Recovery of carotenoids from shrimp waste in organic solvents

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Recovery of carotenoids from shrimp waste in organic solvents

N.M. Sachindra

^*

, N. Bhaskar, N.S. Mahendrakar

Department of Meat, Fish and Poultry Technology, Central Food Technological Research Institute, Mysore 570 013, India Accepted 8 July 2005

Available online 29 August 2005

Abstract

Shrimp waste, which is produced in large quantities in the Indian seafood processing industries, is one of the important sources of natural carotenoids. Studies were carried out to assess the extractability of shrimp waste carotenoids in diﬀerent organic solvents and solvent mixtures and to optimize the extraction conditions for maximum yield. A 50:50 mixture of isopropyl alcohol and hexane gave the highest (43.9lg/g waste) carotenoid extraction yield compared to acetone, methanol, ethanol, isopropyl alcohol, ethyl acetate, ethyl methyl ketone, petroleum ether, and hexane individually and to a mixture of acetone and hexane. Extraction conditions such as percentage of hexane in the solvent mixture of isopropyl alcohol and hexane, ratio of solvent to waste and number of extractions was optimized using a statistically designed experiment. The optimized conditions for maximum yield of carotenoids were 60%

hexane in solvent mixture, solvent mixture to waste ratio of 5:1 in each extraction and three extractions. A regression equation for predicting the carotenoid yield as a function of three processing variable (hexane % in solvent mixture, solvent-to-waste ratio and number of extractions) was derived by statistical analysis, and a model with predictive ability of 0.98 was obtained.

1. Introduction

The seafood processing industry is one of the major food processing industries in India. In the year 2003–04, 129,785 ton of frozen shrimps were produced (MPEDA, 2004). Processing of shrimp invariably generates solid waste in the form of head and body carapace. As the waste generation from processing of Indian shrimps ranges from 48% to 56% of the total weight depending on the species (Sachindra et al., 2005a), it can be estimated that the solid waste generation in Indian shrimp processing industries would be around 125,000 to 150,000 ton per annum.

The major components (dry weight basis) of shrimp waste are protein (35–50%), chitin (15–25%), minerals (10–15%) and carotenoids (Sachindra, 2003). At present, a small quantity of this waste is used in the dry form as an ingre- dient in animal feed and for the production of chitin/

chitosan. However, large quantities of this byproduct are being wasted, resulting not only in the loss of valuable components, but also in environmental pollution.

Shrimp waste is one of the important natural sources of carotenoid (Shahidi et al., 1998). The carotenoid content in the wastes from Indian shrimps was found to vary from 35 to 153 lg/g depending on the species, the major pigment being astaxanthin and its esters (Sachindra et al., 2005a). The recovery of these valuable components from the waste would improve the econom- ics of the shrimp processing plant. Since, at present the shrimp waste generated in Indian shrimp processing industries is not commercially exploited for the recovery of valuable components, it forms one of the cheapest raw materials for recovery of carotenoids. The extracted carotenoids would be a cheaper alternative than syn- thetic carotenoids in aquaculture feed formulations and in surimi based products.

Methods are available for extraction of pigments from crustacean wastes using vegetable oils (Anderson, 1975;

Spinelli and Mahnken, 1978; Chen and Meyers, 1982;

doi:10.1016/j.wasman.2005.07.002

* Corresponding author. Tel.: +91 821 2514840; fax: +91 821 2517233.

E-mail address:[email protected](N.M. Sachindra).

www.elsevier.com/locate/wasman

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Shahidi and Synowiecki, 1991; Sachindra and Mahendra- kar, 2005). Use of organic solvent for recovery of carotenoids from shrimp waste has been limited to analytical purposes only (Britton, 1985; Meyers and Bligh, 1981).

Solvents such as acetone (Mandeville et al., 1991; Masato- shi and Junji, 1999; Sachindra et al., 2005b) and alcohol (Kozo, 1997) have been used for extraction of carotenoids from crustacean waste.Li et al. (2005)extracted carotenoids from ﬁsh eggs using acetone, and the carotenoids in acetone extract was phase separated by using methyl tertiary butyl ether.Charest et al. (2001)used alcohol as cosolvent in supercritical CO2extraction of astaxanthin from crawﬁsh shells. Selective extraction of astaxanthin from crustacean waste has been attempted by supercritical carbon dioxide extraction technique (Lopez et al., 2004). A method has been patented for extraction of carotenoids from shrimp waste using a solvent mixture (Sachindra et al., 2001). Several organic solvents have been permitted for use in food industries as carrier or extraction solvents. Some of the organic solvents permitted for use in food industries are acetone, benzyl alcohol, ethyl acetate, hexane, isopropanol, methanol, methyl ethyl ketone, and ethanol, although levels of use depends on the type of foods in which they are used (Food and Drug Regulation, 2005).

There are no reports on optimization of extraction conditions for recovery of carotenoids from shrimp waste using organic solvents, and the information is limited on the effect of different solvents and solvent mixtures on extractability of carotenoids. There is a need to develop a suitable extraction method with optimum carotenoid yield from shrimp waste, which can be applied on a commercial scale. Hence, studies were conducted to determine the yield of carotenoids from shrimp waste in different organic solvents and their mixtures, and optimization of conditions for solvent extraction of carotenoids by a statistically designed experiment.

2. Materials and methods 2.1. Preparation of shrimp waste

Shrimp waste from processing of Penaeus indicus, comprising of head and carapace, was collected from a shrimp processing plant situated at Mangalore, India and transported to the laboratory under frozen (4 C) conditions and stored at20C until use. The mate- rial was thawed in running water before use and homogenized in a laboratory mixer.

2.2. Yield of carotenoids in diﬀerent organic solvents/

solvent mixtures

Carotenoids in the homogenized shrimp waste were extracted using diﬀerent organic solvents and solvent

mixtures by the method of Simpson and Haard (1985) as explained bySachindra et al. (2005a). The polar solvents used were acetone, methanol, ethyl methyl ketone, isopropyl alcohol (IPA), ethyl acetate and ethanol; while the non-polar solvents used were petroleum ether and hexane. The solvent mixtures were prepared by mixing equal quantities of a polar and non-polar solvent. The solvent mixtures used were acetone and hexane, and IPA and hexane (Sachindra et al., 2001). Carotenoids in 20 g of sample were extracted by homogenizing the sample with 50 ml of solvent. The extract was filtered and the residue was repeatedly extracted with fresh solvent and the filtrate collected until the filtrate was color- less. The solvent extracts were pooled together and in the case of extracts in polar solvents, they were phase separated with an equal quantity of petroleum ether.

The petroleum ether extract was repeatedly washed with an equal quantity of 0.1% saline to remove traces of polar solvents, if any, then dried with 25 g of sodium sul- phate, filtered, flushed with nitrogen for 5 min, and then evaporated under vacuum at 40 C using a rotary flash evaporator. In the case of carotenoid extract in petroleum ether, hexane and solvent mixture, the addi- tion of petroleum ether for phase separation was avoided and the extracts were directly washed with saline, dried and concentrated. The resulting carotenoids concentrate was taken up in petroleum ether and made up to 100 ml, and the absorbance of the appropriately diluted extract was measured at 468 nm using a Spec- tronic 21 spectrophotometer. The yield of the carotenoids was calculated as astaxanthin (Simpson and Haard, 1985) using the following equation:

Carotenoid yield ðlg astaxanthin=g sampleÞ

¼A_{468 nm}V_extractDilution factor 0.2Wsample

;

whereAis absorbance,Vis volume of extract, 0.2 is the A468of 1lg/ml of standard astaxanthin andWis weight of sample in grams.

2.3. Optimization of conditions for solvent extraction of carotenoids

The conditions for extraction were optimized with respect to hexane percentage in the hexane–IPA solvent mixture (X1), solvent-to-waste ratio (X2) and number of extractions (X3) using the Box–Behnkan experimental design (Box and Behnken, 1960). The experimental design was developed using the software STATISTICA (Statsoft. Inc, 1999). The experimental design determines the eﬀect of a combination of process variables (factors) and their interactions on the response variable. The experimental design involved three factors (X1,X2,X3), each at three equidistant levels (1, 0, +1), and the response variable was the carotenoid yield (Y) (Table 1). In total, 15

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combinations of factors were used. The levels of three factors were selected based on preliminary experiments.

The extraction of carotenoids and the determination of their concentration was carried out as explained earlier.

2.4. Statistical analysis

All of the statistical analyses were carried out using the software STATISTICA (Statsoft. Inc, 1999). The analysis of variance technique and DuncanÕs multiple range tests were used to determine the significant difference in yield between different solvents and for mean separation, respectively, at 95% confidence (p60.05) level. The effect of each factor and their interactions on the carotenoids yield was assessed by ANOVA technique. The optimization data was analyzed for determination of regression coefficients to arrive at the regression equation. A regression model containing 10 coefficients, including linear and quadratic effect of factors and linear effect of interactions, was assumed to describe relationships between response (Y) and the experimental factors (X1,X2,X3) as follows:

Y ¼b₀þX³

i¼1

b_iX_iþX³

i¼1

b_iiX²_i þX²

i¼1

X³

j¼iþ1

b_ijX_iX_j; ð1Þ

whereb0is the constant coefficient,biis the linear coefficient of main factors,biiis the quadratic coefficient for main factors andbijis the second order interaction coefficient. The response variable was assigned at low and high of the observed values for a desirability of 0 and 1, respectively, to get the overall desirability. The desirability function to get optimum carotenoid yield was fitted by the least square method using the software. The 3D response graph and profile for predicted values and desirability level for factors were plotted using the software (Statsoft. Inc, 1999).

3. Results and discussion 3.1. Yield of carotenoids

The solvent extracted carotenoid was in the form of a paste with an orange-red color. The highest carotenoid yield (43.9 lg/g waste) from waste was obtained when the carotenoids were extracted with a mixture of IPA and hexane, followed by IPA (40.8 lg/g) and acetone (40.6 lg/g) (Table 2). The lowest carotenoid yield was obtained with two non-polar solvents, petroleum ether (12.1lg/g) and hexane (13.1lg/g). The extraction yield differed significantly (p60.05) between solvents. Even though 50:50 mixtures of IPA and hexane gave significantly (p60.05) higher yield than IPA alone, no significant (pP0.05) difference was observed in carotenoid yield between acetone and the 50:50 mixture of acetone and hexane. Although these observations are with respect to the wastes fromP. indicus, which has the lowest level of carotenoids among the marine shrimps from In-

Table 1

Independent factors, their coded and actual levels, and combination of independent factors for optimization experiment

Run no. Hexane % in the solvent (X1) Solvent-to-waste ratio (v/w) (X2) Number of extractions (X3)

Coded level Actual level Coded level Actual level Coded level Actual level

1 1 10 1 2 0 3

2 +1 80 1 2 0 3

3 1 10 +1 8 0 3

4 +1 80 +1 8 0 3

5 1 10 0 5 1 1

6 +1 80 0 5 1 1

7 1 10 0 5 +1 5

8 +1 80 0 5 +1 5

9 0 45 1 2 1 1

10 0 45 +1 8 1 1

11 0 45 1 2 +1 5

12 0 45 +1 8 +1 5

13 0 45 0 5 0 3

14 0 45 0 5 0 3

15 0 45 0 5 0 3

Table 2

Yield of carotenoids from shrimp waste in diﬀerent solvents and solvent mixtures

Solvent/solvent mixture Yield (lg/g waste) (WWB)^*

Acetone 40.6 ± 1.6^a

Methanol 29.0 ± 3.3^b

Ethyl methyl ketone 36.8 ± 1.9^c

Isopropyl alcohol (IPA) 40.8 ± 3.0^a

Ethyl acetate 36.9 ± 2.9^c

Ethanol 31.9 ± 2.2^d

Petroleum ether 12.1 ± 1.8^e

Hexane 13.1 ± 0.9^e

Acetone:hexane (50:50) 38.5 ± 1.0^ac

IPA:hexane (50:50) 43.9 ± 0.7^f

* WWB – wet weight basis; values are means ± SD (n= 6); values with different superscript (a–f) differ significantly (p60.05).

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dian waters (Sachindra et al., 2005a), similar observa- tion may be made with waste from other species of shrimps.

Britton (1985)recommended the use of water miscible polar organic solvents, usually acetone, methanol or ethanol, for extraction of carotenoids from tissues containing water.Delgado-Vargus et al. (2000)discussed the advantages and disadvantages of various organic solvents for extraction of carotenoids and suggested that polar solvents are generally good extraction media for xanthophylls but not for carotenes. For wet tissues, use of non-polar solvents is not recommended as their pene- tration through the hydrophobic mass that surrounds the pigment is limited (Delgado-Vargus et al., 2000).De Ritter and Purcell (1981)postulated that complete extraction of carotenoids from plant tissues could be achieved with samples of low moisture content by use of slightly polar plus non-polar solvents. In the present study, the increased extraction yield of carotenoids by the mixture of IPA and hexane may be due to the reason that along with xanthophylls, increased amount of carotenes are also extracted due to the inclusion of a non-polar solvent in the extraction medium.

Even though acetone is used as a common extraction medium for carotenoids, the present study indicated that IPA is also a good extraction medium for carotenoids from shrimp waste. Further it is stated that, when IPA or a mixture of IPA and hexane was used for oil extraction, more antioxidants were extracted and oils with ex- tended stability were obtained (Procter and Bowen, 1996). Shrimp waste is known to contain antioxidants (Li et al., 1998); thus, the use of IPA and hexane for extraction of carotenoids may improve their stability during storage.

3.2. Optimization of conditions for carotenoid extraction The extraction with 50:50 a mixture of IPA and hexane at a solvent-to-waste ratio of 2.5:1 (v/w) gave a higher carotenoid yield than other solvents as observed in Table 2. In order to determine the combined eﬀect

of different levels of hexane in the solvent mixture (X1), solvent-to-waste ratios (X2) and number of extractions (X3) on carotenoid yield (Y), optimization experiments were conducted. All of the three factors namely, hexane % in solvent mixture, solvent level to waste, and number of extractions, and the interaction between X1 andX2,X1 andX3, andX2 andX3 (p60.05) had a significant effect on the carotenoid yield. A significant (p60.05) lack of fit indicates that there is still some statistically significant variability left that cannot be ac- counted for by the factors and their interactions (Table 3).

The regression coefficients for the main effects and their interactions are obtained by the regression analysis of the optimization experiment data to fit a suitable regression equation for carotenoid yield as a function of linear and quadratic effects of main factors and the linear-by-linear interaction effects. Using the regression coefficients for factors and their interactions the regression equation(1)can be written as

Y ¼b₀þb_iX1þb_iX2þb_iX3þb_iiX1²þb_iiX2²þb_iiX3² þb_ijX1X2þb_ijX1X2þb_ijX2X3;

ð2Þ whereYis the carotenoids yield, andb0,bi,bii,bijare the regression coeﬃcients of factors and their interactions as explained in Section 2.4. The regression equation (Eq.

(3)) was derived, for the carotenoid yield as a function of the three main eﬀects and their interactions, using Eq. (2) and the regression coeﬃcients. The regression equation obtained was

Y ¼ 0.44366þ0.21985X1þ2.11016X2þ13.65674X3 0.00135X1²0.07938X2²1.25022X3²

þ0.00659X1X20.02276X1X30.29520X2X3.

ð3Þ The regression equation (Eq. (3)) was used to arrive at the predicted value of carotenoid yield at each combination of processing variables (factors). The closeness of the observed and the predicted carotenoid yield

Table 3

ANOVA for the carotenoid yield (Y) as a function of hexane % in the hexane–IPA mixture (X1), solvent-to-waste ratio (X2) and number of extractions (X3) and their interactions

Factor Sum of squares Degrees of freedom Mean sum of squares Fvalue

Hexane % in solvent mixture (X1) 49.20 2 24.60 722.60^*

Solvent-to-waste ratio (X2) 39.96 2 19.98 586.95^*

Number of extractions (X3) 519.92 2 259.96 7636.37^*

Interaction

X1·X2 1.91 1 1.91 56.23^*

X1·X3 10.15 1 10.15 298.16^*

X2·X3 12.55 1 12.55 368.62^*

Lack of ﬁt 14.99 3 4.99 146.75^*

Pure error 0.068 2 0.034

* p60.05.

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(R²: +0.9765; slope: 0.9766) can be noted in Fig. 1, which indicates that this regression equation can be used to determine the carotenoid yield at diﬀerent levels of three factors, which are inﬂuencing the carotenoid yield.

The response surface graph (Fig. 2) of the eﬀect of hexane % in the solvent mixture and solvent-to-waste ratio when the number of extractions was kept at 3 shows that increasing the hexane % in the solvent mixture to above 60% did not improve the carotenoid yield further. The response surface graph (Fig. 3) of the eﬀect of hexane %

in combination with the number of extractions at a constant solvent-to-waste ratio of 5:1 (v/w), indicates that the carotenoid yield was highly inﬂuenced by the change in number of extractions. It is stated that when tissues contain a large amount of water, the ﬁrst extraction with polar solvents may remove little pigment, but as they dry the tissues, the carotenoid yield increases in the subsequent extractions (Britton, 1985). In the present study, as the extraction medium is a mixture polar and non-polar solvent, the polar solvents remove the water in tissues which will aid in the extractability of pigments in non- polar solvents in subsequent extractions.

The desirability function to obtain an optimum carotenoid yield was fitted by the least square method assign- ing the carotenoid yield (lg/g) at the observed low (20.25) and high (41.72) values for a corresponding desirability of 0 and 1, respectively, and the profiles were plotted. These desirability profiles show which levels of predictor (X1,X2 and X3) variables produce the most desirable predicted responses on the dependent variable (Y). The profiles for predicted response and the desirability level for factors (Fig. 4) indicate that 60% hexane in the solvent mixture, a solvent-to-waste ratio of 5 and 3 extractions give optimum carotenoid yield at an optimum desirability score of 0.90294. These profiles suggest that an increase in the hexane level in the solvent mixture, solvent-to-waste ratio and number of extractions above the optimized levels will not increase the yield significantly. The recovered carotenoids in the solvent mixture can be concentrated by removing the IPA by phase separation and recovery of hexane from the resultant extract.

PY = 0.9766 OY + 0.8191 R² = 0.9765

20 25 30 35 40 45

Observed yield (OY), μg/g waste

Predicted yield (PY), μg/g waste

Fig. 1. Observed vs. predicted carotenoid yield.

Fig. 2. Response surface graph for carotenoid yield from shrimp waste as a function of hexane % in the solvent mixture and solvent-to-waste ratio (number of extractions = 3).

Fig. 3. Response surface graph for carotenoid yield from shrimp waste as a function of hexane % in the solvent mixture and number of extractions (solvent-to-waste ratio = 5).

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4. Conclusion

Use of a mixture of polar and non-polar solvents, namely IPA and hexane, for extraction of carotenoids from shrimp waste produces the highest yield. The optimized conditions for the solvent extraction of carotenoids from shrimp waste were found to be 60% hexane in the solvent mixture of IPA and hexane, a solvent-to-waste ratio of 5 in each extraction and 3 extractions. The use of IPA and hexane instead of normally used acetone is ben- eﬁcial in the large-scale extraction of carotenoids from shrimp waste, as the cost of IPA and hexane is lower than that of acetone and the yield of carotenoids is higher.

Although the results obtained are for the waste from the speciesP. indicus, it would be applicable to waste from other species of shrimps. The residue available after carotenoid extraction may be used for the preparation of chitin/chitosan, thus having an integrated approach for eﬃcient utilization of shrimp waste.

Acknowledgments

Authors thank Dr. V. Prakash, Director, CFTRI for his encouragement during the investigation.

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