Nano Astaxanthin improves shrimp color, contributes to increasing market value
The color of shrimp depends on the presence of carotenoid pigments (mainly astaxanthin) and has a significant impact on their market value. In this study, we observed that the visual appearance of color in black tiger shrimp (Penaeus monodon), as assessed using a commercial grading scale, does not always correlate well with the total functional amount of carotene. We also observed that the appearance was influenced by the color of the shrimp tank. When shrimp were kept in black or white tanks for 28 days, although they had similar average total astaxanthin content (29–33 μg/g tail), shrimp in black tanks had a much more orange/red color when cooked compared to shrimp cultured in white tanks. Pigments are concentrated mainly in the cephalothorax, abdominal epidermis, and the extra-abdominal skeleton. Light microscopy revealed a more uniform distribution of pigment in the epidermis of brightly colored shrimp compared with the concentrated pigment region in lighter-colored shrimp. Non-esterified astaxanthin was the major carotenoid present in all body sites of shrimp from the black tank (50%) with the remainder being made up of mono- and diester astaxanthin. However, for shrimp from white tanks, non-esterified astaxanthin accounted for only 12–13%, with mono-esters accounting for about 60% of the total present. In a separate test, we demonstrated that environmental color changes cause a rapid change in shrimp coloration. When shrimp were transferred from white tank to black tank, scores increased significantly within 1 hour without any change in astaxanthin content. Color improvement continued to increase for 168 h. Transferring shrimp from a black tank to a white tank resulted in a decrease in scores, but the change was much slower. This work suggests that, provided shrimp have adequate levels of astaxanthin in their diets, it may be possible to improve their overall appearance (raw and cooked) by color correction at harvest.
1.INTRODUCTION
The color of shrimp is one of the most important attributes in determining a consumer’s purchasing decision for shrimp and thus significantly influences their market value. In Australia, cooked black tiger shrimp (Penaeus monodon) is usually assessed for color using a subjective color grading chart with a score of 1 to 12, reflecting low to high pigmentation (Aqua-Marine Marketing). Pty Ltd., Kippa-Ring, Queensland). Shrimp with a higher score are usually valued at A$2–4/kg more than shrimp with a lighter color and typically, shrimp with a poor color cannot be sold at acceptable market prices and are frozen. and hold until the demand is higher. The color of shrimp depends largely on the amount of astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) present in the external tissues; especially in the exoskeleton and in the epidermis between the abdominal muscles and the exoskeleton (Menasveta et al., 1993; Boonyaratpalin et al., 2001). Astaxanthin is commonly present in free and esterified (mono- and di-ester) forms with fatty acids (Okada et al., 1994). These carotenoids can also be present as carotenoid-proteins, especially in the exoskeleton, and when they separate from the protein, they change color from blue to red, as can be seen when shrimp are cooked. (Britton et al., 1981).
Small amounts of other carotenoids including lutein and zeaxanthin have also been reported (Pan et al., 2001; Sachindra et al., 2005). In addition to pigmentation, carotenoids have also been suggested to play a role in several important functions in crustaceans such as providing active provitamin A (Miki et al., 1982), increasing stress tolerance (Torrissen, 1984; Chien et al., 2003) and in various developmental and differentiation processes, as summarized by Linan-Cabello et al. (2002).
The source of astaxanthin in shrimp, as in other animals, is from the diet and in wild shrimp the majority of astaxanthin comes from conversion from several other ingested carotenoids such as zeaxanthin and β-carotene. (Katayama et al., 1971). Therefore, the presence of astaxanthin in shrimp depends on its availability from feed in the environment as well as on its ability to be absorbed, transported, and subsequently deposited at specific anatomical sites. For farmed shrimp, the addition of synthetic astaxanthin to the feed is an additional cost and it is often necessary to adjust the amount and timing of the addition to achieve the desired pigment level (Chien and Jeng, 1992). It is often difficult for producers to predict the optimal concentration of feed required due to the contribution of the algal flora in the pond, as well as the apparent genetic coloration of individual individuals and obvious banding differences between species. shrimp.
As part of our study to determine the optimal pigmentation of farmed shrimp, we measured the total astaxanthin content in a large number of shrimp and observed that this concentration did not always correlate well. with realistic colors. We suggest that this difference in appearance may depend on the location of pigments in different body regions of shrimp (Sachindra et al., 2005). In addition, it is known that shrimp have the ability to change their external appearance to blend with the color of the environment, through the movement of pigments in pigment cells in the epidermis layers below the exoskeleton ( Fingerman, 1965; Robison and Charlton, 1973). Morphological and physiological color changes have been described in crustaceans in association with slow and rapid changes due to environmental or hormonal factors, respectively (Rao, 1985; Melville-Smith et al. , 2003). Such movement of pigments in raw shrimp can directly affect the color of cooked shrimp. In the study described here, we report on the changes in pigmentation seen in individuals in groups of shrimp, the location of pigments in different body compositions, and how they are affected. by ambient light.
2.material and method
Shrimp used in this study were obtained from the experimental herd and from commercial farms. Shrimp were not evaluated for molting stage and shrimp of different weights were used. However, for specific comparative trials, shrimp were similar in weight.
2.1 Experimental shrimp
Experimental shrimp (P. monodon) were obtained from herds maintained at the CSIRO, Marine and Atmospheric Research (CMAR) laboratories in Cleveland. Until necessary for the experiments, shrimp were housed in 2500 L fiberglass tanks. Seawater was pumped through the tanks at a rate of 1.2 L.min−1 to maintain the water temperature and salinity times. at about 28°C and 32‰ respectively. Photoperiod before the start of the experiment was maintained at 12 am: 12 pm. Shrimp were fed commercial pellets (Lucky Star, Taiwan Hung Kuo Industrial Pty Ltd.) twice daily. The actual content of astaxanthin was determined and found to be approximately 40 mg of astaxanthin per kg of feed.
2.2 Commercial frozen shrimp
Whole cooked, frozen black tiger shrimp (weighing approximately 20–50 g) were obtained from two commercial farms in Queensland.
2.3 Test 1 — effect of environment color
Black tiger shrimp P. from the same herd were reared together in black lined tanks in the laboratory and fed a basic commercial shrimp feed containing 40 mg astaxanthin/kg feed (feed and other details as shown). mentioned above). The intensity of light (Iso-Tech ILM350, light meter) at a distance of 10 cm from the water is about 10lx. On day 0, half of the shrimp (n=5) were randomly picked and transferred to a white-lined tank and maintained in the same light intensity as before. All shrimp were continued to be fed the same commercial feed and light conditions were maintained constant for each group. On day 28, shrimp were removed from both tanks and placed in a mixture of seawater and ice for 5 min until death, after which they were removed and boiled in filtered seawater for 2 min. The average weight of shrimp after cooking was 9.27± 0.99 g. Although the mean weight of shrimp in the black tank was lower than that of the shrimp in the white tank (8.10 ± 1.20 and 10.43 ± 1.51 g, respectively), these differences were not significant and reflect reflect the results of randomization on day 0. Shrimp were photographed and then frozen and kept at -20°C until analysis.
2.4. Test 2 – speed of color change
Black tiger shrimp P. monodon of similar size from the same flock and reared together in black-lined tanks were randomly separated into black- or white-lined tanks at CMAR, Cleveland for 28 days (n=96). During this time, each animal was fed an identical diet containing 40 mg of astaxanthin/kg of feed. On the start day of the experiment, at 9 am, shrimp were transferred from their tank to the tank of the opposite color. Then, at each time point 0, 1, 3, 6, 12, 24, 72, 168 and 336 h, 6 shrimp were removed and placed in a mixture of seawater and ice for 5 min until death. They are then removed and boiled in filtered seawater for 2 minutes and then cooled in ice. Shrimp were photographed and frozen and kept at -20°C until required for analysis. The lighting procedure was continued throughout the experimental period, which means that the shrimp at 12 o’clock were subjected to darkness for about 4 h.
2.5. Subjective assessment of shrimp color
All shrimp, commercial and experimental, were evaluated for color by display under standard fluorescent lighting, on a large table in the food processing laboratory set at an air temperature of 10 °C. Subjective assessment of color was performed using a grading scorecard (1 to 12 for lightest to darkest) for P. monodon (Aqua-Marine Marketing Pty Ltd., Kippa-Ring, Queensland ). Seven panelists were used for Experiment 1 and 14 panelists were used for Experiment 2. Although panelists were not trained to evaluate color of shrimp but they were experienced in different food attributes (including color) and were given anchor points for extremes.
2.6. Relationship between scoring and astaxanthin content
Cooked commercial shrimp (mean weight= 34.9 g [range 22 to 51 g], n= 61) color graded according to the subjective assessment described in Section 2.5 used to determined total astaxanthin content and body distribution in shrimp at different levels (see Figures 2 and 3). Upon arrival at the laboratory, frozen shrimp were thawed and, in this case, scored using a 3-member panel to reach consensus on individual shrimp. The shrimp were then sorted in order of scores and the initial assessment was confirmed. Each shrimp was then dissected into the exoskeleton, cuticle, cephalothorax, and digestive gland to extract astaxanthin.
2.7. carotene analysis
Shrimp were weighed and then dissected as follows. All procedures, including extraction, were performed under low-light conditions in a dark laboratory. In most cases, and unless otherwise mentioned, comparisons between groups of shrimp were made on pigments present in the ‘tail’, which includes the epidermis of the abdominal muscles along with the ventral exoskeleton. , including uropods and telson. This is done by removing the head and then separating the exoskeleton from the abdominal muscles. The epidermal pigment layer is carefully separated from the muscle so as not to include any components from the gastrointestinal tract. In all cases, except for the investigation of pigment distribution (Table 1), the abdominal muscle was not removed because it did not contribute to pigmentation in shrimp. When indicated, carotene analyzes were also performed on individually dissected components such as the ventral exoskeleton, abdominal epidermis, head, and digestive glands. Each tissue was weighed, chopped, and then extracted three times with 20 mL of acetone at 2°C, allowed to stand overnight between each extraction. The residue left after this exhaustive extraction process is essentially pigment-free. The pooled extract was adjusted to a total volume of 60 mL and 10 mL of H2O and 5 mL of n-hexane were added, mixed well, and left to separate. The upper layer was removed and the lower layer was washed twice with 5 mL H2O and 5 mL n-hexane. The combined upper layers containing the pigments were evaporated to dryness under nitrogen and then re-dissolved in 20 mL of n-hexane. The concentration of astaxanthin in the extract was determined by measuring the absorbance at 477 nm, using a molar extinction factor of 2172 in a 1 cm cuvette. The Astaxanthin standard was obtained from Sigma Chemical Co. St Louis, MO, USA and the lutein standard from Extrasynthese, Genay, France. From previous analyses, we know that astaxanthin and its esters account for about 95% of total carotenoids and therefore lesser substances such as lutein are not indicated but should contribute to the total. All samples were stored in the dark, under nitrogen at −20 °C until further analysis was required.
Results for astaxanthin content are expressed as μg/g wet weight of the shrimp component or as a percentage of the pigment distribution in the individual ingredients. For each individual shrimp, the total astaxanthin (μg) extracted from each anatomical site was determined and the relative distribution expressed on a percentage basis.
2.8. Separation of astaxanthin and astaxanthin esters by HPLC
The n-hexane carotene extracts were transferred to brown sample vials for HPLC separation on a Waters system consisting of a Model 600 pump, Model 717 autoinjector and model 996 photodiode array detector. fixed at 477 nm. The system is controlled by Waters Empower software (2003). Separation was achieved using a Luna 5μm C18 (2) 100 Å 250 mm×4.6 mm column (Phenomenex #00G-4252-E0) fitted with a protective case (Phenomenex #AJ0-4287). The solvent gradient system, with a flow rate of 1.5 mL/min, is as follows: solvent A (methanol:H20, 80:20,v/v); solvent B, (ethylacetate) essentially as described by Wade et al. (2005).
2.9. microscope
Portions of the epidermis from the first abdominal segment of each shrimp were carefully removed from the abdominal muscles after removal of the outer shell. Cell plates were placed on a microscope slide and covered with physiological saline under the slide. Pigment cells were viewed with an Olympus microscope, Model BH2, and images were obtained with a Nikon Coolpix camera, Model 995, using 10 × 4 magnification. The distance between the centers of the chromatophores was measured with micrometer by stage and mean interval (±SE) was found to be 380±9.21 μm for shrimp weighing 20 to 30 g.
2.10. Statistical analysis
A confidence level of 5% was used to compare significant differences between means (p≤0.05) using Paired Student’s t-test (Microsoft Excel, XP). For the data presented in Figure 1, we examined the relationship between shrimp weight and shrimp width at the 2nd ventral segment with linear, quadratic and cubic models. Using the Akaike Information Criteria (AIC), the linear model was selected as the best fit model for the dataset because it has the lowest AIC value (Sakamoto et al, 1986).
3.RESULT AND DISCUSS
3.1. Relationship between scoring and astaxanthin content
Determination of carotene content in whole shrimp can provide a satisfactory indicator of carotene levels in feeds suitable for growth or health, but it may not necessarily be the best performance method. to visualize the color of shrimp, as some pigments may be located in regions and organs such as digestive glands, and therefore do not contribute to appearance. In addition, we need to deal with the relatively wide range of shrimp weights encountered in these trials. We reasoned on the basis of weight and visual area relationships, as measured by body width, that pigment content, expressed as abdominal muscle weight plus extra-abdominal skeleton , providing a satisfactory set of units. In support of this, we found a linear relationship (R2= 0.623) between the width at the second ventral segment (and thus represents surface area) and shrimp weight (Figure 1). On this basis, we expressed the weight of the extracted pigment (μg) per wet weight of the abdominal muscle plus the extra-abdominal exoskeleton.
However, in the course of our research to determine the optimal concentration of pigments in feed to achieve beautiful color, we measured the total astaxanthin content (expressed on the basis of abdominal and skeletal muscle weight). ventral bone) in a large number of shrimp and observed that the number was not well related to the external appearance. This was further demonstrated by measuring the astaxanthin content in shrimp that were subjectively classified into different categories using a color grading chart. The average grading score of shrimp ranged from 6 to 11. The relationship between the grading score and the total astaxanthin content is shown in Figure 2. It can be seen that the color grading score or the appearance is not related. closely to the measured astaxanthin content. For example, at point 9, the astaxanthin content ranged from 5 to 25 μg/g of shrimp tail. Therefore, there must be some other explanation for the observed difference.
3.2. Pigment distribution in shrimp
The distribution of total astaxanthin in the components is presented in Figure 3 for each score from 6 to 11 (shrimp range obtained). Essentially, total astaxanthin was almost uniformly distributed among the periosteum, abdominal epidermis, and cephalothorax (epidermis and outer layers are not separated), with only 1–2% present in the gastrointestinal tract. These findings are essentially similar to those observed by Negre-Sandargues et al., 1993 and Paibulkichakul et al., 2008. Pigment distribution was not effectively changed across the scores obtained. investigate here. It should be noted that in other situations when shrimp are about to molt, the percentage of total astaxanthin present in the exoskeleton is markedly reduced as the pigment is mobilized back to the epidermis as has been observed in lobsters. Western red (Wade et al., 2005).
3.3. Effect of background color on astaxanthin distribution in raw shrimp
The shrimp, which were previously kept together under identical conditions, changed their appearance after they were separated and were then reared in either black or white tanks, even though they were fed the same food. food rations. These differences can be seen in undercooked shrimp; those from the black tank were dark green/brown compared to the lighter color of those from the white tank. The total astaxanthin content (Table 1) of shrimp cultured in black or white tanks for 28 days was not significantly different (33.3 μg/g shrimp tail for the black tank vs 29.1 μg/g shrimp tail for the black tank) white, p≥0.05) and will not result in any noticeable color difference. However, there was a marked difference in their cooked color (Figure 4), with shrimp cultured in black tanks being more orange/red in color than shrimp cultured in white tanks. Close examination of the outer shell/cuticle (Figure 5) revealed that the dark colored shrimp had a more evenly distributed pigment than the lighter colored shrimp, which had a dense pigment density. thickened in small spots (pigment cells).
The distribution of astaxanthin in different body compositions was also determined in shrimp after 28 days under each background color condition (Table 1). It was clear that shrimp cultured in black tanks had a higher percentage of total astaxanthin at cephalothorax (42.7%) (p≤0.05) than shrimp cultured in white tanks (34.6%), despite the expression class. ventral skin was higher (p≤0.05) in animals from white tanks (39.3% vs 31.9%).
The main carotenoid pigments in shrimp that contribute to their color are astaxanthin (non-esterified) and astaxanthin ester (mono- or di-ester), along with small amounts of other carotenoids such as lutein and β-carotene (Okada). et al., 1994; Boonyaratpalin et al., 2001). We investigated the distribution of these pigments in three different locations, ventral epidermis, ventral exoskeleton and cephalothorax, for shrimp reared in black or white tanks for 28 days (Table 2. ). Essentially, we found that regardless of body location, the percentage distribution of individual pigments was very different depending on whether the shrimp were kept in black or white tanks. For shrimp cultured in black tanks, astaxanthin, in its non-esterified form, accounts for about 50% of the total carotenoids. The remainder is mainly composed of astaxanthin mono-esters and astaxanthin di-esters in equal proportions (about 20% each), along with a small amount of lutein (5–7%). However, for shrimp cultured in white tanks, the distribution is very different. Non-esterified astaxanthin was greatly reduced, accounting for only about 12% of the total carotene content. In contrast, astaxanthin mono-esters increased from about 20% to 60% of the total. The ratio of astaxanthin di-ester was not significantly changed.
Therefore, this study suggests that the form of astaxanthin present in the peripheral tissues of shrimp depends on the background light conditions. Therefore, it is proposed that in the dark, a large proportion of astaxanthin mono-esters are hydrolyzed, while under light conditions, free astaxanthin is esterified with a fatty acid to form a mono-ester. The specific fatty acids involved in this process are of particular interest and are currently being investigated. Although our findings were mainly related to background color, they appear to differ from those reported by You et al. (2006) who studied the effects of different light sources and lighting patterns on shrimp color and growth rate. Using Litopenaeus vannamei, they observed that body coloration was best in the shrimp subjected to the highest light intensity and suggested that high astaxanthin was a requirement under these conditions to minimize potential damage from the sun’s rays. ultraviolet. The reason for this increase in pigmentation remains unclear but may be related to greater growth of phytoplankton, bacteria, etc. in water, and thus, more readily available astaxanthin precursors, under stronger light conditions (Tseng et al., 1998). In addition, the efficiency of absorption and deposition processes may be higher in shrimp cultured under brighter conditions.
In a controlled environmental study with juvenile rock lobster (Jasus edwardsii), Stuart et al. (1996) could not demonstrate any significant effect of the substrate color (black stone or white gravel) on the color of the exoskeleton. This study observed a year-long growth period under specific conditions and concluded that any anecdotal differences reported by commercial lobster fishers could be the result of result of genetic differences.
3.4. Color change rate
In the previous trial, shrimp were maintained under the indicated background color conditions for 28 days before being evaluated. The following test was performed to determine the rate of color change as this could significantly affect the commercial application of the process. In this trial, shrimp were maintained under the specified environmental color conditions for 28 days before changing the conditions at 0h. At this time, shrimp from the white tank had a total astaxanthin content (p≥0.05) similar to that of the shrimp from the black tank (32.1± 2.20 μg/g of tail and 36.7± 2.16 μg/g/g). g corresponding shrimp tail). To obtain more precise data (via increased numbers) on the astaxanthin content of shrimp from these two tanks, all shrimp used for more than 168 hours were analyzed. The overall values were 35.1± 1,096 μg/g tail for shrimp transferred to the white tank and 36.7± 1,108 μg/g of tail for those transferred to the black tank (see Figure 6). Although the total pigment content of the two groups of shrimp was similar at 0 h, there was a big difference in the color of raw and cooked shrimp. When graded by participants, cooked shrimp from the white tank were significantly lighter in color (p≤0.001), with an average score of about 7.0 ± 0.306, compared with 10.8 ± 0.225 for shrimp from black tank (Figure 6).
However, within 1 hour of transferring live shrimp from the white tank to the black tank, the pigmentation increased markedly and when the shrimp were cooked, the shrimp’s red color was rated by panel members as 8.8 ± 0.147. . This was different from the shrimp removed from the white tank at time 0 (p= 0.0148). With further time, this trend continues, rising to around 10 points at 72 and 168 hours after the move. The transfer of shrimp from the black tank to the white tank also resulted in a change in color, where the shrimp were lighter in color, but the difference in the first 1 hour (p=0.189) and 12 hours (p=0.024) was not much as has been observed with the migration from white to black pools. By 168 hours, however, the score had dropped from about 11 to about 7.5, when changing from black to white.
These findings indicate that the color changes quite rapidly and is likely the result of changes in the chromatophores as indicated above (Figure 5). It is expected that the color change will occur fairly quickly because shrimp need to adapt to different environments through camouflage (Fingerman, 1965). Light microscopy of the dissected cuticles taken from the first abdominal segment of shrimp indicated that indeed, overall morphological changes occurred in the short time when the shrimp were transferred from the environment. from one medium to another (Figure 7). Investigation of the epidermis of shrimp maintained in the white tank until 0h time point (Figure 7a) revealed small, densely packed pigment cells. When similar cuticles were observed from shrimp that were transferred to the black tank and left for 3 h or 7 days (Figures 7c and e, respectively), there was dispersion of pigment throughout the chromatophores. pigment, by day 7, pigment occupies most of the surface, located in astrocytes. The sequence in Figure 7 (b, d and f) shows that the opposite occurred when shrimp were transferred from the black tank to the white tank. Disperse pigment (Figure 7b) was slightly more concentrated after 3 h (Figure 7d) and tightly concentrated after 7 days (Figure 7f). We have shown that the degree of esterification of astaxanthin appears to be dependent on the background color and it is possible that it is the altered chemical form that determines its location in the chromatophores.
Figure 7. Light microscopy of cuticle detached from the first ventral segment of shrimp showing pigment cells at different time points after switching between tanks (from white to black; a, 0h, c, 3h, e, 7 days and black to white ; b, 0h, d, 3h and f, 7 days). However, the overall magnification varied between individual images, with the average distance between the centers of the chromatophores being about 380 μm.
4.CONCLUSION
Pigmentation is an important commercial trait for shrimp marketing. Although natural and artificial pigments are an essential component of feed formulations and are adjusted to optimize shrimp coloration, large color variations have been observed. From this study, we conclude that at least part of the reason for this variation is the shrimp’s ability to camouflage and hide or reveal the pigment astaxanthin through movement within the chromatophores in the chromatophores. their epidermis. We suggest that in the light environment, the pigment is concentrated and the shrimp appear lighter than when the shrimp are in the darker environment, where the pigment is more dispersed. Importantly, it has been proposed that the chemical form of astaxanthin changes as it travels within epidermal pigment cells, becoming esterified with fatty acids or alternatively, hydrolysed to free astaxanthin, as it migrates. from the condensed state to the dispersed state (respectively). Given that shrimp darken fairly quickly in the presence of a black or dark background, this method provides the shrimp industry with a simple means of increasing shrimp pigmentation and reducing variability without additional feed costs. Therefore, consideration of background color conditions can have a significant impact on the market value of shrimp.
R.K. Tume, A.L. Sikes, S. Tabrett , D.M. Smith
a CSIRO, Food Futures National Research Flagship
b CSIRO, Food and Nutritional Sciences, PO Box 3312, Tingalpa DC, Queensland 4173, Australia
c CSIRO, Marine and Atmospheric Research, Cleveland, Queensland 4163, Australia