Diffusion of organochlorine (OCPs) and cypermethrin pesticides from rohu (Labeo rohita) internal organs to edible tissues during ice storage: a threat to human health.

The migration of organochlorine pesticides (OCPs) and cypermethrin residues from internal organs to edible tissues of ice-held Labeo rohita (rohu) was investigated in this study. The liver (246 µg/kg) had the highest level of ∑OCP residues, followed by the gills (226 µg/kg), intestine (167 µg/kg), and muscle tissue (54 µg/kg). The predominant OCPs in the liver and gut were endosulfan (53-66 µg/kg), endrin (45-53 µg/kg), and dichloro-diphenyl-trichloroethane (DDT; 26-35 µg/kg). The ∑OCP residues in muscle increased to 152 µg/kg when the entire rohu was stored in ice, but they decreased to 129 µg/kg in gill tissues. On days 5 and 9, the total OCPs in the liver increased to 317 µg/kg and 933 µg/kg, respectively. Beyond day 5 of storage, total internal organ disintegration had led to an abnormal increase in OCP residues of liver-like mass. Despite a threefold increase in overall OCP residues by day 9, accumulation of benzene hexachloride (BHC) and heptachlor was sixfold, endrin and DDT were fourfold, aldrin was threefold, and endosulfan and cypermethrin were both twofold. Endosulfan, DDT, endrin, and heptachlor were similarly lost in the gills at a rate of 40%, while aldrin and BHC were also lost at 60 and 30%, respectively. The accumulation of OCP residues in tissues has been attributed to particular types of fatty acid derivatives. The study concluded that while pesticide diffusion to edible tissues can occur during ice storage, the levels observed were well below the allowable limit for endosulfan, endrin, and DDT.

Trace metals in commercial seafood products (canned, pickled and smoked): Comparison, exposure and health risk assessment.

This study used inductively coupled plasma and mass spectrometry, followed by microwave digestion, to assess the concentration of six trace metals (Cr, Ni, As, Cd, Hg, and Pb) in three canned products (tuna in oil [TIO], sardine in oil [SIO], and mackerel in oil [MIO]), two pickled products (prawn pickle [PP] and fish pickle [FP]), and one smoked product (masmin) collected from Tuticorin market, southern India. Trace metal (TM) levels in canned, pickled, and smoked fish varied from 0.01 to 1.48, 0.009 to 0.94, and 0.08 to 4.13 mg/kg, respectively. The concentration of TMs in the seafood was observed in the following order: As > Cr > Pb > Ni > Cd > Hg. Regarding their accumulation in the seafood products, masmin showed the highest levels followed by SIO, MIO, TIO, FP, and PP. The results revealed that smoked products showed higher TM concentrations, followed by canned and pickled products. The level of TMs in seafood products was below the maximum residue limit set by the European Commission (EC/1881/2006) and the Food Safety and Standards Authority of India (FSSAI). Risk assessment for consumer health implied that the evaluated target hazard quotient (THQ < 1), hazard index (HI < 1), and target cancer risk (TCR < 1.E-04) values did not exceed their guideline values and did not cause non-carcinogenic and carcinogenic health impacts through seafood product consumption. The pollution index (Pi) values of TMs in seafood products ranged from 0.01 to 0.7, with As and Cd showing the lowest and highest values, which were below 0.7, indicating their safety for consumption. The metal pollution index (MPI) for TMs in seafood ranged from 9.E-05 to 0.007, with the lowest and highest values associated with PP and TIO products. The MPI value for seafood products was below 1, signifying low TMs accumulation, suggesting the safety of seafood for consumption. The maximum seafood meal consumption limit (CRmm) was calculated, showing that adults can safely consume > 16 meals/month without chronic or acute toxicity. The results of this study suggest that the accumulation of TMs in the analyzed seafood product was below the health guidelines and pollution index values, making it suitable for smooth domestic and international export as well as safe consumption.

Potentially toxic metals in seawater, sediment and seaweeds: bioaccumulation, ecological and human health risk assessment.

This study assesses the bioaccumulation, ecological, and health risks associated with potentially toxic metals (PTMs), including Pb, Hg, Cd, As, and Cr in Hare Island, Thoothukudi. The results revealed that the concentration of PTMs in sediment, seawater, and S. wightii ranged from 0.095 to 2.81 mg kg-1, 0.017 to 1.515 mg L-1, and 0.076 to 5.713 mg kg-1, respectively. The highest concentrations of PTMs were found in the S. wightii compared to seawater and sediment. The high bioaccumulation of Hg and As in S. wightii suggests that it can be used as a bioindicator for these elements in this region. The ecological risk indices, which include individual, complex, biological, and ecological pollution indices, suggest that Hare Island had moderate contamination with Hg and Cd. However, there are no human health risks associated with PTMs. This study examines the current ecological and health risks associated with PTMs and emphasizes the importance of regular monitoring.

First report on the exposure and health risk assessment of organochlorine pesticide residues in Caulerpa racemosa, and their potential impact on household culinary processes.

Seaweeds are widely consumed as natural seafood in various Asian countries. Chemical contaminants, such as pesticide residues (PRs), can contaminate it due to its high bio-accumulation nature. Limited research exists on the presence of PRs in edible seaweeds, their decrease in levels during cooking processes, and the evaluation of hazard indices and associated health risks to humans. This study investigated the effects of different cooking methods on the levels of organochlorine pesticides in Caulerpa racemosa seaweed. It also assessed the potential health risks associated with consuming seaweed by estimating daily intake, hazard quotient, and hazard index. The PRs were reduced after different cooking methods. The impact of thermal cooking on PRs in C. racemosa was found to be notably beneficial. The PRs decreased following MWC, boiling, and steam cooking. Several PRs were analyzed, and endrin, DDT, endosulfan, and cypermethrin were found to be the most prevalent. The HQ and HI values for raw and cooked seaweeds were found to be below one, suggesting that the PRs in C. racemosa pose no risk to consumers of seaweed. In summary, thermal cooking proves to be an efficient method for minimizing PRs, while the cooking of seaweeds ensures a high level of safety during consumption.

Spatial distribution and seasonal variation of pesticide residues in the sediment, seawater, and edible seaweeds: Environmental and human health risk assessment.

This study evaluates the distribution of pesticide residues (PRs) and assesses the ecological and human health risks posed by them from seawater, sediment and edible seaweeds in the Gulf of Mannar. Results showed that hexachlorocyclohexane, heptachlor, aldrin, endrin and endosulfan were the predominant PRs. The maximum concentration of PRs was observed in the monsoon and post-monsoon seasons. Their concentrations in sediment exceeded interim sediment quality guidelines, indicating significant risks to benthic organisms. Except for endrin, the presence of other organochlorine pesticides (OCPs) was below the maximum residual limit. Human health risk assessment revealed that the estimated daily intake of PRs through seaweeds was below the acceptable daily intake. The hazard quotient and hazard ratio were <1, implying that the study seaweeds do not pose significant health risks. The presence of endrin in edible seaweeds, however, indicates safety concerns for consumers and the continued use of banned OCPs in agriculture.

Effect of antibiotics and sanitizers on Salmonella biofilms associated with seafood contact surfaces.

This study investigates the effect of antibiotics and sanitizers on biofilm forming Salmonella isolated from different seafood contact surfaces. Four Salmonella were isolated from 384 swab samples collected from various contact surfaces of fishing boats, fish landing centres and seafood processing plants. One out of four isolates was from the fishing boat (FB I -1) other three isolates were from the seafood processing plant (FPPII -4, FPPII- 5, FPPI-3). The ability of Salmonella to form biofilms on different contact surfaces (HDPE, stainless steel, wood, glass, tiles) was tested with the microbial load on different incubation days, and a higher count was observed on day five. The effect of sanitizer viz., sodium hypochlorite (20, 50, 100, 200 mg/l) and iodophor (2, 5, 10 mg/l) on the biofilm formed on different seafood contact surfaces were investigated. A reduction of 2-3 log was observed on surfaces of HDPE and stainless steel when they were treated with a minimum of 5 mg/l of iodophor or 20 mg/l of sodium hypochlorite after a contact time of 5 min. Antibiotic resistance of biofilm forming Salmonella was tested for different classes of antibiotics (penicillin, ß-lactams, quinolones, macrolides, aminoglycosides, phenol drugs, sulfonamides, cephalosporin). All four isolates showed intermediate resistance to ciprofloxacin, a quinolone drug. Only one isolate FB I -1 (fishing boat deck) expressed resistance to more drugs, viz., ßlactams (AMC, AMP, penicillin G), macrolides (AZM) and nitrofurantoin (NIT). These findings shall help the seafood processors to mitigate the formation of Salmonella biofilms on various seafood contact surfaces with different sanitizers and the antibiotic resistance of biofilm forming Salmonella shall give knowledge on human clinical treatments. With this study, we shall recommend the regulatory authorities control the contamination level of fish handling areas.

Prevalence of biofilm forming Salmonella in different seafood contact surfaces of fishing boats, fish landing centres, fish markets and seafood processing plants.

The prevalence of biofilm forming Salmonella on different seafood contact surfaces was investigated. Out of 384 swab samples, 16.14 % and 1 % were confirmed biochemically and molecularly as Salmonella respectively. One out of four isolates was from the boat deck, and three were from the seafood processing plant. Salmonella was more prevalent in January, June, and September months. Different assays investigated the biofilm forming ability of isolates. Two out of four isolates have shown strong biofilms, and the others were moderate biofilm formers by microtitre plate assay. In the CRA assay, three isolates showed 'rdar' morphotype, and one showed 'bdar' morphotype. All isolates were positive for gcpA gene (~1700 bp), a critical gene found in Salmonella biofilms. The microbial load of Salmonella biofilms on different contact surfaces were determined, stainless steel and HDPE were found prone to biofilms. With this, a suitable mechanism shall be formulated to control the biofilms of Salmonella.

Bioaccumulation of organochlorine pesticide residues (OCPs) at different growth stages of pacific white leg shrimp (Penaeus vannamei): First report on ecotoxicological and human health risk assessment.

Pesticide residues (PRs) in farmed shrimps are concerning food safety risks. Globally, India is a major exporter of pacific white leg shrimp (P. vannamei). This study was undertaken to analyze PRs in the water, sediments, shrimps, and feed at different growth stages to evaluate the ecotoxicological and human health risks. PRs in the seawater and sediments ranged from not detected (ND) to 0.027 µg/L and 0.006-12.39 µg/kg, and the concentrations were within the maximum residual limits (MRLs) and sediment quality guidelines prescribed by the World Health Organization and Canadian Environment Guidelines, respectively. PRs in shrimps at three growth stages viz. Postlarvae, juvenile, and adults, ranged from ND to 0.522 µg/kg, below the MRLs set by Codex Alimentarius Commission and European Commission. Most of the PRs in water, sediments, and shrimps did not vary significantly (p > 0.05) from days of culture (DOC-01) to DOC-90. The hazard quotient (HQ) and hazard ratio (HR) were found to be < 1, indicating that consumption of shrimps has no noncarcinogenic and carcinogenic risks. PRs in shrimp feed ranged from ND to 0.777 µg/kg and were found to be below the MRLs set by EC, which confirms that the feed fed is safe for aquaculture practices and does not biomagnify in animals. The risk quotient (RQ) and toxic unit (TU) ranged from insignificant level (ISL) to 0.509 and ISL to 0.022, indicating that PRs do not pose acute and chronic ecotoxicity to aquatic organisms. The study suggested no health risk due to PRs in shrimps cultured in India and exported to the USA, China, and Japan. However, regular monitoring of PRs is recommended to maintain a sustainable ecosystem.

Accumulation potential of heavy metals at different growth stages of Pacific white leg shrimp, Penaeus vannamei farmed along the Southeast coast of Peninsular India: A report on ecotoxicology and human health risk assessment.

This study compared the heavy metal concentration in water, sediment, and shrimp at different growth stages of culture and subsequently evaluated the ecotoxicological and human health risk status. Total trace element concentration in the water, sediment and shrimp ranged from not detected (ND) (Hg) to 91.05 (Fe) µg/L, 0.01 (Hg) to 19, 246.33 (Fe) mg/kg, and ND (Hg) to 13.98 (Fe) mg/kg, respectively. Toxic metals such as, Cd, Hg, and Pb in shrimps ranged from ND to 2.11 mg/kg, ND to 0.158 mg/kg, ND to 0.088 mg/kg, and ND to 0.469 mg/kg, respectively. Toxic heavy metals at all the growth stages of shrimps (days of culture (DOC)-01 to DOC-90) were found below the maximum residual limit (MRL) of 0.5 mg/kg set by the European Commission (EC). Similarly, Cu, Zn, and As concentrations in shrimp were also far below the MRLs of 30 mg/kg, 100 mg/kg, and 76 mg/kg set by the World Health Organization and Food Safety and Standard Authority of India, respectively. The concentration of heavy metals increased from DOC-01 to DOC-90 and was positively correlated with the length and weight of the shrimps (p < 0.05). The risk assessment was estimated for both Indians and Americans and found no carcinogenic (lifetime cancer risk (LCR) < 10-4) and non-carcinogenic (THQ and TTHQ<1) health risks through consumption of shrimp cultured in this region. The hazard quotient (HQdermal < 1), hazard index (HI < 1), and LCR (<10-4) values of the heavy metals indicated that the dermal absorption might not be a concern for the local fishermen and marine fish/shrimp farmworkers. Water and sediment quality indices were applied to assess the surface water and sediment quality, and their results were found nil to low levels of heavy metal contamination at all the sampling sites. All heavy metals studied in sediments were < effect range low (ERL) and < threshold effect level (TEL), indicating no adverse biological effects on aquatic organisms. Therefore, regular monitoring of the shrimp aquaculture system throughout the crop will provide evidence of heavy metals bioaccumulation in shrimps. This research will provide baseline data to help farmers establish the optimal aquaculture practices and regulatory authorities to formulate legislation and strategies to reduce heavy metal biomagnification in shrimps from farm to fork.

Effect of household culinary processes on organochlorine pesticide residues (OCPs) in the seafood (Penaeus vannamei) and its associated human health risk assessment: Our vision and future scope.

Food safety is crucial in today's competitive trading market, as it directly affects human health and promotes seafood exports. The effects of thermal processing (boiling, frying, grilling, and microwave cooking) on pesticide residues (PR) in P. vannamei were assessed. The PR in raw and processed shrimp ranged from 0.007 to 0.703 µg/kg for uncooked/raw, not detected (ND) to 0.917 µg/kg for boiled, ND to 0.506 µg/kg for fried, ND to 0.573 µg/kg for grilled and ND to 0.514 µg/kg for microwave cooked shrimps. The Endrin, endosulfan sulfate, and heptachlor were predominant PR found in the raw and processed shrimp. The PR content in raw and cooked shrimps were below the maximum residue limits (MRL) set by the Codex Alimentarius Commission (2021) and the European Commission (86/363/1986 and 57/2007). The estimated daily intake (EDI) of PR from raw and processed shrimps were below the ADI prescribed by CAC. The hazard quotient (HQ) and hazard ratio (HR) values were <1, indicating no non-carcinogenic or carcinogenic health implications through shrimp consumption. The estimated maximum allowable shrimp consumption rate (CRlim) suggests an adult can eat >100 shrimp meals/month, which is over the USEPA's (2000)recommendation of >16 meals/month without health issues. The Effect of thermal processing was detected in the following order: boiling < grilling < frying < microwave cooking. The processing factor (PF < 0.7), paired t-test (t < 0.05), Tukey post hoc (p < 0.05) test, Bray-Curtis similarity index, and matrix plot exhibited that all the four thermal processing methods have a considerable impact on pesticides in the processed shrimps. But frying (59.4%) and microwave cooking (60.3%) reduced PR far beyond boiling (48.8%) and grilling (51.3%). Hence, we recommend frying and microwave processing are better methods for minimizing PR in seafood than boiling or grilling.

Distribution and ecological risk assessment of heavy metals using geochemical normalization factors in the aquatic sediments.

Thamirabharani river acquires a noticeable quantity of sewage and agriculture waste from local inhabitants. The distribution of heavy metals in the surface sediments of the Thamirabharani river was analyzed using Inductively Coupled Plasma- Mass Spectrometry (ICP-MS) to study the ecological risks. The heavy metal concentrations in the sediments ranged from 0.098 ± 0.03(Cd) to 159.181 ± 13.36 mg kg-1 (Fe). The Cd, Zn, Ni, Fe, and Mn concentrations in the sediments were above the US Environmental Protection Agency-Sediment Quality Guidelines. The fact that Cd, Co, and Cu concentrations at sites 4 and 5 exceeded the background values (BGVs) of 0.2, 13, and 32 mg kg-1 suggests anthropogenic activity, notably in the downstream of the river. The sediment contaminated with Cd is more evident, particularly in the estuarine region. The potential ecological risk index (150

Dietary intake of trace elements from commercially important fish and shellfish of Thoothukudi along the southeast coast of India and implications for human health risk assessment.

The concentrations of eight trace elements (chromium, cobalt, copper, zinc, arsenic, cadmium, mercury and lead) in14 commercially important fish and shellfish collected from Thoothukudi along the southeast coast of India was investigated using inductively coupled plasma mass spectrometry in order to assess the health risks associated with their consumption. The concentration of trace elements ranged from 0.001 to 39.5 µg/g. The estimated weekly intake of cadmium in seven fish and shellfish (0.0081-0.0996 mg/kg body weight) were above the provisional tolerable weekly intake set by the Joint FAO/WHO Expert Committee on Food Additives. The risk assessment analysis indicated that there was non- carcinogenic risk upon lifetime consumption of rock crab, C. natator (TTHQ >1) and carcinogenic risks upon lifetime consumption of S. jello, P. semisulcatus, P. sanguinolentus C. natator, Uroteuthis duvaceli, Sepia pharaonis and Cistopus indicus due to cadmium exposure indicating a potential health risk to the exposed consumers.

Trace element concentrations in the organs of fish along the southeast coast of India.

Trace element pollution in the marine system is a global concern as the exposure of marine organisms to this pollution results in bioaccumulation and further transfer of the trace elements to humans through food chain. In the present study, the distribution of trace elements, namely chromium, cobalt, nickel, iron, copper, zinc, arsenic, cadmium, mercury, and lead, in gills, bone, liver, and muscle of eight commercially important fish collected along the southeast coast of India was analyzed using an inductively coupled plasma mass spectrometer. The liver was the main organ of accumulation for copper, zinc, arsenic, cadmium, and mercury; bone for chromium, cobalt, and lead; gills for copper; and muscle for arsenic and mercury. The concentration of toxic trace elements such as arsenic, cadmium, mercury, and lead in the edible portion of fish was lower than the recommended International Legislation limits, indicating that the fish of this region are safe for consumption.

Heavy metal concentration in reef-associated surface sediments, Hare Island, Gulf of Mannar Marine Biosphere Reserve (southeast coast of India): The first report on pollution load and biological hazard assessment using geochemical normalization factors and hazard indices.

In this study, reef-associated surface sediment samples were collected from Hare Island in the Gulf of Mannar Marine Biosphere Reserve, Bay of Bengal and analyzed for heavy metal concentration. The sediment quality was evaluated based on the geochemical, biological, and ecological hazard indices. The mean concentration of heavy metals in the sediments ranged from 0.02 (Cd) to 26,262.87 mg/kg (Fe). Except for Cd and Hg, all other elements were found to be below the sediment quality guidelines and contamination level. The biological and ecological hazard (BEHI) revealed that most sediment samples (80%) fell under the low-risk category with 9% probability of toxicity to the marine flora and fauna. The overall contamination level of heavy metals in Hare Island suggested that the sediment could be grouped under low-risk category. Hence, this study recommends the need for a routine monitoring program in this region to maintain a clean and sustainable ecosystem in future.

Risk assessment of organochlorine pesticides in seaweeds along the Gulf of Mannar, Southeast India.

Bioaccumulation of organochlorine pesticides (OCPs) in red, green, and brown seaweeds from the Gulf of Mannar was investigated using gas chromatography-mass spectrometry (GC-MS). The concentration of OCPs in all the seaweeds was in the order of endosulfan > endrin > HCH > DDT > aldrin > heptachlor > methoxychlor. The concentrations of HCH, endrin, endosulfan, and DDT in Sargassum wightii and Gelidiella acerosa, and HCH and endosulfan in Gracilaria verrucosa were above the limits prescribed by the European Commission. However, in Ulva lactuca, except for endosulfan, all other OCPs were below the permissible level. The bioaccumulation of OCPs in seaweeds was in the order of S. wightii > G. acerosa > Gracilaria verrucosa > U. lactuca. The results of comparative analysis of the levels of the investigated pesticides against the standard human health risk assessment revealed that pesticide exposure from seaweeds is hazardous to human health and the marine environment of the Gulf of Mannar.

Human health risk assessment of heavy metals in aquatic sediments and freshwater fish caught from Thamirabarani River, the Western Ghats of South Tamil Nadu.

Industrialization and advancements in agriculture are increasingly the cause of environmental concerns and need to be addressed. This study was carried out at four sampling sites, viz. Manimuthar (site 1), Tirunelveli (site 2), Srivaikuntam (site 3), and Authoor (site 4), along the Thamirabarani river. The heavy metal concentration in fish and sediments were ranged from 0.001 to 9.505 mg kg-1 and 0.294 to 106.25 mg kg-1. The lifetime cancer risk (LCR) values for children and adults were found to be above the acceptable threshold value (ATV) and children were found to be more susceptible to health risks. For all sites except site 4, the LCR of toxic metals except Cd were within the ATV (10-6-10-4); the value of Cd was above the ATV, and poses a high cancer risk to the downstream inhabitants. This study advises routine heavy metal monitoring on aquatic organisms along these sites to implement regulatory standards.

Concentrations of trace elements in the organs of commercially exploited crustaceans and cephalopods caught in the waters of Thoothukudi, South India.

Concentrations of trace elements (arsenic, cadmium, mercury, and lead) in tissues (muscle, gills, and digestive gland) of three commercially exploited crustaceans (Portunus sanguinolentus, Charybdis natator, and Penaeus semisulcatus) and three cephalopods (Doryteuthis sibogae, Sepia pharaonis, and Cistopus indicus) were examined. The animals were captured in the waters of Thoothukudi, and the tissues of six individuals of each species were analyzed using ICP-MS. The highest concentrations of arsenic (16.5 µg/g) and mercury (0.052 µg/g) were recorded in the digestive gland of C. natator, and cadmium (69.9 µg/g) and lead (0.351 µg/g) in the digestive gland of S. pharaonis. The edible portion of the crustaceans and cephalopods contained lower concentrations of trace elements, and these were below allowable limits set by the European Union. The edible parts of the investigated samples are safe for human consumption, but accidental contamination of the edible tissues with material from the digestive glands could lead to concerns relating to metal toxicity.

A single enzyme PCR-RFLP protocol targeting 16S rRNA/tRNAval region to authenticate four commercially important shrimp species in India.

Food authenticity is an issue of major concern for food authorities, as mislabeling represents one of the major commercial frauds. In this study, a novel PCR-RFLP protocol was developed as a tool to authenticate four shrimp products of commercial importance belonging to the family, Penaeidae, viz. Litopenaeus vannamei, Penaeus monodon, P. semisulcatus and Fenneropenaeus indicus. PCR amplification was performed targeting 16S rRNA/tRNAval region having an amplicon size of 530bp using the specific primers for shrimps, 16S-Cru4/16S-Cru3. Subsequent restriction analysis with a single restriction enzyme, Tsp5091, yielded distinct RFLP pattern for each species of shrimps having fragment sizes below 150bp. The unique RFLP patterns were also obtained in processed shrimp products without any degradation or alteration in the major fragments. The method was also validated with commercial shrimp products. Thus, the developed protocol can be performed within 8h using a single enzyme to authenticate four shrimp products of commercial significance.

Effect of additives in the shelflife extension of chilled and frozen stored Indian octopus (Cistopus indicus).

In this study, the effect of commercial additives viz. cafodos and altesa employed to treat Indian octopus (Cistopus indicus) was examined during chilled and frozen storage. Shelf lives of treated and untreated octopus in ice were 6 and 8 days, respectively in ice. Treated and untreated frozen octopus had a shelf life of 40 days. Autolytic and microbiological changes were not controlled by the additives, as evidenced through rapid reduction in non-protein nitrogen (NPN) and α-amino nitrogen (α-AN) compounds; as well as accumulation of water soluble ammoniacal nitrogen and total volatile base- nitrogen (TVB-N) compounds. Loss of texture and colour were the major quality defects noticed in treated octopus as a result of enhanced protein solubility. Therefore, the additives approved for use in octopus neither enhanced the shelf life nor improved the sensory quality.

Antioxidant activities of squid protein hydrolysates prepared with papain using response surface methodology.

Squid protein hydrolysates (SPH) were prepared from the Indian squid Loligo duvauceli using papain. Response surface methodology (RSM) was used for optimization of hydrolysis conditions, including temperature, time, and the enzyme-substrate ratio using DPPH radical scavenging activity as a response. The amino acid composition of SPH was compared with raw squid muscle. In vitro antioxidant activities were evaluated based on reducing power, metal chelation, ABTS, hydroxyl radical, and superoxide anion radical scavenging assays. SPH exhibited good ABTS radical scavenging activities of 96.50±0.90%, superoxide anion radical scavenging activities of 96.4±0.89%, reducing powers of 0.71±0.02, moderate hydroxyl radical scavenging activities of 64.03±2.11%, and metal chelating activities of 52.04±1.02%. In vivo antioxidant activities determined using a sardine minced model system showed 42% reduction in formation of secondary oxidative products as thiobarbituric acid reactive substances (TBARS), almost equivalent to reduction by ascorbic acid of 41.42% at 400 ppm.