The evaluation of the three edible tissues of dead adult Chinese mitten crabs (Eriocheir sinensis) freshness in harvest season, based on the analysis of TVBN and biogenic amine
© The Author(s) 2016
Received: 20 May 2016
Accepted: 29 September 2016
Published: 3 November 2016
This study was carried out to evaluate the quality of the three edible tissues (muscles, hepatopancreas and gonads) of the male and female Chinese mitten crab (Eriocheir sinensis) in natural harvest season, based on total volatile base nitrogen test (TVBN) and biogenic amines analysis at intervals time of 3 h extended for 24 h during 30 °C storage (a mean ambient temperature in harvest season). In addition, the relationship between the value of TVBN and storage time or main biogenic amine was evaluated. Results showed that TVBN level of female gonad reached the recommended limit (30 mg/100 g) at 3 h, and the times of reaching the recommended limit were 15 h in both sexes muscles, while in male and female hepatopancreas reached the recommended limit at times of over 24 and 24 h respectively. Moreover, putrescine was highest in amount in all biogenic amines analyzed. We suggested that TVBN value could be as a good indicator for Chinese mitten crab freshness. According to analysis for biogenic amine accumulated in edible tissues of crab, it was possible that dead crab poisoning may be caused by putrescine. Dead Chinese mitten crabs for over 24 h, should be not recommended for eating or processing.
KeywordsChinese mitten crab Total volatile base nitrogen test Biogenic amines Freshness Food safety
The Chinese mitten crab (Eriocheir sinensis) is a traditional seafood source in China, where it supports an important aquaculture industry yielding high annual production, worth approximately $1.25 billion (Veilleux and de Lafontaine 2007; Cheng et al. 2008). The muscles and hepatopancreas and gonads are the most prized parts of the crab. These tissues are also usually considered as edible tissues of Chinese mitten crabs. The preferred crabs are captured during the fall, and are autumn delicacy in Shanghai cuisine and eastern China, as they have full gonads prior to reproduction (Cheng et al. 2008). The average ambient temperature in this season is 30 °C, which is highest temperature among the average of season temperature in the whole year. Chinese mitten crabs are usually cooked alive, which are similar to cooking Lobsters. However, the edible tissues of Chinese mitten crabs collected at autumn are often processed and stored in order to meeting the need of human consummation at any time. Although, these crabs (normal adult crabs and precocious crabs) collected in harvest season were generally processed after cooking, but uncooked crabs could also be processed (Gillespie et al. 1983). Moreover, for acquiring high profit, dead and deteriorating crabs were possibly also collected for processing by processors. Therefore, it is very important to evaluate the quality of crab in order to reduce the risk of eating crab.
At the present, there are two main methods of assessing seafood quality to determine its freshness. These are sensory and non-sensory methods. Sensory methods rely mostly on appearance, odors, texture and taste of the seafood whether to be acceptable or rejected while non-sensory methods use physical, biochemical, chemical and microbiological means (Huss 1995). Among the non-sensory methods, total volatile nitrogen (TVBN) test is known as an important and popular test method for evaluating various seafood freshness, such as crab, fish and mullet etc., and is well correlated with sensory analysis (Zamir et al. 1998; Özogul and Özogul 2000; Özyurt et al. 2009; Zhang et al. 2012). For example, Özyurt et al. (2009) found that TVBN values of red mullet (Mullus barbatus) and goldband goatfish (Upeneus moluccensis) were well correlated with sensory analysis.
TVBN is mainly composed of ammonia and primary, secondary and tertiary amines. The TVBN content of sea food increases as putrefaction progresses since ammonia is produced during storage as a result of the deamination of amino acids. Min et al. (2007) has also confirmed the quantities of biogenic amines (BAs) concentration are highly correlated with the TVBN concentration in beef, pork, and chicken during storage. So far it has been accepted that the quantities of BAs are also to be considered as a marker of the level of microbiological contamination in food (Vinci and Antonelli 2002a, b; Min et al. 2004a). BAs are organic compounds with a low molecular weight that are formed through the enzymatic decarboxylation of specific amino acids in various foods during storage (Hernandez-Jover et al. 1996; Min et al. 2004b). Especially, BAs are produced in foods, where high levels of protein are present, for example in seafood (Vinci and Antonelli 2002a, b). The BAs that are often found in foods including putrescine (PUT), cadaverine (CAD), histamine (HIM), tyramine (TYM), serotonin (SER), spermine (SPM) and spermidine (SPD). PUT is formed from ornithine, CAD from lysine, HIM from histidine, TYM from tyrosine, SER from TRM, SPM from PUT and SPD from SPM (Halasz et al. 1994; Chen et al. 2002). In addition, dopamine (DA) and octopamine(OA) have been found in spoilage sea food (Özyurt et al. 2009; Hernandez-Jover et al. 1996). Among BAs, HIM has been more frequently implicated in food poisoning, and PUT and CAD are known to enhance histamine poisoning (Chen et al. 2002). Histamine scrombroid/toxicity accounted for 35% of the documented seafood-associated outbreaks (Center for Science in the Public Interest 2009). Therefore, although there are no any reports about analysis of biogenic amine of dead crab and deteriorating crab collected at autumn, people commonly considered that an amount of histamine contained in dead crab and deteriorating crab, and which eventually resulted in the people fear of eating them.
In the current study, it is the first time to evaluate the quality of the edible of both the male and female Chinese mitten crab in 24 h after dead at 30 °C based on TVBN test. Moreover, we have further investigated the changes of biogenic amine of Chinese mitten crab edible tissues during 24 h of storage, and the relationship between the value of TVBN and storage time or main biogenic amine was evaluated.
The adult normal Chinese mitten crabs were collected from the Aquaculture Technology Extension Station in Shanghai Chongming (China), and were immediately brought back to the laboratory. The average of carapace length, carapace width and body weight were 59.92 ± 6.00, 64.25 ± 2.02 mm and 122.72 ± 14.63 g for male respectively, and 57.11 ± 6.32, 60.12 ± 3.41 mm and 95.53 ± 15.14 g for female respectively. On arrival, the crabs were executed by −80 °C refrigerator storage for 2 h. Each dead crab was individually packaged by plastic bag, and all of them were stored in a cabinet with the temperature controlled at 30 ± 1 °C. The three edible tissues of crab including muscle (the whole body muscle), hepatopancreas and gonad were collected for future analysis at intervals time of 3 h extended for 24 h. Each analysis was performed in triplicate.
Determination of total volatile base nitrogen (TVBN)
TVBN was determined with a Kjeltec System 2300 (Foss Tecator, USA) (AOAC 1995). Bases from the weighed samples were distilled in the presence of magnesium oxide. The distillate was collected in a solution of excess boric acid in the presence of indicators (methyl red mixed with bromocresol green) and titrated with 0.1 N hydrochloric acid. The TVBN value was expressed as mg N/100 g sample as described by Xing et al. (2012).
Biogenic amine analysis
Biogenic amine analysis was carried out according to Min et al. (2007) and Ding et al. (2006). Two grams of the sample was weighed into a 50 mL polypropylene conical tube and homogenized in 10 mL of 0.4 M perchloric acid. The homogenized sample was centrifuged for 10 min at 3000 rpm and rinsed with supernatant into a 25 mL bottle through filter paper. The extraction was repeated with 10 mL of 0.4 M perchloric acid solution, mixed thoroughly with a vortex mixer and centrifuged as above. Supernatants were combined and adjusted to 25 ml with 0.4 M perchloric acid solution.
The alkalinity of a 1 mL sample extract was adjusted using 100 μL of 2 mol/L sodium hydroxide solution. A 300 μL saturated sodium bicarbonate was added as a buffer. A 2 mL dansyl chloride solution (10 mg/mL acetone) was added and incubated at 40 °C for 45 min in the dark. Residual dansyl chloride was removed by adding 100 μL ammonia (25%). After 30 min, dansylated extract was adjusted to 5 mL with 0.1 mol/L ammonium acetate/acetonitrile (1:1), and filtered through a 0.45 μm syringe filter. Ten microliters of a filtered sample was injected to the HPLC with a diode array detector (LC-10Avp series; Shimadzu, Kyoto, Japan) equipped with a C18 column (4.6 × 150 mm,shim-pack, Shimadzu, Kyoto, Japan). Gradient elution program was used with a mixture of 0.1 M ammonium acetate as solvent A and acetonitrile as solvent B. Both solvents were vacuum filtered by a membrane filter (47 mm PTFE 0.45 μm, Shanghai ANPEL Co., Ltd.) and degassed with an ultra-sonicator. The flow rate was 1 mL/min. The order of the gradient was shown as follows: 45% solvent A and 55% solvent B for 0–7 min, 50% solvent A and 50% solvent B for 7–25 min, 10% solvent A and 90% solvent B for 25–35 min, 45% solvent A and 55% solvent B for 35–45 min. The column temperature was 40 °C. The amounts of the dansyl derivatives of the biogenic amines were quantified by a measurement of the UV-absorption at 254 nm.
Statistical analysis was performed using the SPSS software (Chicago, USA; Version 17.0). The data of relative expression of the gene which was run in different duplications and levels were used in the statistical analysis. Data represented the mean ± S.E. Statistical significance was determined using one-way analysis of variance and post hoc Duncan multiple range tests. P < 0.05 indicated statistical significance. Pearson’s correlation coefficients were used to determine the relationship between BAs and TVBN.
Results and discussion
Pearson’s correlation coefficients between volatile basic nitrogen time and PUT concentration versus TVBN
Muscles ♂ (♀)
Hepatopancreas ♂ (♀)
Gonads ♂ (♀)
This study found that the TVBN contents of muscle and hepatopancreas of both male and female, and male gonad at 0 h were generally in the range of 7.9–9.63 mg/100 g. However, the TVBN content of female gonad (27.50 mg/100 g) was higher than that of other tissues on day 0. Besides rich proteins, the higher beginning value of TVBN may be also one of the reasons that the value of TVBN value for female gonad was earliest reaching recommended limit, compared to other tissues. Chomnawang et al. (2007) have reported that TVBN content of hybrid catfish fillet samples on day 0 at 4 °C was in the range of 15.73 and 16.91 mg/100 g. Gormley et al. (2003) found that TVBN of raw whiting fillet, mackerel fillet and salmon portions chilled on day 0 at 4 °C was 13.7, 15.9 and 17.0 mg/100 g respectively. Yamanaka (1990) reported that TVBN of saury pike stored at 5 °C was 5.5 mg/100 g during storage period on day 0. These results revealed that the beginning contents of TVBN were different in different animals and tissues. Moreover, above researchers suggested that the higher levels of TVBN in muscles at day 0 of storage may be related to fish muscle undergoing some deterioration during handling (Benner et al. 2003). However, for female gonad, the higher TVBN values at 0 h might be referring to handing or biological enzymes activity. Further researches are needed to explain the reason of the higher TVBN values at 0 h.
Among the nine biogenic amines, only PUT, CAD and OA were measured in crab edible tissues. In addition, the types of biogenic amines measured and their levels were different among the kinds of tissues. PUT could be measured in all tissues. CAD could only be found in male muscle and hepatopancreas, and female muscle, whereas OA could be found in hepatopancreas and gonad. So far, using the quantity of BAs as an index for the freshness of meat has been tried by many researchers. Mietz and Karmas (1977) proposed a sum of concentrations of HIM, PUT, CAD, SPM and SPD could be as an index of freshness for canned tuna. Later, Veciana-Nogues et al. (1997) reported that the TYM content increased significantly during tuna storage and the authors suggested that TYM content was the sum of the concentrations of HIM, CAD, TYM and PUT. Min et al. (2007) have confirmed that the content of PUT, CAD, and TYM could be as a good index for evaluating fresh beef, pork, and chicken meat freshness during storage. All of above results demonstrated that PUT and CAD were common biogenic amines, and they could be an index for evaluating freshness for various food products. Similarly, Benner et al. (2003) also found PUT and CAD are suitable for indicating decomposition in penaeid shrimp, especially for PUT. The present study, only PUT was measured in all tissues and was always higher in amount than other biogenic amines. For example, the average of PUT levels in male and female muscle, hepatopancreas and gonad at 24 h was 16.28 μg/g, 22.88 and 13.7 μg/g respectively. In comparison to that, the average amount of CAD in both sexes muscle at 24 h was 8.01 μg/g (Fig. 2a). In the hepatopancreas, the average content of CAD in male and female was 6.18 μg/g, and that of OA was 6.36 μg/g (Fig. 2b). In the gonads of both males and females, the average amount of OA was only 4.15 μg/g (Fig. 2c). Figure 2a shows that only Put and CAD could be measured in muscle. As storage time increased, the PUT levels in both male and female increased from 4.16 and 3.78 (0 h) to 15.33 and 17.22 μg/g (24 h) respectively. Compared to the 0 h, the significant increase of PUT concentration in male and female occurred on 18 and 6 h respectively (p < 0.05). However, the CAD levels of both male and female significant increase could be found at 12 and 6 h respectively. In the male hepatopancreas (Fig. 2b), the concentration of PUT and CAD significant increased at 24 and 21 h respectively (p < 0.05). The levels of PUT in both male and female gonads obviously increased with storage time increased, but significant increase was found at 12 h for male and 18 h for female (Fig. 2c). These PUT and CAD data generally agree with previous research by Shakila et al. (1995), which showed overall increases in PUT and CAD concentrations in Indian shrimp (Parapenaeopis stylifera) stored at 0, 5 and 30 °C. Benner et al. (2003) study showed that decomposition of the shrimp progressed more rapidly with time at progressively higher temperatures. Compared to the 0 and 12 °C, the times of PUT and CAD significant increase were earlier in higher temperatures (24 and 36 °C). This study also found that OA could be found in hepatopancreas and gonads of both sexes. However, there are no significant differences in OA levels among different storage time for both male and female (p > 0.05) (Fig. 2b, c).
In addition, the relationship between the value of TVBN and storage time or PUT was evaluated. In all organs, a high correlation was found between TVBN and storage time (Table 1). Pearson’s correlation coefficients of TVBN in all organs with time were higher than 0.94. Meanwhile, PUTs in muscle and gonad of both sexes and male hepatopancreas were positively correlated with TVBN (Pearson’s correlation coefficients >0.60) (Table 1). As TVBN, the PUT could also be as an indicator for decomposition in above organs of dead Chinese mitten crab, especially muscles and gonads.
Because biogenic amines as heat stable compounds have been reported, cooking or prolonged exposure to heat will not eliminate the toxin (Tapingkae et al. 2010). We suggested that biogenic amine, especially for PUT should be the most comprehensive chemical indicator for decomposition in dead Chinese mitten crab.
Based on the analysis of TVBN, we suggested three edible tissues of both sexes of Chinese mitten crab shouldn’t be used for food after dead for over 24 h at higher temperature. In all of the edible tissues, the female gonad was earliest decomposition tissue compare to other tissues at higher temperatures. TVBN value could be as a good indicator for Chinese mitten crab freshness. According to analysis of biogenic amine, we suggested that the edible tissues of dead adult Chinese mitten crab poisoning may be the result of PUT accumulation. As for whether HIM involved in this dangerous, it would need be judged by a serials of details analysis, such as sample type, storage condition, and various environmental microflora.
XZ carried out biogenic amine analysis, participated in the design of the study, performed the statistical analysis and drafted the manuscript. JB carried out determination of total volatile base nitrogen and drafted the manuscript. YX conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
We thank Dr. Nicholas Silva for proof reading this manuscript. This study was supported by a grant from the National Nature Science Foundation of China (No. 31272677).We gratefully acknowledge the grants from the Innovation Program of Shanghai Municipal Education Commission (Grant No. 11YZ151), the innovation research group who develop projects in the universities in Shanghai (nutrition, feed, and environment of animal aquaculture), and the Excellent Academic Leaders of Shanghai (Grant No. 12xd1402700).
All authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- [AOAC]Officials Methods of Analysis (1995) Automated method, 940.25 16th ed. Ch. 35:6Google Scholar
- Barrento S, Marques A, Teixeira B, Anacleto P, Vaz-Pires P, Nunes ML (2009) Effect of season on the chemical composition and nutritional quality of the edible crab Cancer pagurus. J Agric Food Chem 57:10814–10824View ArticlePubMedGoogle Scholar
- Benner RA Jr, Staruszkiewicz WF, Rogers PL, Otwell WS (2003) Evaluation of putrescine, cadaverine, and indole as chemical indicators of decomposition in penaeid shrimp. J Food Sci 68:2178–2185View ArticleGoogle Scholar
- Center for Science in the Public Interest (2009) Outbreak alert! analyzing foodborne outbreaks 1998–2007,11th Ed., Center for Science in the Public Interest, Washington, DCGoogle Scholar
- Chen MT, Lin YS, Tsai HT, Kuo HL (2002) Efficiency of hurdle technology applied to raw cured meat (Si-raw) prcocessing. Asian-Aust J Anim Sci 15:1646–1652View ArticleGoogle Scholar
- Cheng YX, Wu XG, Yang XZ, Hines AH (2008) Current trends in hatchery techniques and stock enhancement for Chinese mitten crab, Eriocheir japonica sinensis. Asian Fish Sci 16:377–384Google Scholar
- Chomnawang C, Nantachai K, Yongsawatdigu J, Thawornchinsombut S, Tungkawachara S (2007) Chemical and biochemical changes of hybrid catfish fillet stored at 4°C and its gel properties. Food Chem 103:420–427View ArticleGoogle Scholar
- Ding ZP, Liu CQ, Di Chen, Tang YF, Yong KL, Fang XM (2006) Determination of ten biogenic amines in fishery products by high performance. J Instrum Anal 25:59–62Google Scholar
- Gillespie NC, Smith LG, Burke JB (1983) Freezing of whole uncooked mud crabs (Scylla serrata). Food Technol Aust 35:370–372Google Scholar
- Gormley TR, Redmond GA, Fagan JD (2003) Freeze-chill applications in the food industry. New Food 2:65–67Google Scholar
- Halasz A, Barath A, Simon-Sarkadi L, Holzapfel W (1994) Biogenic amines and their production by microorganisms in food. Trends Food Sci Technol 5:42–49View ArticleGoogle Scholar
- Hernandez-Jover T, Izquierdo-Pulido M, Veciana-Nogues MT, Vidal-Carou MC (1996) Ionpair high-performance liquid chromatographic determination of biogenic amines in meat and meat products. J Agric Food Chem 44:2710–2715View ArticleGoogle Scholar
- Huss HH (1995) Quality and quality changes in fresh fish, Rome: FAO Fisheries Technical Paper No. 348Google Scholar
- Lehane L, Olley J (2000) Histamine fish poisoning revisited. Int J Food Microbiol 58:1–37View ArticlePubMedGoogle Scholar
- Marks HS, Anderson CR (2005) Determination of putrescine and cadaverine in seafood (finfish and shellfish) by liquid chromatography using pyrene excimer fluorescence. J Chromatogr A 1094:60–69View ArticleGoogle Scholar
- Mietz JL, Karmas E (1977) Chemical quality index of canned tuna as determined by high-pressure liquid chromatography. J Food Sci 42:155–188View ArticleGoogle Scholar
- Min JS, Lee SO, Jang A, Lee M, Kim Y (2004a) Quantitative analysis of biogenic amines in raw and processed foods of animal origin on Korean domestic market. Asian-Aust J Anim Sci 17:1764–1768View ArticleGoogle Scholar
- Min JS, Lee SO, Jang A, Lee M, Kim Y (2004b) Production of biogenic amines by microflora inoculated in meats. Asian-Aust J Anim Sci 17:1472–1478View ArticleGoogle Scholar
- Min JS, Lee SO, Jang A, Jo C, Park CS, Lee M (2007) Relationship between the concentration of biogenic amines and volatile basic nitrogen in fresh beef, pork, and chicken meat. Asian-Aust J Anim Sci 20:1278–1284View ArticleGoogle Scholar
- Özoğul F, Özoğul Y (2000) Comparision of methods used for determination of total volatile basic nitrogen (TVB-N) in rainbow trout (Oncorhynchus mykiss). Turk J Zool 24:113–120Google Scholar
- Özyurt G, Kuley E, Özkütük S, Özogul F (2009) Sensory, microbiological and chemical assessment of the freshness of red mullet (Mullus barbatus) and goldband goatfish (Upeneus moluccensis) during storage in ice. Food Chem 114:505–510View ArticleGoogle Scholar
- Robson AA, Kelly MS, Latchford JW (2007) Effect of temperature on the spoilage rate of whole, unprocessed crabs: Carcinus maenas, Necora puber and Cancer pagurus. Food Microbiol 24:419–424View ArticlePubMedGoogle Scholar
- Shakila RJ, Vasundhara TS, Rao DV (1995) Rapid quality assessment of shrimps during storage by monitoring amines. J Food Sci Technol (Mysore) 32:310–314Google Scholar
- Staruszkiewicz WF, Barnett JD, Rogers PL, Benner RA Jr, Wong LL, Cook J (2004) Effects of on-board and dockside handling on the formation of biogenic amines in mahimahi (Coryphaena hippurus), skipjacktuna (Katsuwonus pelamis), and yellowfintuna (Thunnus albacares). J Food Prot 67:134–141PubMedGoogle Scholar
- Tapingkae W, Tanasupawat S, Parkin KL, Benjakul S, Visessanguan W (2010) Degradation of histamine by extremely halophilic archaea isolated from high salt-fermented fishery products. Enzyme Microbial Technol 46:92–99View ArticleGoogle Scholar
- Tsai YH, Chang SC, Kung HF, Wei CI, Hwang DF (2005) Histamine production by enterobacteraerogenes in sail- fish and milk fish at various storage temperatures. J Food Prot 68:1690–1695PubMedGoogle Scholar
- Veciana-Nogues MT, Marine-Font A, Vidal-Carou MC (1997) Biogenic amines as hygienic quality indicators of tuna. Relationships with microbial counts, ATP-related compounds, volatile amines, and organoleptic changes. J Agric Food Chem 45:2036–2041View ArticleGoogle Scholar
- Veilleux E, de Lafontaine Y (2007) Biological synopsis of the Chinese mitten crab (Eriocheir sinensis). Can Manuscr Rep Fish Aquat Sci 2812, vi + 45pGoogle Scholar
- Vinci G, Antonelli ML (2002a) Biogenic amines: quality index of freshness in red and white meat. Food Control 13:519–524View ArticleGoogle Scholar
- Vinci G, Antonelli ML (2002b) Biogenic amines: quality index of freshness in red and white meat. Food Control 13:519–524View ArticleGoogle Scholar
- Wu XG, Chang GL, Cheng YX, Zeng CS, Lu JF (2010) Effect of dietary phospholipid and highly unsaturated fatty acids on the gonadal development, lipid class and fatty acid composition of precocious of Chinese mitten crab (Eriocheir sinensis). Aquac Nutr 16:25–36View ArticleGoogle Scholar
- Xing SH, Zhang XS, Dediu L, Ma CY, Fu ZT (2012) Shelf life modeling of tilapia in the cold chain. J Food Agric Environ 10:257–260Google Scholar
- Xu YS, Xia WS, Kim JM (2009) Biogenic and volatile amines in Chinese mitten crab (Eriocheir sinensis) stored at different temperatures. Int J Food Sci Technol 44:1547–1552View ArticleGoogle Scholar
- Yamanaka H (1990) Polyamines as potential indices for freshness and decomposition of saury pike (Cololabis saira) and scallop (Patinopecten yessoensis). In: Torry Research Station, Chilling and freezing of new fish products. Aberdeen, pp 123–129Google Scholar
- Zamir R, Qasim R, Ullah A (1998) Changes in physical and chemical constituents of crab meat during storage at refrigerator temperature (7 ± 2 °C). Pak J Pharm Sci 11:27–33PubMedGoogle Scholar
- Zhang JB, Yang XZ, Fan P, Zhao LL, Wang C, Yang ZG, Hong YH, Cheng YX (2012) Changes in total volatile basic nitrogen and biogenic amines in two common species of marine fish at high temperature. Acta Hydrobiol Sin 36:284–290Google Scholar