Oxidative Stability and Browning Development of Semi-dried Shrimp (Acetes chinensis) with Different Salt Contents and Packaging Methods Stored at Refrigerated Temperature

Abstract

Lipid oxidation progress in semi-dried shrimp (Acetes chinesis) products during cold storage was studied. Samples with low salt content and without vacuum package exhibited the highest level of lipid oxidation and browning development. High salt content samples showed lower TBARS and 1-penten-3-ol concentration during whole storage period than low salt samples. Vacuum package showed some effects in retarding lipid oxidation particularly for low salt content sample. Vacuum package could also preserve astaxanthin at the end of storage. Browning development in this semi-dried shrimp product during storage was not due to astaxanthin loss. The mechanism of the browning requires further investigation.

Introduction

Acetes chinesis is a kind of small marine shrimp widely caught in the coast of China. It is reported that approximately 300,000 tons of Acetes chinesis are made into dried products or shrimp sauce annually in China (Cao et al., 2010). Traditionally, fresh raw Acetes chinesis caught from ocean is cooked in brine and dried to make dried salted product. However, excessive salt intake could lead to hypertension and cardiovascular disease (Hendriksen et al., 2014). In addition, product with relatively higher moisture content (>35%) tastes better with stronger umami flavor than dried product with lower moisture content. New semi-dried product with low salt and high moisture content is gaining popularity in the market as it is preferred by more and more consumers. The semi-dried product has high water activity and is required to be stored in the freezer to prevent spoilage and quality deterioration for long term storage. Nevertheless, this product is often temporally stored at refrigerated temperature or room temperature for convenience in the market or consumption at home.

Semi-dried Acetes chinesis products can show discoloration or browning and off-smell by observation if stored at abused temperature for some period. The browning and off-smell are not desirable and can negatively influence the consumers' perception of the quality of this product. Browning in this kind of product or other food products is often a very complex chemical reaction and involves different pathways. Current studies have demonstrated that it could be explained by Maillard reaction or lipid oxidation mechanism (Zamora and Hidalgo, 2005; Lu, et al., 2015). Maillard reaction typically occurs between reducing sugar and amino acid or protein. Similar reaction could also occur between lipid oxidation derived carbonyls and amines, amino acid or protein (Zamora and Hidalgo, 2005; Zamora and Hidalgo, 2016). On the other hand, off-smell could also be partially due to the secondary lipid oxidation products generated during storage.

Lipid oxidation is one major concern for the quality of dried seafood products during storage (Qiu et al., 2019). Other than storage temperature, salt content and packaging method could also have effects on the lipid oxidation progress of this product, which might influence the browning and off-flavor eventually. Effect of salt on the lipid oxidation of seafood products during storage is controversial (Vidal et al., 2017; Mariutti and Bragagnolo, 2017). Vacuum package is often used in the food industry to retard lipid oxidation in food products. Related research on lipid oxidation progress for this semi-dried product during storage is scarce. The main objective of this study is to investigate the oxidative stability and browning development of two products with different salt contents and packaging methods stored at refrigerated temperature. Our ultimate goal is to investigate the browning development mechanism of this product during storage, which is beyond the scope of this study.

Materials and Methods

Materials    Semi-dried shrimp (Acetes chinesis) products with low and high salt contents were purchased from local market in Xiamen, China. Astaxanthin was from Aladdin (Shanghai, China). 1-penten-3-ol and 4-methyl-1-pentanol were from Sigma-Aldrich (St. Louis, MO). HPLC grade acetonitrile and methanol were from Merck (Darmstadt, Germany). All other chemicals used in this study were of analytical grade.

Storage    Semi-dried products were vacuum packaged or sealed without vacuum and stored at 4 °C in a refrigerator. There were four groups including low salt vacuum packaged sample (LSV), low salt sample without vacuum package (LS), high salt vacuum packaged sample (HSV), high salt sample without vacuum package (HS). Samples were taken out for analysis weekly during 6 weeks of storage.

Surface color measurement    The surface color of samples was measured using a colorimeter (WSC-S; INESA Scientific Instrument Co., Ltd, Shanghai, China). L^* (white), a^* (red), and b^* (yellow) values were measured. Three readings per sample taken by turning 45 degree angle turns were averaged. The colorimeter was calibrated using white and black tiles before measurement.

Chemical analysis

Moisture, water activity and salt content analysis    Moisture content was determined by the AOAC method (2002). Water activity was measured using Pawkit water activity meter (Pullman, WA). Salt content analysis was also based on AOAC method (2002). Briefly, 1 g ground shrimp (Acetes chinesis) sample was mixed with 9 mL water and a certain amount of 0.1 M AgNO₃ solution to completely precipitate the Cl⁻ in the solution. Then 20 mL HNO₃ was added and boiled gently in a hot plate for approximately 15 min. After cooling, it was titrated with 0.1 N NH₄SCN solution.

Lipid extraction by Bligh and Dyer method    Lipid from semi-dried shrimp was extracted by Bligh and Dyer method (Bligh and Dyer, 1959). Briefly, sample was ground to powder by IKA A10 basic mill followed by homogenization with methanol, chloroform and water using a lab dispenser (Fluko Equipment Shanghai Co., Ltd). After phase separation by centrifugation, the chloroform layer which contained lipid was kept for analysis. For each treatment, triplicate lipid extractions were performed and analyzed for peroxide value (PV) and astaxanthin content.

PV analysis    PV analysis was based on a method described by Shantha and Decker (Shantha and Decker, 1994). Briefly, lipid extract was evaporated to dryness under nitrogen and mixed with 10 mL chloroform/methanol (7:3 v/v) in a sealed test tube. Then 50 µL NH₄SCN solution and 50 µL FeCl₂ solution were added, mixed and incubated at room temperature for exactly 5 min. Absorbance of the solution was measured by a spectrometer at 500 nm against a blank. A standard curve prepared by FeCl₃ solution was made on each week of analysis. Results were reported as meq peroxides/kg lipids.

Determination of astaxanthin content    Lipid extract was evaporated to dryness under nitrogen. 1 mL of methanol was added and mixed. This solution was filtered through a 0.22 µm syringe filter before HPLC analysis. Analysis was performed with a Waters HPLC equipped with a UV-Vis detector at 476 nm (Waters, Milford, MA, USA). An Alltima C18 (250*4.6 mm, 5 µm) reversed phase column was used. The mobile phase consisted of methanol: acetonitrile (6:2) at a flow rate of 0.8 mL/min. The injection volume was 5 µL and running time was 10 min. External standard curves were made to calculate the astaxanthin concentration. Results were reported as µg/g sample.

Thiobarbituric acid reactive substances (TBARS) analysis    TBARS analysis was based on the method described by Bedinghaus and Ockerman (1995) with slight modification. Briefly, 5.0 g of ground samples were mixed with 2.5 mL 0.5% propyl gallate solution and 50 mL 20% ice-cold trichloroacetic acid and 1.6% phosphoric acid for 2 min by a lab dispenser, followed by mixing with 50 mL ice-cold distilled water for 1 min. The mixture was centrifuged at 4000×g for 5 min. 5 mL of the supernatant was mixed with 5 mL of 0.02 M thiobarbituric acid solution in a glass test tube and heated in a boiling water bath for 35 min, cooled and centrifuged at 4000×g for 3 min. The absorbance of the supernatant was measured at 532 nm. 1,1,3,3-tetraethoxypropane was used to prepare the standard curve. TBARS was expressed as mg malonaldehyde equivalents/kg (mg MA eq/kg) sample.

1-penten-3-ol analysis    1-penten-3-ol is one typical volatile compound that is generated from lipid oxidation in seafood products. 1-penten-3-ol was analyzed by SPME-GC-FID. Briefly, 2 g of ground sample was weighed into 20 mL SPME vial. 100 µL of 4-methyl-1-pentanol solution (6 µg/mL) was added as internal standard. The vial containing the sample was tightly sealed and kept at 50 °C for 20 min to allow the volatiles in the headspace to reach equilibrium. A DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA) was inserted into the vial to absorb volatile compounds for 20 min at 50 °C. The fiber was then retrieved and inserted into Shimadzu GC 2010plus equipped with FEMEWAX capillary column (30 m × 0.25 mm, 0.25 µm film) for 1 min in splitless mode (Shimadzu, Tokyo, Japan). The injector and the detector temperature were set at 250 °C and 280 °C respectively. Nitrogen was used as the carrier gas. The column temperature was set at 35 °C for 5 min, 35 to 100 °C at 10 °C/min, 100 to 250 °C at 20 °C/min and held there for 5 min. Quantification of 1-penten-3-ol was performed based on the response factor against 4-methyl-1-pentanol.

Statistical analysis    All data were shown as the mean value ± standard deviation of triplicate measurements. Data from the different treatments were subjected to analysis of variance using SPSS software (version 19.0; SPSS Inc., Chicago, IL). Difference between treatments were evaluated based on a least squares difference (LSD) method (P<0.05).

Results and Discussion

Moisture, water activity, salt content and browning development of samples during refrigerated storage    Moisture content of LS and HS samples were 43.1% and 52.8% respectively. Water activity of LS and HS were 0.86 and 0.83 respectively. Salt content of LS and HS samples were 5.5% and 11.9% respectively. Moisture and water activity did not change much during the refrigerated storage. Browning development of this semi-dried product was analyzed by both instrument analysis and also direct observation. The b^* value is often used to indicate the yellowness of the product. There was no significant difference between samples with high or low salt contents at the beginning of storage (P>0.05) (Figure 1). On week 3, low salt samples had significantly higher b^* values than high salt samples. At the end of storage, the ranking order of b* in all samples was as follows: LS^a≥LSV^ab≥HS^bc≥HSV^c. LS also showed the highest level of browning development by direct observation which was in agreement the instrument analysis, but LSV samples seemed to have significantly lower level of browning than LS (Figure S1). HS and HSV samples did not show obvious browning.

Fig. 1.

Change of b* in semi-dried Acetes chinensis during 6 weeks storage at 4 °C. Data points and error bars represent means ± standard deviations (n=3). LS: low salt sample without vacuum package; LSV: low salt vacuum packaged sample; HS: high salt sample without vacuum package; HSV: high salt vacuum packaged sample.

Fig. S1.

Browning development of semi-dried Acetes chinensis during storage on week 0, 3, and 6 at 4 °C. LS: low salt sample without vacuum package; LSV: low salt vacuum packaged sample; HS: high salt sample without vacuum package; HSV: high salt vacuum packaged sample.

Primary lipid oxidation in semi-dried shrimp (Acetes chinensis) during refrigerated storage    Primary lipid oxidation was indicated by change of PV in samples during the storage period. There was no significant difference in PV between LS and HS samples on week 0 (Figure 2). On week 1, LV showed the highest PV among all the samples (P<0.05). PV decreased significantly in all the samples after week 2 and maintained low values till the end of storage. HS and HSV both had higher PV than LS and LSV on week 4 and 6, while vacuumed packaged samples exhibited lower PV than normally packaged samples (P<0.05) on week 5.

Fig. 2.

Change of peroxide value (PV) in semi-dried Acetes chinensis during 6 weeks storage at 4 °C. Data points and error bars represent means ± standard deviations (n=3). LS: low salt sample without vacuum package; LSV: low salt vacuum packaged sample; HS: high salt sample without vacuum package; HSV: high salt vacuum packaged sample.

Lipid hydroperoxides are initially formed when lipids in seafoods are oxidized. As lipid oxidation progresses further, more hydroperoxides are formed and the PV is relatively higher. At the same time, these hydroperoxides can also decompose into secondary lipid oxidation compounds such as aldehydes, ketones, hydrocarbons, alcohols, etc. The relative rate between formation and decomposition of hydroperoxides determined the changing trend of PV. The lower PV after week 2 observed in the present study could be due to the fast decomposition of hydroperoxides. Analysis of secondary lipid oxidation would provide more information of the oxidative stability of the products, which was showed next.

Secondary lipid oxidation in semi-dried shrimp during refrigerated storage    During the later stage of lipid oxidation, hydroperoxides break down into secondary lipid oxidation products which can be measured by a simple spectrometer method TBARS analysis. These secondary lipid oxidation compounds often have negative impact on the sensory quality of the products even at low concentration (Jacobsen, 2015). Throughout the storage period, TBARS in HS was significantly lower than LS (Figure 3). TBARS in all samples started to slowly increase after week 2, corresponding to the decrease of PV, and reached the highest level on week 5. Vacuum package decreased TBARS for low salt samples on week 1 and 5.

Fig. 3.

Change of Thiobarbituric acid reactive substances (TBARS) in semi-dried Acetes chinensis during 6 weeks storage at 4 °C. Data points and error bars represent means ± standard deviations (n=3). LS: low salt sample without vacuum package; LSV: low salt vacuum packaged sample; HS: high salt sample without vacuum package; HSV: high salt vacuum packaged sample.

In order to further confirm the TBARS analysis result, a specific volatile secondary lipid oxidation compound 1-penten-3-ol was quantified. 1-penten-3-ol was mainly originated from n-3 long chain unsaturated fatty acid oxidation and was also reported to have burnt, meaty, paint-like, grassy-green smell (Dehaut et al., 2014; Venkateshwarlu et al., 2004). 1-penten-3-ol was found to be the one of the most noticeable compounds detected in rancid sardine oil (Society and Fisheries, 1980) and fish oil enriched milk samples (Qiu et al., 2018). In the present study, high salt content sample had lower 1-penten-3-ol concentration than low salt content sample throughout the storage period (P<0.05). Vacuum package showed inhibitory effect on the formation of 1-penten-3-ol for high salt content sample on week 4. Corresponding to TBARS, 1-penten-3-ol analysis results further confirmed the TBARS results that high salt samples had lower level of secondary lipid oxidation.

Fig. 4.

Change of 1-penten-3-ol in semi-dried Acetes chinensis during 6 weeks storage at 4 °C. Data points and error bars represent means ± standard deviations (n=3). LS: low salt sample without vacuum package; LSV: low salt vacuum packaged sample; HS: high salt sample without vacuum package; HSV: high salt vacuum packaged sample.

Change of astaxanthin in semi-dried shrimp during refrigerated storage    Astaxanthin is usually the major carotenoid in shrimp (Niamnuy et al., 2008). Astaxanthin is also easy to be oxidized and degraded during storage as an endogenous antioxidant. Astaxanthin concentration decreased significantly after one week storage and maintained a general stable value to the end of storage. On week 0, high salt sample seemed to have higher astaxanthin concentration but not statistically significant. The differences of astaxanthin concentration between high salt and low salt samples were not significant throughout the storage. Vacuum packaged samples showed higher astaxanthin concentration than samples without vacuum package on week 6. HSV had also higher astaxanthin concentration than LSV (P<0.05). Degradation of astaxanthin was reported to be responsible for the color loss of dried shrimp (Niamnuy et al., 2008). In semi-dried Acetes chinensis, HS and LS did not show significant differences in astaxanthin concentration, but LS had a much higher degree of browning than HS during the storage. Astaxanthin could cause the loss of red color but was not the major reason of browning in the LS samples.

Comparison of lipid oxidation and browning development in semi-dried shrimp with different salt contents and packaging methods during refrigerated storage    In the present study, there was lipid oxidation progress in semi-dried Acetes chinesis particularly after week 4 during refrigerated storage as confirmed by change of TBARS and 1-penten-3-ol analysis results. Many studies showed that salt can have prooxidant effects (Guillén et al., 2004; Souza and Bragagnolo, 2014;). In contrast, the present study showed that secondary lipid oxidation and 1-penten-3-ol concentration was significantly lower in high salt samples. Nevertheless, it could be also due to the relatively lower lipid content in HS (2.01%) than LS (2.42%). It has to be pointed out that samples were not prepared in the lab due to some difficulty in preparation. More detailed study is required to investigate the relationship between salt content and lipid oxidation.

Vacuum package retarded lipid oxidation as evidenced by the secondary lipid oxidation results for low salt content sample and also higher concentration of astaxanthin in all the vacuum packaged samples at the end of storage. Astaxanthin is an endogenous antioxidant, which could be consumed first when the lipid oxidation occurs. Vacuum package did not show strong inhibitory effect in the secondary lipid oxidation for the high salt content sample as it underwent lower level of lipid oxidation. Vacuum package did inhibit the browning development particularly for low salt content samples.

Many studies showed that non-enzymatic browning in food products was often caused by lipid oxidation or Maillard reaction. Thanonkaew et al. (2006) studied the mechanism of yellow pigmentation development in squid during frozen storage. They found that the yellow pigmentation could be caused by aldehydic lipid oxidation products and the amines on phospholipids headgroups. Significant increase of browning intensity was also observed in herring fillet during drying process, which could be caused by reaction between carbonyl compounds generated from lipid oxidation and free amino acids such as lysine (Shah, et al., 2009). On the other hand, Maillard reaction between reducing sugar and amino acid led to the browning discoloration of Japanese common squid during air-drying (Geng et al., 2015). In another study, both lipid oxidation and Maillard reaction could cause browning during the production of Antarctic krill meal (Lu et al., 2013). Browning of salted mullet roe products during storage at room temperature was also ascribed to these two reactions (Rosa et al., 2012).

Fig. 5.

Change of astaxanthin in semi-dried Acetes chinensis during 6 weeks storage at 4 °C. Data points and error bars represent means ± standard deviations (n=3). LS: low salt sample without vacuum package; LSV: low salt vacuum packaged sample; HS: high salt sample without vacuum package; HSV: high salt vacuum packaged sample.

In the present study, samples with low salt contents had higher secondary lipid oxidation products and higher degree of browning, suggesting lipid oxidation had some effects on the browning development. Lipid oxidation in vacuum packaged samples was inhibited in some way and also showed lower degree of browning development particularly for low salt samples by observation. It should be pointed out that samples might have much more significant lipid oxidation and browning development if stored at room temperature, which might provide more clear information to correlate the lipid oxidation progress and browning development. The mechanism of browning development is complex. A study of the change of reducing sugar and amino acids would also help to discover the mechanism in the future as Maillard reaction also plays an important role in the browning development particularly at higher temperature.

Conclusions

Low salt content sample without vacuum package had the highest degree of lipid oxidation and also the browning development. Samples with high salt content showed oxidative stability than samples with low salt content during refrigerated storage, but this might be also due to its relatively lower lipid content. Vacuum package retarded lipid oxidation and also browning development of the low salt semi-dried product. Browning development of this product might be due to the progress of lipid oxidation or Maillard reaction, but not astaxanthin concentration. Further research is required to discover the browning mechanism.

Acknowledgements    This work was supported by the National Key Research and Development Program of China (Grant No. 2018YFD0901004) and Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, P.R. China (Grant number NYJG201508).

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