Effect of refrigerated storage on the biochemical, microbial and functional properties of processed amur common carp (Cyprinus carpio haematopterus) meat products

Introduction

India has witnessed a significant transformation in its fisheries sector over the past two decades. Inland fish production increased remarkably from 2.823 million metric tons (MMT) in 2000-01 to 13.11 MMT in 2023-24, marking nearly a 400% rise (DAHDF, 2023) [1] . This expansion is largely attributed to the rapid growth of aquaculture, which involves the cultivation and harvesting of fish in freshwater ecosystems such as lakes, ponds, rivers, and reservoirs. Between 2014 -15 and 2021-22, the sector recorded an impressive average annual growth rate of 8.61%. Inland fisheries now account for 75% of India's total fish production, while marine fisheries contribute the remaining 25%, reaffirming the strategic importance of inland aquaculture to national fish output. With an extensive inland water surface area of 314,400 km², India ranks among the top countries globally in terms of inland fishery resources (DAHDF, 2023) [1]. In recent years, attention has turned to the development of value-added fishery products such as surimi, a refined fish protein concentrates widely used in imitation seafood. Traditionally, marine fish like Alaska pollock and threadfin bream have been the preferred sources for surimi. However, with increasing raw material costs and supply limitations, the focus has shifted toward freshwater carps as promising alternatives due to their high availability, low production cost, and favourable processing characteristics. Several Asian carp species, including grass carp, silver carp, bighead carp, and black carp, are currently being explored for surimi production, particularly in China and Southeast Asia. Research by Endoo and Yongsawatdigul (2014) [2] highlighted the gel-forming potential of Asian carps, although studies on their gelation properties compared to marine species remain limited. In India, Indian major carps such as Catla catla, Labeo rohita, and Cirrhinus mrigala dominate inland aquaculture due to their adaptability to lowinput systems. These species contributed approximately 5.4 MMT to the country's inland fish production in 2023 (FAO, 2013) [3] . However, emerging carp species like the Amur common carp (Cyprinus carpio haematopterus) are gaining recognition for their superior growth performance and economic potential. Introduced by Karnataka Veterinary, Animal and Fisheries Sciences University (KVAFSU), Bidar, the Amur carp is a genetically improved strain of common carp characterized by 27% faster growth, delayed sexual maturation, greater feed conversion efficiency, and lower disease susceptibility (Vijayakumar et al., 2020) [4] . Field trials across various ecological zones, including hilly areas like Meghalaya, demonstrated consistent growth and adaptability (Das, 2017; Sanjay Kumar, 2017) [5,6] . India's total fish production reached 18.402 MMT in 2023-24, a substantial increase from 9.579 MMT in 2013-14. Inland fisheries contributed 13.9 MMT to this total, with Karnataka alone producing 1.225 MMT of carp in 2023 (GoI PIB, 2025) [7] . While specific production data for Amur carp is not yet officially reported, its cultivation is expanding, particularly in Karnataka's seasonal water bodies. The processing qualities of Amur carp make it especially suitable for surimi production. Its firm, white flesh is rich in myofibrillar proteins essential for gelation and textural integrity in surimi products. The low fat and oil content reduce the risk of lipid oxidation, enhancing shelf life and sensory quality. Its mild flavor allows for versatility in surimi-based applications. Moreover, the Amur carp's resilience and compatibility with sustainable farming practices make it an environment friendly choice. Lingaraju et al., 2025 [8] noted that Amur carp combines economic viability, processing efficiency, and desirable sensory traits, making it a superior raw material for large-scale surimi production. Given these attributes, the Amur carp holds significant promise for enhancing the sustainability and profitability of India's value-added fish product sector.

The aim of this study is to evaluate the biochemical, microbial, and functional changes in processed Amur common carp meat products during refrigerated storage. It investigates how storage duration affects quality parameters such as lipid oxidation, protein stability, and microbial load across various surimi-based formulations.

Materials and Methods

Farm raised freshwater Amur common carp was procured from a local fish farm located around Karnataka, India. The average length and weight of fish used in this study was measuring a standard-length of 35.24 cm, and average weight of fish 836.34 gm respectively.

Surimi Preparation

The fish was dressed, and fillets were separated from the both sides of the fish using a sharp knife. The fillets were fed to the reciprocating type meat picking machine. Picked meat was used in 4 batches. One batch of Picked meat (unwashed) was used as control (Amur Carp Minced Meat-ACMM). Minced meat was prepared from picked meat, mixed with cryoprotectants at the rate of sugar 4%, sorbitol 4%, sodium polyphosphate 0.2% and ascorbic acid 0.5%. Fish meat was packed in polyethylene bags (500g) frozen at -35 o C by Plate freezer for ninety minutes and stored at -20 o C (Amur Carp Surimi-ACS). A part of minced meat was transferred to a bowl chopper, mixed and ground with dilute alkali solution sodium bicarbonate at rate of 0.2%, and washed thoroughly in chilled water, dewatered to prepare High protein amur surimi (HPAS). HPAS was mixed with cryoprotectants, packed and stored at -20 o C. The last batch of minced meat was converted to Amur Carp Flesh Powder (ACFP) by suitable modification (Majewska et al., 2025) [9] and stored.

Biochemical Analysis

Biochemical quality indicators were also evaluated during refrigerated storage. The pH of fish meat was measured using a digital pH meter after homogenizing 5 g of sample with 45 ml of distilled water (AOAC, 2010) [10] . Total volatile base nitrogen (TVB-N), which reflects the degree of spoilage, was determined by Conway's micro-diffusion technique (Conway, 1962) [11] . Peroxide value (PV), indicative of lipid peroxidation, was estimated by extracting fat with chloroform, followed by reaction with potassium iodide and titration with sodium thiosulphate (Jacobs, 1951) [12] . Free fatty acid (FFA) content was determined by titrating lipid extracts with sodium hydroxide using phenolphthalein as an indicator and was expressed as a percentage of oleic acid (Pearson, 1976) [13] . All analyses were conducted in triplicate, and results were expressed as mean ± standard deviation.

Microbiological Analysis Total Plate Count (TPA)

The total plate count was determined according to the procedure described in APHA (2000) [14] . Enumeration of bacterial load was done using plate count agar (Hi-media Laboratories, Mumbai) by following spread plate technique. About 25 g of the sample prepared from Amur carp minced meat, surimi, flesh powder and high protein amur surimi sample was homogenized aseptically with 225 ml of sterile physiological saline (0.85% NaCl) for 5 min in a sterilized pestle and mortar. Appropriate dilutions were made from the homogenate, and 0.1 ml was placed on sterile total plate count (TPC) agar plates. The plates were incubated at 37 °C for 24 to 48 hours. The number of colonies were counted and expressed as number of colony forming units (CFU) per gram of sample.

Functional Characteristics Estimation of non-protein nitrogen (NPN)

Non-protein nitrogen content was determined by the method as described by Velankar and Govindan (1958) [15] . About 3.0 g of meat sample was macerated with 10 ml of 20% TCA for 5 minutes using a pestle and mortar. The slurry was allowed to settle at refrigerated temperature (6-80 C) for 30 min. The slurry was filtered and made up to 50 ml with distilled water and an aliquot of 5 ml was taken for nitrogen estimation by Kjeldahl method. The NPN content was expressed as mg N/100 g meat.

Protein Solubility

Solubility of proteins in extraction buffer (Phosphate buffer 50 mM, pH 7.5, 1.0 M NaCl) was determined for fresh and frozen stored samples. This buffer containing 1.0 M NaCl is termed as high ionic strength buffer. About 5 g of meat was homogenized with extraction buffer (EB) in the ratio of 1:10 (meat: EB) for 2 minutes using a homogenizer (Ultra Turrax T25, Janke and Kunkel, Staufen, Germany). The homogenate was centrifuged at 12000 × g at 4 ℃ for 15 min using high speed refrigerated centrifuge (Sorvall Legand XTR centrifuge, Thermo Fisher Scientific, New Hampshire, USA). The protein content of the supernatant was determined by Kjeldahl method. The solubility of the protein was expressed as percentage of total protein in the meat.

Statistical Analysis:

All the experiments were performed in triplicates and expressed as the mean ± standard deviation (n = 3). One-way Analysis of Variance (ANOVA) followed by Tukey's test in SPSS statistics software (version 27.00) were performed to test the significance differences through the storage time.

Results and Discussion

This study evaluated the biochemical, microbial, and functional stability of Amur common carp (Cyprinus carpio haematopterus) processed into four products ACMM, ACS, ACFP, and high-protein surimi (HPAS) during 28 days of refrigerated storage. Key quality indicators (pH, TVB-N, TBA, PV, FFA, TPC, NPN and SSN) were assessed to monitor spoilage, oxidation, and protein degradation.

Biochemical Analysis

The initial pH of all samples ranged from 6.67 to 6.81. HPAS showed the highest pH due to the use of dilute alkali (NaHCO₃) during alkaline washing, which neutralizes endogenous acids and stabilizes muscle proteins (Chotimarkorn & Yongsawatdigul, 2009) [16] . A gradual decline in pH was observed in all treatments over 28 days, particularly in ACMM and ACFP, which are more susceptible to microbial activity and proteolytic breakdown (Ojagh et al., 2010) [17] . In contrast, ACS and HPAS maintained higher pH stability, consistent with reduced microbial substrates following surimi processing and the effect of cryoprotectants in suppressing pH-lowering microbial metabolism (Benjakul et al., 2001) [18] . [19] . HPAS maintained the lowest TVB-N values (7.81 mg/100g), validating that alkalinewashed surimi removes non-protein nitrogenous compounds, reducing spoilage potential (Liu et al., 2013) [20] . The TVBN levels in Amur carp surimi and flesh powder reached 10.08% and 9.46%, respectively. These findings are consistent with the acceptability threshold of 20 mg/100g for fish products (ICMSF, 2011) [21] . [22] . Surimi products (ACS and HPAS) exhibited much lower PVs (4.44 and 2.91 meq O₂/kg, respectively), due to removal of lipid substrates during washing, which is known to improve oxidative stability (Park et al., 2006) [23] . ACFP remained the most stable (1.44 meq O₂/kg) due to low water activity and reduced enzymatic and microbial activity (Rawdkuen et al., 2008) [24] . The thiobarbituric acid reactive substances (TBA-RS) values showed a statistically significant increase (p<0.005) across all product categories during the storage period, reflecting the progression of lipid oxidation. Initially, the highest TBA value was observed in ACMM (0.40 mg MDA/kg), while HPAS exhibited the lowest (0.12 mg MDA/kg), suggesting superior lipid stability in HPAS likely due to better washing efficiency or endogenous antioxidant retention (Zhang et al., 2022) [25] . All samples showed marked oxidative advancement by day 7, with HPAS rising to 0.44 mg MDA/kg and ACMM reaching 0.62 mg MDA/kg. At the day 14, ACMM and ACFP further escalated to 0.71 and 0.66 mg MDA/kg respectively, indicating increased susceptibility of flesh powder to lipid peroxidation (Chakraborty et al., 2009) [26] . An accelerated oxidation phase was evident on day 21, as ACMM, ACFP, and HPAS displayed converging TBA values between 0.75-0.92 mg MDA/kg, suggesting depletion of protective factors and intensified oxidative stress (Murthy et al., 2021) [27] . On day 28, the peak TBA values were recorded ACMM at 1.24, ACFP at 0.97, ACS at 0.96, and HPAS at 0.85 mg MDA/kg. Although HPAS started with lower TBA levels, its relatively high end-point oxidation implies that even highly processed surimi types are vulnerable over time, depending on storage conditions and antioxidant dynamics. These results align with established understanding that TBA values correlate with lipid degradation in fish-based systems and demonstrate the need for targeted preservation strategies in surimi products (Janero, 1990; Shahidi, 1990) [28,29] . [30] . The FFA level of minced meat was 2.42%, while it was reduced to 1.50% in surimi. Surimi's lower TBA values are attributed to the reduction of pro-oxidants like sarcoplasmic proteins and lipids (Zhang et al., 2022) [25] . Alkaline washing also improves antioxidant capacity by promoting the removal of oxidation catalysts (Park et al., 2006) [23] . Negligible FFA levels were observed in Amur carp flesh powder and high protein surimi. After four weeks of refrigerated storage, an increase in FFA levels was noted. Minced meat exhibited the highest FFA percentage at 3.95%, followed by surimi at 2.53%. However, both high-protein surimi and Amur carp flesh powder maintained FFA levels within acceptable limits, recording 1.01% and 0.47% of oleic acid, respectively. HPAS and ACFP maintained significantly lower FFA accumulation (<1.1%), consistent with previous findings showing reduced lipid hydrolysis in low-fat or washed surimi products (Gómez-Guillén & Montero, 2005) [31] . [17,21] . [32] . ACMM had the highest initial NPN, a reflection of endogenous enzymatic hydrolysis and retained sarcoplasmic proteins (Maqsood & Benjakul, 2011) [33] . ACFP's lower NPN compared to ACMM and surimi products is attributed to heat treatment, which reduces enzymatic activity (Gokoglu et al., 2004) [34] . In contrast, HPAS and ACS had significantly lower NPN content due to repeated washing, which removes water-soluble nitrogenous substances (Benjakul et al., 2003) [35] .

Fig 1:

Changes in the non-protein nitrogen during refrigerated storage

Salt-Soluble Nitrogen (SSN)

Salt-Soluble Nitrogen (SSN), an indicator of myofibrillar protein functionality, showed a gradual decrease across all treatments. ACMM declined from 80.19% to 62.16%, consistent with denaturation and aggregation during storage (Shahidi, 1994) [36] . Surimi products retained higher SSN, particularly HPAS (75.46% to 69.62%), due to the alkaline solubilization and cryoprotectants, which preserve protein integrity and functionality (Park et al., 2006; Wang et al., 2022) [23,37] . ACFP showed minimal change, possibly due to low proteolytic activity in the dry state (Rawdkuen et al., 2008) [24] .

Conclusion

The study demonstrates that processed Amur common carp meat products exhibit varying degrees of biochemical, microbial, and functional stability during refrigerated storage, with high-protein surimi (HPAS) consistently showing superior performance. Surimi-based formulations, particularly HPAS and ACS, maintained lower levels of lipid oxidation, protein degradation, and microbial proliferation compared to minced meat and flesh powder, confirming the efficacy of washing and cryoprotectant treatments. These findings support the suitability of Amur carp for value-added processing and reinforce its potential as a sustainable and high-quality raw material for India's surimi industry.

References

1. Department of Animal Husbandry, Dairying and Fisheries (DAHDF). Handbook on Fisheries Statistics 2023. Ministry of Fisheries, Animal Husbandry & Dairying, Government of India; 2023.
2. Endoo N, Yongsawatdigul J. Comparative study on chemical and gel-forming properties of surimi from freshwater and marine fish during frozen storage. Food Appl Biosci J. 2014;2(3):192-202.
3. Food and Agriculture Organization (FAO). Global Aquaculture Production statistics. Rome: FAO; 2013.
4. Vijayakumar S, Khavi M, Atnur VS. Comparative study on growth performance of amur common carp and Indian major carps in ponds integrated with and without poultry farm. J Entomol Zool Stud. 2020;8(1):26-29.
5. Das S. Performance of an improved breed of common carp -Amur (Hungarian strain) in the North-Eastern hill state of Meghalaya, India. Indian J Fish. 2017;64:33-38.
6. Kumar S. Aquaculture trials of Amur carp in hilly regions of North-East India. Mumbai: Central Institute of Fisheries Education; 2017.
7. Press Information Bureau, Government of India (GoI PIB). Casting Nets, Catching Success: India's fisheries on the rise. GoI; 2025.
8. Lingaraju GP, Bojayanaik M, Chavan SD, Fernandes DJ. Changes in proximate composition and physicochemical properties of fresh Amur carp (Cyprinus carpio haematopterus) during ice storage. J Adv Biol Biotechnol. 2025;28(6):227-34.
9. Majewska E, Twarda-Clapa A, JędrzejczakKrzepkowska M, Kamińska-Dwórznicka A, ZakłosSzyda M, Białkowska AM. Antifreeze proteins produced by Antarctic yeast from the genus Glaciozyma as cryoprotectants in food storage. PLoS One. 2025;20(3):e0318459.
10. AOAC International. Official Methods of Analysis. 18th ed. Washington, D.C.: Association of Official Analytical Chemists; 2010.
11. Conway EJ. Microdiffusion Analysis and Volumetric Error. 5th ed. London: Crosby Lockwood and Son Ltd; 1962.
12. Jacobs MB. The Chemical Analysis of Foods and Food Products. 2nd ed. New York: Van Nostrand; 1951.
13. Pearson D. The Chemical Analysis of Foods. 7th ed. London: Churchill Livingstone; 1976.
14. American Public Health Association (APHA). Standard Methods for the Examination of Water and Wastewater. 23rd ed. Washington, D.C.: APHA; 2000.
15. Velankar NK, Govindan TK. A preliminary study of the distribution of non-protein nitrogen in some marine fishes and invertebrates. Proc Indian Acad Sci B. 1958;47(4):202-209.
16. Chotimarkorn C, Yongsawatdigul J. Influence of washing and pH on gelation characteristics of surimi. Food Chem. 2009;114(1):120-126.
17. Ojagh SM, Rezaei M, Razavi SH, Hosseini SM. Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. Food Chem. 2010;120(1):193-198.
18. Benjakul S, Sumpavapol P, Visessanguan W, Tanaka M. Effect of freezing and frozen storage on muscle proteins of some tropical fish. Food Chem. 2001;82(2):269-277.
19. Hosseini SV, Rezaei M, Zandi M, Farahmandfar R. Effect of washing cycles on chemical composition and gel properties of surimi from silver carp. Iran J Fish Sci. 2010;9(3):508-519.
20. Liu R, Zhao SM, Xiong SB, Xie BJ. Effect of washing cycles on functional properties of surimi from grass carp. J Food Sci. 2013;78(4):E422-E429. 21. International Commission on Microbiological Specifications for Foods (ICMSF). Microorganisms in Foods 7: Microbiological Testing in Food Safety Management. New York: Springer; 2011.
21. Shahidi F, Zhong Y. Lipid oxidation and improving the oxidative stability in fish muscle. Crit Rev Food Sci Nutr. 2005;45(7-8):479-494.
22. Park JW, Lanier TC, Regenstein JM. Fish protein functionality: Oxidative stability during surimi processing. Crit Rev Food Sci Nutr. 2006;46(5):439455.
23. Rawdkuen S, Benjakul S, Kishimura H. Effect of drying on lipid oxidation and shelf life of fish powders. Food Chem. 2008;110(1):66-72.
24. Zhang X, Zhang T, Wang Q, Qiu H, Zhou Y. Effect of washing times on the quality characteristics and protein oxidation of silver carp surimi. Foods. 2022;11(16):2397. Available from: https://doi.org/10.3390/foods11162397
25. Chakraborty A, Dora KC, Sarkar S, Chowdhury S. Shelf-life of surimi prepared from Tilapia during frozen storage. Asian J Anim Sci. 2009;4(1):18-21.
26. Murthy LN, Phadke GG, Jeyakumari A, Ravishankar CN. Effect of added calcium and heat setting on gel forming and functional properties of Sardinella surimi. J Food Sci Technol. 2021;58:427-436.
27. Janero DR. Malondialdehyde and thiobarbituric acidreactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med. 1990;9(6):515-540.
28. Shahidi F. Evaluation of warmed-over flavor and oxidative rancidity in meat products. Proc Int Congr Meat Sci Technol. 1990;9:1008-1013.
29. Maqsood S, Benjakul S. Lipid oxidation and lipolysis in fish and fish products: A review. Food Chem. 2010;120(3):675-686.
30. Gómez-Guillén MC, Borderias J, Montero P. Lipid oxidation and antioxidant protection in fish muscle products. Trends Food Sci Technol. 2005;16(1112):560-567.
31. Chaijan M, Benjakul S, Visessanguan W. Changes in protein degradation and quality of sardine mince during iced storage. Food Chem. 2006;99(4):656-663.
32. Maqsood S, Benjakul S. Effect of pH and washing cycles on the gel properties and biochemical changes of surimi from threadfin bream. Food Chem. 2011;126(3):1141-1149.
33. Gokoglu N, Yerlikaya P, Cengiz EI. Effects of cooking methods on the proximate composition and mineral contents of rainbow trout. Food Chem. 2004;84(1):1922.
34. Benjakul S, Sumpavapol P, Visessanguan W, Tanaka M. Effect of freezing and frozen storage on muscle proteins of some tropical fish. Food Chem. 2003;82(2):269-277.
35. Shahidi F. Lipid oxidation and improving the oxidative stability. Chem Soc Rev. 1994;23(1):23-31.
36. Wang H, Liu J, Zhang X. Effects of sarcoplasmic protein removal on surimi gel properties. J Food Sci. 2022;87(5):2104-2111.