Nano astaxanthin improves shrimp color, contributing 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), when assessed using a commercial grading scale, does not always correlate well with total function. amount of carotene. We also observed that the appearance was influenced by the color of the shrimp tank. When shrimp were raised in black or white tanks for 28 days, although they had similar average total astaxanthin content (29–33 μg/g shrimp tail), shrimp raised in black tanks had a much more orange/red color when cooking compared to shrimp raised in white tanks. Pigments are concentrated mainly in the cephalothorax, abdominal epidermis and abdominal exoskeleton. Light microscopy revealed a more uniform pigment distribution in the epidermis of the more vividly colored shrimp compared to concentrated pigmentation in the lighter colored shrimp. Non-esterified astaxanthin was the major carotenoid present in all body sites of shrimp from black tanks (50%) with the remainder being made up of mono- and diester astaxanthin. However, for shrimp from white tanks, non-esterified astaxanthin accounts for only 12–13%, with mono-esters accounting for about 60% of the current total. In a separate experiment, we demonstrated that changes in environmental color caused rapid changes in shrimp color. When shrimp were transferred from white tanks to black tanks, scores increased significantly within 1 hour without any change in nano astaxanthin content. Color improvement continued to increase for 168 h. Transferring shrimp from black tanks to white tanks 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 diet, it may be possible to improve their overall appearance (raw and cooked) by adjusting color at harvest.
1.INTRODUCE
The color of shrimp is one of the most important attributes in determining consumers’ shrimp purchasing decisions and thus significantly affects their market value. In Australia, cooked black tiger shrimp (Penaeus monodon) are typically evaluated for color using a subjective color grading chart with scores ranging from 1 to 12, reflecting low to high pigmentation (Aqua-Marine Marketing Pty Ltd., Kippa-Ring, Queensland) . Shrimp with higher scores are typically priced at A$2–4/kg more than lighter-colored shrimp, and typically, poorly colored shrimp cannot be sold at an acceptable market price and are frozen. and hold until 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 usually present in free and esterified forms (mono- and di-ester) 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 clearly seen when shrimp is 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 provitamin A activity (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 dietary and in wild shrimp the majority of astaxanthin originates 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 food in the environment as well as its ability to be absorbed, transported and subsequently deposited in specific anatomical locations. For farmed shrimp, the addition of synthetic astaxanthin to feed is an additional cost and it is often necessary to adjust the amount and timing of supplementation to achieve the desired pigmentation level (Chien and Jeng, 1992). It is often difficult for producers to predict the optimal feed concentration required due to the contribution of the pond’s algal biota, as well as the apparent genetic coloration of each individual and the apparent banding differences between species. shrimp.
As part of our research to determine the optimal pigmentation of farmed shrimp, we measured the total astaxanthin content in a large number of shrimp and observed that this content did not always correlate well. with realistic colors. We speculate that this difference in appearance may depend on the location of the pigments in different body regions of the shrimp (Sachindra et al., 2005). In addition, it is known that shrimp have the ability to change their appearance to blend with the color of the environment, through the movement of pigments in chromatophores in the epidermal layers below the exoskeleton ( Fingerman, 1965; Robison and Charlton, 1973). Morphological and physiological color changes have been described in crustaceans in relation to 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 pigmentation changes seen in individuals within groups of shrimp, the location of the pigments in different body components, and how they are affected by environmental light.
2.Materials and methods
Shrimp used in this study were obtained from experimental stocks and from commercial farms. Shrimp were not evaluated for molting stage and shrimp of different weights were used. However, for specific comparison tests, the shrimp had similar weights.
2.1 Experimental shrimp
Experimental shrimp (P. monodon) were obtained from stocks maintained at the CSIRO, Marine and Atmospheric Research (CMAR) laboratories in Cleveland. Until needed for experiments, shrimp were kept in 2500 L fiberglass tanks. Seawater was pumped through the tanks at a rate of 1.2 L.min−1 to maintain water temperature and salinity at times respectively at about 28°C and 32‰. Photoperiod before the start of the experiment was maintained at 12 h light: 12 h dark. Shrimp were fed commercial pellets (Lucky Star, Taiwan Hung Kuo Industrial Pty Ltd.) twice daily. The actual content of astaxanthin has been determined and found to be approximately 40 mg of astaxanthin per kg of feed.
2.2 Commercial frozen shrimp
Whole frozen, cooked black tiger prawns (weight approximately 20–50 g) were obtained from two commercial farms in Queensland.
2.3 Experiment 1 — effect of environmental color
P. tiger prawns from the same colony were raised 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 described). mentioned above). The light intensity (Iso-Tech ILM350, light meter) at a distance of 10 cm from the water surface is about 10lx. On day 0, half of the shrimp (n= 5) were randomly captured and transferred to white-lined tanks and maintained in an environment with the same light intensity as before. All shrimp 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 is 9.27± 0.99 g. Although the average weight of shrimp in black tanks was lower than that of shrimp in white tanks (8.10 ± 1.20 and 10.43 ± 1.51 g, respectively), these differences were not significant and reflected reflects the results of randomization on day 0. Shrimp were photographed and then frozen and kept at −20°C until needed for analysis.
2.4. Test 2 – speed of color change
Similar sized P. monodon shrimp from the same stock and housed together in black-lined tanks were randomly assigned to either black-lined or white-lined tanks at CMAR, Cleveland for 28 days (n= 96). During this period, each animal was fed an identical diet containing 40 mg astaxanthin/kg of feed. On the day the experiment started, at 9:00 a.m., shrimp were transferred from their tanks to tanks 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 sea water and ice for 5 min until death. They were then removed and boiled in filtered seawater for 2 minutes and then cooled in ice water. Shrimp were photographed and frozen and kept at −20°C until required for analysis. The lighting procedure was continued throughout the experimental period, which meant that the shrimp at 12 o’clock were subjected to approximately 4 hours of darkness.
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 a food processing laboratory set to an air temperature of 10 °C. Subjective assessment of color was performed using a grading scorecard (1 to 12 for lightest to darkest color) 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 the panelists were not trained to judge color of shrimp but they were experienced for different food attributes (including color) and were given anchor points for the extremes.
2.6. Relationship between scoring score and astaxanthin content
Cooked commercial shrimp (mean weight= 34.9 g [range 22 to 51 g], n= 61) graded by color according to the subjective assessment described in Section 2.5 were used for Determine the total astaxanthin content and distribution on the shrimp body at different levels (see Figures 2 and 3). Upon arrival at the laboratory, the frozen shrimp were thawed and, in this case, scored using a three-member panel to reach consensus on each shrimp. The shrimp were then sorted in order of score and the initial rating was confirmed. Each shrimp is then dissected into its exoskeleton, cuticle, cephalothorax and digestive glands to extract astaxanthin.
2.7. carotenoid analysis
Shrimp were weighed and then dissected as follows. All procedures, including extraction, were performed under low light intensity conditions in a dark laboratory. In most cases, and unless otherwise mentioned, comparisons between shrimp groups are made on the pigments present in the ‘shrimp tail’, which includes the cuticle of the abdominal muscles along with the abdominal exoskeleton , including uropods and telsons. This is done by removing the head and then separating the exoskeleton from the abdominal muscles. The epidermal pigment layer was 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 does not contribute to pigmentation in shrimp. When indicated, carotenoid analyzes were also performed on individually dissected components such as the abdominal exoskeleton, ventral epidermis, head, and digestive glands. Each tissue was weighed, chopped, and then extracted three times with 20 mL of acetone at 2°C, left overnight between each extraction. The shrimp residue remaining after this comprehensive extraction process is essentially free of pigment. The pooled extract was adjusted to a total volume of 60 mL and 10 mL H2O and 5 mL n-hexane were added, mixed well, and allowed to phase 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 gas and then redissolved in 20 mL of n-hexane. The concentration of nano astaxanthin in the extract was determined by measuring the absorbance at 477 nm, using a molar extinction coefficient of 2172 in a 1 cm cuvette. Astaxanthin standard was obtained from Sigma Chemical Co. St Louis, MO, USA and lutein standard from Extrasynthese, Genay, France. From previous analyzes we know that nano astaxanthin and its esters make up approximately 95% of total carotenoids and therefore lesser ones such as lutein are not assigned but would contribute to the total. All samples were stored in the dark, under nitrogen at −20 °C until further analysis was required.
Nano astaxanthin content results are expressed as μg/g wet weight of the shrimp ingredient or as a percentage of pigment distribution in individual ingredients. For each individual shrimp, the total nano astaxanthin (μg) extracted from each anatomical site was determined and the relative distribution was expressed on a percentage basis.
2.8. Separation of nano astaxanthin and astaxanthin esters by HPLC
The n-hexane carotenoid extracts were transferred to brown sample vials for HPLC separation on a Waters system consisting of a Model 600 pump, a Model 717 autoinjector, and a Model 996 photodiode array detector.Nano Astaxanthin was determined. determined at a wavelength of 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 cartridge (Phenomenex #AJ0-4287). The gradient solvent system, with a flow rate of 1.5 mL/min, was as follows: solvent A (methanol:H20, 80:20,v/v); Solvent B, (ethylacetate) was essentially as described by Wade et al. (2005).
2.9. microscope
Portions of the cuticle from the first abdominal segment of each shrimp were carefully removed from the abdominal muscle after removing the outer shell. The cell sheets were placed on a microscope slide and covered with physiological saline under the slide. Chromatophores 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 chromatophores was measured with micrometers in stages and the mean (±SE) distance was found to be 380± 9.21 μm for shrimp weighing between 20 and 30 g.
2.10. Statistical analysis
A 5% confidence level was used to compare significant differences between means (p≤0.05) using a paired Student’s t-test (Microsoft Excel, XP). For the data presented in Figure 1, we tested the relationship between shrimp weight and shrimp width at the second abdominal segment with linear, quadratic, and cubic models. Using the Akaike Information Criterion (AIC), the linear model was selected as the best fitting model for the data set because it had the lowest AIC value (Sakamoto et al, 1986).
3.Results and Discussion
3.1. Relationship between scoring score and nano astaxanthin content
Determination of carotenoid content in whole shrimp can provide a satisfactory indication of the carotenoid level in the feed suitable for growth or health, but it may not necessarily be the best method of expression. to visualize the color of the shrimp, as some pigments may be located in areas and organs such as the digestive glands, and therefore do not contribute to appearance. Additionally, we need to contend with the relatively wide range in shrimp weights encountered in these trials. We argue on the basis of visual area and weight relationships, assessed by body width, that pigment content, expressed on the weight of the abdominal muscles plus the abdominal exoskeleton , providing a satisfactory set of units. In support of this, we found a linear relationship (R2= 0.623) between second abdominal width (and thus surface area) and shrimp weight (Figure 1). On this basis, we expressed the weight of the extracted pigment (μg) per the wet weight of the abdominal muscle plus the abdominal exoskeleton.
However, during research to determine the optimal concentration of pigments in feed to achieve good color, we measured the total astaxanthin content (expressed on the basis of the weight of the abdominal muscles and skeleton). extraabdominal bones) in a large number of shrimp and it was observed that number did not correlate well with appearance. This was further demonstrated by measuring astaxanthin content in shrimp that had been subjectively visually classified into different categories using a color grading chart. The average grading score of shrimp ranged from 6 to 11. The relationship between grading score and total astaxanthin content is shown in Figure 2. It can be seen that color or appearance grading score is not related closely to the measured nano astaxanthin content. For example, in claim 9, the astaxanthin content ranged from 5 to 25 μg/g shrimp tail. Therefore, there must be some other explanation for the observed difference.
3.2. Pigment distribution in shrimp
The distribution of total nano astaxanthin in the ingredients is presented in Figure 3 for each score from 6 to 11 (range obtained). Essentially, total astaxanthin is distributed almost evenly between the abdominal exoskeleton, abdominal epidermis, and cephalothorax (the epidermis and exoskeleton are not separate), with only 1–2% present in the digestive gland. These findings are essentially similar to those observed by Negre-Sandargues et al., 1993 and Paibulkichakul et al., 2008. Pigment distribution did not vary effectively across the measured scores. investigation here. It should be noted that in other situations when shrimp are about to molt, the percentage of total astaxanthin present in the exoskeleton decreases markedly as the pigment is mobilized back into the cuticle as has been observed in lobsters. Western red (Wade et al. , 2005).
3.3. Effect of background color on nano astaxanthin distribution in raw shrimp
The shrimp, which had previously been raised together under identical conditions, changed their appearance after they were separated and then raised in black or white tanks, even though they were fed the same food. food rations. These differences are clearly visible in uncooked shrimp; those from black tanks were dark green/brown compared to the lighter color of those from white tanks. The total nano astaxanthin content (Table 1) of shrimp grown in black or white tanks for 28 days was not significantly different (33.3 μg/g shrimp tails for black tanks vs. 29.1 μg/g shrimp tails for shrimp tanks). white, p≥0.05) and will not result in any noticeable differences in color. However, there was a distinct difference in their cooked color (Figure 4), with shrimp raised in black tanks being more orange/red than shrimp raised in white tanks. Close examination of the outer shell/cuticle (Figure 5) shows that darkly colored shrimp have more evenly distributed pigmentation than lighter colored shrimp, which have dense pigmentation. concentrated in small spots (pigment cells).
The distribution of nano astaxanthin in different body components was also determined in shrimp after 28 days under each background color condition (Table 1). It is clear that shrimp raised in black tanks have a higher percentage of total nano astaxanthin in the cephalothorax (42.7%) (p≤0.05) than shrimp raised in white tanks (34.6%), although the epidermis Abdominal dermis 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 esters (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 studied 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 position, the percentage distribution of individual pigments was very different depending on whether the shrimp were kept in black or white tanks. For shrimp raised in black tanks, astaxanthin, in its non-esterified form, makes up about 50% of the total carotenoids. The remainder mainly consists of equal parts astaxanthin mono-esters and astaxanthin di-esters (about 20% each), along with a small amount of lutein (5–7%). However, for shrimp raised in white tanks, the distribution is very different. Non-esterified astaxanthin was greatly reduced, accounting for only about 12% of the total carotene. In contrast, astaxanthin mono-ester increased from about 20% to 60% of the total. The proportion of astaxanthin di-ester did not change significantly.
Therefore, this study suggests that the form of nano astaxanthin present in the peripheral tissues of shrimp depends on the background light conditions. Therefore, it is proposed that in dark environments, a large proportion of astaxanthin mono-esters are hydrolyzed, whereas in 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 studied. Although our findings mainly concern 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 the color and growth rate of shrimp. Using Litopenaeus vannamei, they observed that body color was best in shrimp exposed to the highest light intensities and suggested that high astaxanthin is a requirement under these conditions to minimize potential damage from the rays. ultraviolet. The reason for this increase in pigmentation is unclear but may be related to greater growth of phytoplankton, bacteria, etc. in water, and thus astaxanthin precursors are more available, under brighter light conditions (Tseng et al., 1998). Additionally, the efficiency of absorption and deposition may be higher in shrimp grown under brighter conditions.
In a controlled environmental study with juvenile rock lobsters (Jasus edwardsii), Stuart et al. (1996) were unable to demonstrate any significant influence of substrate color (black rock or white gravel) on exoskeleton color. This study observed a year-long growth period under specific conditions and concluded that any anecdotal differences reported by commercial lobster fishers are likely result of genetic differences.
3.4. Color change rate
In the pretest, shrimp were maintained under the specified background color conditions for 28 days before being evaluated. The following test was performed to determine the rate of color change as this can 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 time 0:00. At this time, shrimp from the white tank had similar total astaxanthin content (p≥0.05) to shrimp from the black tank (32.1± 2.20μg/g shrimp tail and 36.7± 2.16 μg/ g corresponding shrimp tail). To obtain more accurate data (through increased counts) on the astaxanthin content of shrimp from these two tanks, all shrimp used over 168 hours were analyzed. The overall values were 35.1± 1.096 μg/g shrimp tails for shrimp transferred to white tanks and 36.7± 1.108 μg/g shrimp tails for those transferred to black tanks (see Figure 6). Although the total pigment content of the two groups of shrimp was similar at 0 h, there was a large difference in the color of raw and cooked shrimp. When graded by participants, cooked shrimp from white tanks were significantly lighter in color (p≤0.001), with an average score of approximately 7.0 ± 0.306, compared to 10.8 ± 0.225 for shrimp from white tanks. black tank (Figure 6). .
However, within 1 hour after transferring live shrimp from the white tank to the black tank, the pigmentation increased significantly and when the shrimp was cooked, the red color of the shrimp 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, increasing to about a 10 point at 72 and 168 hours after transfer. Transferring shrimp from black to white tanks also resulted in a change in color, with shrimp becoming lighter in color, but the difference during the first 1 hour (p= 0.189) and 12 hours (p= 0.024) did not occur. as much as was observed with the move from white to black tanks. However, by 168 hours, the score had dropped from about 11 to about 7.5, when changing from black to white tanks.
These findings indicate that color changes quite rapidly and is likely the result of changes in pigment cells as indicated above (Figure 5). It is thought that color change will occur quite rapidly because shrimp need to adapt to different environments through camouflage (Fingerman, 1965). Light microscopy of dissected cuticles taken from the first abdominal segment of the shrimp indicated that indeed, gross morphological changes occurred over a short period of time when the shrimp were transferred from the medium. one environment to another (Figure 7). Investigation of the cuticles of shrimp maintained in white tanks until time 0h (Figure 7a) revealed small, densely packed pigment cells. When similar cuticle layers were observed from shrimp that had been transferred to black tanks and left for 3 h or 7 days (Figure 7c and e, respectively), there was pigment dispersion throughout the chromatophores. pigment, by day 7, pigment occupies most of the surface, located in stellate melanocytes. The sequence in Figure 7 (b, d and f) shows that the opposite occurred when shrimp were transferred from black tanks to white tanks. The dispersed pigment (Fig. 7b) was slightly more concentrated after 3 h (Fig. 7d) and tightly concentrated after 7 days (Fig. 7f). We have shown that the degree of esterification of nano astaxanthin appears to depend on the background color and that it may be the altered chemical form that determines its location in melanocytes.
Figure 7. Light microscopy of cuticle detached from the first abdominal segment of shrimp showing pigment cells at different times after transfer 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, the average distance between the centers of the chromatophores being approximately 380 μm.
4.Conclude
Pigmentation is an important commercial characteristic for shrimp marketing. Although natural and artificial pigments are an essential component of feed formulations and are adjusted to optimize shrimp color, large variations in color 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 nano astaxanthin pigmentation through movement within pigment cells in the their epidermis. We propose that in light environments, pigmentation is concentrated and shrimp appear lighter than when shrimp are in darker environments, where pigmentation is more dispersed. Importantly, it has been proposed that the chemical form of astaxanthin changes as it moves within epidermal melanocytes, becoming esterified with fatty acids or alternatively, hydrolyzing to free astaxanthin, as it moves from the concentrated state to the dispersed state (respectively). Given that shrimp darken fairly quickly when given a black or dark background color, this method provides the shrimp farming industry with a simple means to increase shrimp pigmentation and reduce variation without adding 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