.80 to 21.80 mg/kg).
Contrastingly, Yeasmin et al. (2013) detected formalin in samples dipped into 10–15%
formalin solution for 5 minutes after 40 minutes of washing using tap water.
In the present study, formaldehyde -treated fish samples were fried at 180 °C for 5 minutes to and formaldehyde content was determined before and after frying. The result showed that frying effectively reduced the formaldehyde content from 26.24±0.08 mg/kg to 4.04±0.05 mg/kg. This reduction might be due to the escape of the analytes (volatile nature) during frying process. Exposure to formalin-contaminated fish by consumption causes acute mucus membrane irritation (nose, eye, and throat), ulceration of the gastrointestinal tract, chest or abdominal pain, nausea, vomiting, diarrhea, and gastrointestinal bleeding (Jinadasa et al., 2022). In the case of chronic exposure deleterious effects such as geotaxis damage, and
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various types of cancers including lung, nasal cavity, nasopharyngeal, brain, pancreas, colon, and lymph hematopoietic system have been approved (Kangarlou et al., 2023). Thus, if humans consume high-formaldehyde fish for an extended period of time, they may experience a variety of biochemical and pathological abnormalities, with unknown health consequences.
4.0 Conclusion
Formaldehyde treatment not only reduced bacterial load but also alter the texture of fish muscles that deceiving the consumers as if it is fresh. However, the HPLC method is more sensitive in formaldehyde detection in fish samples compared to spectrometric method.
Further, this study concluded, the formaldehyde treatment caused fish muscle hardening that deceives the consumers in identifying fresh fish along with enhanced shelf life due to reduction in bacterial load. Further, tepid water washing for 10 min. and frying for 5 min.
may reduce the formaldehyde from fish muscles effectively and eliminate the deleterious effect for formaldehyde.
Acknowledgements
The authors are thankful to Dean, College of Fisheries, Central Agricultural University (Imphal), Lembucherra , India, for providing the laboratory facilities to carry out the work.
Authors‟ contributions
Naresh Kumar Mehta- Conception and design, and data analysis and draft preparation Durba Pal- Investigation, data analysis
Ranendra. K. Majumdar- Conceptualization, Review & Validation M. Bhargavi Priyadarshini- Editing of the draft, Review and Visualization Rupali Das- Investigation and analysis
Gangotri Debbarma- Investigation and Analysis Pratap Chandra Acharya- Review and Supervision All authors read and approved the final manuscript.”
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Data availability –Data set generated during research are available from the corresponding author on reasonable request.
Funding
Authors are also greatly thankful for funding support received from the Department of Biotechnology, Govt. of India under the project- Centre of Excellence- DBT-NER/LIVS/05/
2011 Phase II.
Ethics approval - Not applicable.
Competing Interests – “The authors have no relevant financial or non-financial interests to disclose.”
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23 Table 1. Effect of formaldehyde treatment on fish quality parameters
Value are means ± SD, n=3, p> 0.05, value in the same row bearing unlike letters differ significantly.
Parameters Fresh Fish Fish treated with 1%
formaldehyde
Fish treated with 5%
formaldehyde
Fish treated with 10%
formaldehyde
pH 6.48±0.03 a 6.42±0.02 a 6.33±0.03 a 6.17±0.04 b
WHC (%) 69.67±1.51a 67.13±0.46 ab 66.17±1.53 b 59.04±1.28c
Protein solubility (%) 87.34±1.98 a 82.54±0.29 a 75.93±1.25 b 69.33±4.34 c
TPC(log10cfu/g) 5.27±0.14 a 4.98±0.06 b 4.70±0.03 c 4.6±0.05 c
Hardness (N) 193.63±24.42 a 205.39±5.07 ab 284.38±54.63bc 319.71±12.52c
Adhesiveness(N-sec) -0.25±0.09 a -0.52±0.46 a -0.68±0.31 a -0.77±0.30 b
Springiness 0.45 ± 0.09 a 0.47 ± 0.07 a 0.455± 0.06 a 0.51± 0.02 a
Cohesiveness 0.23 ± 0.00 a 0.245 ± 0.02 a 0.267 ± 0.02 a 0.28 ± 0.01 a
Gumminess(N) 45.15 ± 4.63 a 50.31 ± 5.50 a 76.69 ± 21.07bc 89.65± 3.79 c
Chewiness(N) 20.65 ± 9.58 a 23.94 ± 6.56 a 36.08 ± 15.34 a 45.70 ± 2.47 a
24
Table 2. Effect of different washing methods and frying on formalin reduction in fish muscles treated with 10 % formaldehyde.
formaldehyde content (mg/kg fish) Time
(Minutes)
Running water Warm water Saline water washing
Frying
0 26.24±0.08Aa 26.24±0.08Aa 26.24±0.08Aa 26.24±0.08Aa
5 24.07±0.08Ba 20.37±0.08Bb 23.83±0.01Bc 4.04±0.05Bd
2.12 ±0.20 (HM)
10 15.5±0.01Ca 4.34±0.01Cb 18.68±0.01Cc -
15 4.35±0.01Da 0.78±0.01Db 1.26±0.03 (HM)
7.88±0.01Dc 20 2.12±0.01Ea
3.65±0.20 (HM)
0.68±0.01Eb 3.51±0.02Ec 6.4±0.23 (HM)
25 2.08±0.01Ea 0.64±0.01Fb 3.22±0.01Fc
30 2.02±0.01Fa Not detected 3±0.03Gb
35 0.88±0.01G - -
Value are means ± SD, n=3.Capital letters (A,B...) show the difference in column while small letter(a,b...) show the difference in rows. Values carrying different letters differ significantly (p>0.05).
HM- refers to values analyzed through HPLC method while other values were achieved through spectrophotomatric method.
25
Fig. 1 Effect of different concentrations of Formaldehyde treatments on fish muscles formaldehyde contents as estimated by spectrophotometric and HPLC methods.
0 10 20 30 40 50 60 70
fresh fish Fish treated with 1% formalin
Fish treated with 5% formalin
Fish treated with 10% formalin
Formalin (mg/Kg)
Different Treatments
Spectrophotmetric method HPLC Mehtod
26
Fig. 2 (a to d): Colour development due to formaldehyde presence during Nash reagent (spectrophotometric) method and effect of different washing methods and frying on reduction of formaldehyde content in fish muscles.
Fig. 2a Running water washing at different length of time
Fig 2b: Warm water washing at different length of time
Fig 2c: Saline water washing at different length of time
Fig 2d: Frying at 180°C for 5 min.
Fig 2b: Warm water washing at different length of time
Fig. 2a Running water washing at different length of time
27
AUTHOR NAMES AND CREDIT ROLES
AUTHOR CREDIT ROLES
Naresh Kumar Mehta conception and design, and data analysis and draft preparation
Durba Pal Investigation, data analysis, draft editing Ranendra. K. Majumdar Conceptualization, Review & Validation M. Bhargavi Priyadarshini Editing of the draft, Review and
Visualization
Rupali Das Investigation and analysis
Gangotri Debbarma Investigation and Analysis Pratap Chandra Acharya Review and Supervision
'Declarations of interest‟
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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