Lipid stability in the drying of Artemia by several methods

,4quacuhural Engineering 8 1 1 9 8 9 2 9 3 - 3 0 5

Lipid Stability in the Drying of Artemia by Several Methods* Shuh-Rehn Lieu & Kenneth L. SimpsonDcpartmcnt of Food Scicnce and Nutrition. Uni~ ersitv of Rhode Island. Kingston, Rhode Island 1)288 I. USA /Recci~cd 9 May 1989: accepted 24 August 1989!

A BS 7 RA ( T

Brine shri.tI~ were dried by Ji'eeze. vacuzt,'a, am/ hot air dehydration metltods. 7he total Lipid, fittO' acid profiles and cholesterol content were determined in order to estimate the extent of rite lipid degradation after rtte drying processes'. Three ehentical tests of lipid oxidation --peroxide t'alue, 2-rhiobar/gitttric acM gTBA) test. and iodine value--were iwrJ'ornted. Total lipid was.fimnd to hat'e S.~)",, ttml 25% loss in vacttttm cold/tot air-dried Artemia, re.g~ective[.v. T/tese apparent losses were due to the shrinkage l~henomenon during do'ing. This resttlted itl aH incomplete extraction By the Bligh & l)ver method (Can. J. Biochem. Physiol. t,~d. 37 (195()) pp. 9I 1-17). 7he Soxttlet method gave the .same lipid content in the /i'ee=e arid hot air-dried Artemia. The fitttv acid coml~osition oJ"/i'ee=e, VaCllttJTt, alld hot air-~h'ied Artemia ~-ave the same restt[t a.s newly hatched naup[ii. There was no evidence of oxidation in any fittty' ac'id jiom t/to three dt3'ing methods. Hot air drybtg resulted in a 56% loss of c/tolestero[ whereas there was no loss ojc/tolesterol in A rtemia dried hy J?ee=e or vacuttm-dlyitzg. The peroxide test indicated tt sigltificallt change in rite/tot air-dried sa.tple whereas the t'altte was not si,-ni/icantIy dij]'etenr ilt the Jieez.e or vacuum-dried Saml~[e ji'otn the ttmlried control. The iodine vahte was also significantly dtfferent itt the /tot air-dried sample front the ahove dried sa.t[~les. 77re 2-thioharhitttric acid vahtes ]br the Jottr sa.tple.s were nor significantly diJJ~,rent. *Contribution number 2518 RI Agricultural Experiment Station. T-'To whom correspondence shoukl be addressed at: Food Science and Nutrition Research Center, University of Rhode Island. 530 Liberty Lane, West Kingston, Rhode Island 02892, USA. 293

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S.-R. Liou, K. L. Simpson

INTRODUCTION The fatty acid profile of newly hatched nauplii of the brine shrimp A r t e m i a is one of the important factors in maintaining satisfactory survival and growth rates in cultured larval fish and crustaceans (Fujita et al., 1980: Levine & Sulkin, 1984). Recently, adult Arremia were found to have better nutritional value than newly hatched nauplii (Sorgeloos et al.. 1986) and can be used as a transition food (weaning or nursery diet) from nauplii to dry feed (Leger et al., 1986). The lipids of the brine shrimp have been studied with regard to the nutritional value of the fatty acid composition of nexvlv hatched nauplii (Wickins, 1972: Watanabe et al., 1978, 198o; Schauer et a[., 198(: Dendrinos & Thorpe, 1987), and to the degradation of A r t e m i a lipids under storage conditions (Sasaki & Capuzzo, 1984). Dried A r t e m i a have been used as a diet for rainbow trout (Gabaudan et al., 1980). The freeze, vacuum, and drum-dried brine shrimp had good protein digestibility and metabolizable energy values, while the oven-dried samples were much lower. The A r t e m i a mainly reproduce at lower salinities bv releasing Nauplii (ovoviviparous reproduction) and at higher salinities by releasing cysts (oviparous reproduction). Under intermediate salinities, and especially with fertilization, both cysts and biomass are produced. In a well managed pond the biomass must be harvested in order to maintain proper pond dynamics. The cysts are normally dried and the biomass may be used directly as a feed or frozen or dried. If the quality of the dried product can be maintained, this would have a number of advantages over the frozen product. The present project was conducted to determine the lipid loss by various analytical methods of the dried products and to evaluate these methods as predictors of lipid degradation in dried A r t e m i a biomass. MATERIALS AND METHODS

Samples The Reference Artemia Center II (RAC-II) cysts were provided by the Environmental Protection Agency (EPA), Narragansett, Rhode Island, The cysts were hatched in aerated sea water (30-32 ppt salinity; 25-28°C; pH 8-9; under fluorescent lighting). After 24 h, the newly hatched nauplii were retained on a 44 /~m nylon mesh screen, briefly rinsed with distilled water and lightly blotted prior to weighing. Samples

Lipid stability in the dr)'ing of artemia by several methods

295

in triplet were immediately extracted and analvzed as a control or placed in aluminum weighing dishes for the drying process. The drying equipment used included a freeze dryer (Unitrap II, Virtis. Gardiner, New York), a vacuum dryer (Jorma Scientific, Inc.), and a hot air dryer (Excalibur Food Dehydrator). Drying process

Freeze and vacuum drying conditions were modified from Gabaudan et al. (_1980). Approximately 2-5 g of newly hatched nauplii were put into each dish for drying purposes. The drying conditions are presented in Table l. After the drying process, the triplet dried samples were extracted and analyzed immediately. The measurements of water content and water activity

The determination of the water content was modified from the AOAC (1984) air oven method. The water activity was determined by a Beckman hygrometer, hygroline sensor assembly, Model EBS. Approximately 2 g sample was placed in the plastic dish and put into the sample dish sensor assembly. The water activity was recorded when the reading attained equilibrium (about 30 min). Lipid extraction and fatty acids profile

Lipids were extracted from about 1 g of newly hatched or 0"3 ,~ of dried Arternia. Samples were extracted according to the Bligh & Dyer (1959)

technique as modified by Kates (1972). The methyl esters of the fatty

TABLE 1 The Conditions of Freeze. Vacuum. and Hot Air Drying Processes. and the Water Contents and Activities of Dried Artemia

I'rocess

Freeze drying Vacut, m drying Hot air drying

Tempemtttre (°C I

Time (h)

I'ressure (arm)

ll4zter content of final product

- 72 to 25" 50 60

9 10 10

2.5 x 10 -5 0.0 [ 1"00

5-0(0-2/' 5'0 {0.2 ) 5.4 (0'2)

" - 72°C is the temperature of the frozen Artemia; 25°C is the temperature of the heated plate of the freeze dryer. 1'The values in the parentheses are the water activity of the final product.

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S.-R. Liou, K. L. Simpson

acids were prepared with boron trifluoride (BF.). The total lipid and fatty acid methyl esters (FAMEi were determined gravimetrically. The weights of lipid and FAME are presented as milligram lipid (or FAME) per gram dry weight sample. Fatty acid profiles were determined by a gas liquid chromatograph (GLC) equipped with a flame ionization detector and a GP (Guarantee Performance). 10% SP-2330 (Supelco. Inc.) stainless steel column. Quantification was done by a Hewlett Packard integrator (HP 3380A). Identifications were made by comparing the sample peak retention times with those of standards (Supelco, Inc.). Relative retention times based on 20:5w3 fatty acid were also used. Lipid extraction by Soxhlet method About 2 g of the freeze-dried and hot air-dried Artemia were weighed and placed in the sample thimbles. The thimbles were then put in a drying, oven set at 100°C for 5 h in order to dehydrate the residual moisture, cooled to room temperature in a desiccator and reweighed. They were then placed into Butt tubes and connected to the preweighed Soxhlet flask containing 125 ml ethyl ether. Extraction was continued for 16 h. After evaporating off any residual solvent, the crude lipid was dried for 1 h, cooled in a desiccator and weighed. Volatile materials measurement Approximately 30 mg of oil from newly hatched Artemia was placed in a 25 ml pear-shaped flask which was connected to a U-shaped trap through a water vacuum system. The pear-shaped flask was heated i n a 50°C water bath for 10 h. Dry ice in ethanol was used to cool the U trap during the heating period. The pear-shaped flask was weighed before and after heating to measure the volatile materials. Cholesterol determination Total cholesterol, free cholesterol, and cholesterol ester were determined by the method of Courchaine et al. (1959) as modified by Kates (1972). The recovery of this method was 96- 100%. Peroxide, TBA, and iodine values The peroxide value was measured by a modified procedure of the Official and Tentative Methods of the American Oil Chemistry Society, Cd

Lipid stability in the drs.ing ofartemia by severn/method.~

297

8-53 (AOCS, 1973) The results are presented as milliequivalents of peroxide per kilograms of sample. Thiobarbituric acid (TBA) reactive-substances were determined bv a modified procedure of Lemon (1975). The newly hatched and dried nauplii were accuratelv, wei~hed~ t2 ~,, and 0-3 ~,~ respectively ~. Water was added back to the dried samples for making up the moisture loss during the drying, ~ process. The standard curve was. made by usim,~ a 1.1.3.3tetraethoxypropane (TEP) solution. The absorbance was measured at ~o2 nm. Iodine value determinations were made bv use of the method of Yasuda ( 1 ~o I ) as modified by Kates I 1972*

Data analysis One-way classification analysis of variance was calculated for total lipid. FAME, cholesterol, peroxide value, TBA test. and iodine value. Student's t-test was used to determine the significant difference (p ~<0-05) of each datum listed above (Chase & Brown, 1986). The total saturated, unsaturated, monoenoic, dienoic, polyenoic, ,,,,'3 and w6 fatty acids were analyzed by the multivariative analysis of variance method (Srivastava & Carter, 1983), combined with an SAS program to test the differential within a p ~ 0-05 range.

RESULTS A N D DISCUSSION The water content and activity of dried Artemia were around 5°/,, and 0'2. respectively (Table 1). Water activities, higher than 0-4 or lower than 0"2. would increase the lipid oxidation reaction rate (Labuza, 1980 ~. In addition, DeMan (1985) states that the dried or freeze-dried foods which have the greatest storage stability usually have water contents in a range of about 5- 15%. The 5% and 0.2 of water content and activity, which were used in this study of the dried Artemia, should minimize the storage oxidation reaction. The time of freeze drying (9 h) was much less than 60 h used by Gabaudan et al. (1980).

Total lipid, FAME content and fatty acid profile The total lipid and fatty acid methyl ester (FAME) content of newly hatched and freeze, vacuum, and hot air dried Artemia are shown in

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S.-R, Liou, K. L. Simpson

Table 2. The total lipid of freeze dried Artemia ( 176 _+8 mg/g) was the same as newly hatched Artemia (179___9 dry, weight basis), whereas vacuum ( 163 + 5) and hot air-dried ( 134 _+6) samples were 16 mg (8-9%) and 45 mg (25%), respectively, less than the control. The total percentage of saturated, monoenoic, dienoic, polyenoic, unsaturated, w3, and w6 showed no statistical differences among the four samples (Table 3). This loss of lipid under certain drying conditions but with no apparent change in the fatty acid profile might be explained if short chain fatty acids were lost. In order to test this, a heated vacuum system was designed to trap the volatile compounds. After 10 h heating in a 50°C water bath, there was about 2-9% oil loss in total lipid. However. this loss of short chain fatty acids and other volatile materials could not explain the 8"9% and 25% total lipid loss under a vacuum and a hot air-drying process. A Soxhlet extraction was then used as an additional method for determining the total lipid content. The total lipid contents showed the same value for the freeze and hot air-dried Artemia (Table 2). The structure of the dehydrated product is affected by the heat and pressure employed in the drying process. Hamm and Deatherage (1960) have described the effect of heat on protein denaturation. The exposure to temperatures in the range of 20 to 30°C resulted in no apparent effect on the proteins. Drying temperatures greater than 40°C resulted in stable cross linkages between proteins. Porosity is one of the structural characteristics of dehydrated products which can be developed by rapid escape of water vapor into the high "FABLE 2 Tile Amounts of "l'otal Lipid and Fatty Acid Methyl Esters in Newly Hatched. Freezedried, Vacuum-dried, and Hot Air-dried Artemia

Newly hatched ntmplii Total lipid" (Bligh & Dyer, 1959) FAME(after Bligh & Dyer, 1959) Total lipid (Soxhlet)

l')'eeze-dried

Vacuttm-dried

Hot air-dried

179+9a h

176+8a

163+5b

134+6c

1 5 6 + 10a

148+ 15ab

140+4b

114+ lc

--

258 + 15a

--

257 + 8a

"The unit of the total lipid and F A M E is mg/g dry weight sample. hValue within each row, bearing the same letter are not significantly different at p >/0.05. - - N o t determined.

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299

TABLE 3 Fatty Acid Composition of Newly Hatched. Freeze-dried. Vacuum-dried. and Hot Airdried Artemia (area %)"

FAME

:Vew/v hatched

Freeze-dried

Vacuum-dried

ttot air-dried

1-46 0-72 1379 470 066

1-54 079 1402 458 0'69

144 080 1392 474 0-69

138 075 1336 500 0'75

15 : 1 16:1 18:1

1.28 0-46 16-71 31"86

1.44 0-29 17-04 31.53

1.41 0.45 16.95 31'74

1.28 0-44 16-55 32.22

Dienoic 16:2w4 18.2w6 20:2w6

4.3t) 12.24 0" l 6

4.45 1"9.'~7 0.14

4.29 I "~.vv 0-10

4.10 "~ -, --

Polyenoic [8:3w3 18:4w3 20:3w6

1.14 0-21 0-16

1-13 0.20 0-18

1.20 0-23 O" 17

1.19 0.2 I 0' 15

20:3w3/20:4w6 20:5w3 22:4w6 22:6w3

4-69 5" 15 0-24 0-01

4-50 4"94 0-16 0" 10

4"49 5" 1 l ---

4-85 5"23 (1"10 --

21-33a 50-3 la 16.70a 11-60a 78"6 la 6.51a 12.80a 99'93

21.62a 50'30a 16"86a 11.2 la 78-37a 6.37a 12.75a 99-99

21-59a 50-55a 16.61 a 1 l'20a 78.36a 6.54a 12-49a 99-95

21-24a 50.49a 16-49a I 1"73a 78.7 l a 6.63a 12.64a 99-95

Saturated 14:0 15 :() l 6:0 t 8:0 20:0 Monoenoic

14:1

Total Total Total Total Total ~ 3 ~' ~6 Total

saturated mono. di. poly. unsat.

FAME

"k,hlue within each row which bear the same letter are not significantly different at p/> 0.O5. hBoth w3 and w6 values do not include 2 0 : 3 w 3 / 2 0 : 4 w 6 . - - T h e fatty acid was not found.

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S.-R. Liou, K. L. Simpson

vacuum condition (Potter, 1978). The porous product has the advantage of quick rehydration or solubility properties (Potter. 1978). The shrinkage phenomenon, found in hot air or vacuum-dried products, is prevented by freeze drying because there is no liquid phase (Karel, 1975). The apparent lipid loss in hot air-dried Artemia when extracted by the Bligh & Dyer method (1959) was probabl.v due to the denatured protein. For the vacuum-dried Artemia, the lower temperature and pressure gave a better reconstitution property than hot air-dried. The mild heating temperature and sublimation operation for the freeze-dried Artemia gave the best reconstitution property and highest total lipid among the three drying methods. Gabaudan et al. (1980), Tucker et al. ~1980), and Grabner et al. (1981) reported that metabolizable energy value, essential amino acids, and some enzymes activity of Artemia nauplii were not diminished by freeze drying. Cholesterol determination

Table 4 shows that the free cholesterol level was not statistically different in the newly hatched, freeze-dried, and vacuum-dried Artemia. The cholesterol level in the hot air-dried sample was significantly lower. No change was note~l in the cholesterol esters for all treatments. Thus the degradation of total cholesterol in the hot air-dried Artemia was from free cholesterol oxidation rather than from cholesterol esters. The average value (63"8 + 0.8 mg/g oil) of total cholesterol (Table 4) is in agreement with the 60-1 mg/g oil value of sterols of Sasaki & Capuzzo (1984), whereas it is considerably higher than 23"8 mg/g oil value of cholesterol of Gallagher & Brown (1975). TABLE 4 The Amounts of the Total Cholesterol, Free Cholesterol, and Cholestcr~)l Ester in Newly Hatched. Freeze-dried. Vacuum-dried. and Hot Air-dried Artemia

Total cholesterol" Frcc cholesterol Cholesterol ester

Newly hatched

Freeze-dried

Vacuum-dried

~lot-air dried

63'8 + 0"8a t' 32.7+ l . l a 31.l + l-3a

64-4+ 1.5a 32.5+ 1.3a 31-9_+ 0.2a

64.1 +0-1a 30.7+ 1.2a 33.4+ l'2a

60.5 +_0-6b 28-9+0.8b 31.6 +0.8a

"The unit of the total cholesterol, free cholesterol, and cholesterol ester is mg/g oil. ~'Value within each ro~ which bear the same letter are not significantly different at p i> O-05.

Lipid smbi!io' in the drs'ing ofartemia by severa! methods

301

Horvath (1966) found that about 50°,'0 of the cholesterol was destroyed after 6 days heating in air at 98°C. The 25-hvdroxvcholesterol, epimeric 7-hydroxycholesterols, and 5ct-cholestane-3fl.5.6fl-triol of automdation compounds of cholesterol were detected bv Smith et al. (1967) after 1 month heating in air at 65°C. For cholesterol ester autoxidation, some researchers have suggested that the polyunsaturated fatty acid esters of cholesterol are a likely source of organic peroxides action as initiators of cholesterol autoxidation (Boyd, 1962: Smith, 1981). This suggests that cholesterol esters can also undergo autoxidation as a free cholesterol in the C-7 position. However, the alcohol function group (-OH, C-3) of cholesterol ester was inactivated by a fatty acid a w l group. Data shown in Table 4, suggest that cholesterol ester in our system was less susceptible to degradation than the free cholesterol. Cholesterol (cholest-5-en-3fi-ol), the chief sterol of A r t e m i a oil (Teshima & Kanazawa, 1971; Payne & Kuwahara. 1972). has been shown to be required for prawn larval survival (Teshima et al.. 1983).

Peroxide, TBA, and iodine values The peroxides are a general term for the formation of hydroperoxides (ROOH) or primary products of lipid oxidation (Gray, 1978; Melton, 1983). In Table 5, the peroxide values of newly hatched, freeze-dried, and vacuum-dried A r t e m i a were not significantly different from each other. However, the hot air-dried sample had a peroxide value which was 2-3 times higher than the others. The peroxides are intermediate products in the formation of carbonyl and hydroxy compounds (Gray, 1978). They are labile and transitory.

TABLE 5 The Peroxide.TBA, and Iodine Values of Newly Hatched, Freeze-dried,Vacuum-dried. and Hot Air-dried Artemia"

Peroxide valuC' TBA' Iodine value

Newly hatched

Freeze-dried

I&~cuum-dried

blot air-dried

5-4 + 0.5a "~"~+ 0"5a 81-3_+ 1.0a

5.4_+ 0.4a l-6 + 0'6a 81.0_+ l.la

5"5+ 1.4a 1'9 + 0-4a 81.8+ 1-7a

13"3_+3'6b 2.4 + 0-04a 77.4_+ 1.3b

"Value within each row which bear the same letter are not significantly different at p >/0-o5. hThe unit of peroxide value is milliequivalentsperoxide per 1000 g oil. 'The unit of TBA is micromolemaloaldehydeper 100 g wet weightsample.

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Therefore. the determination of p e r o ~ d e value is highly empirical and any change in procedure is reflected in the results. Additionally, it was difficult to determine the accurate endpoint of titration because of the presence of the colored carotenoids canthaxanthin and echinenone (Soejima et al., 1980) in the extracted Artemia oil. Other workers have experienced the same difficulty with other kinds of oil (Hung et al., t980; Hardy et al., 1983 ). T h e newly hatched, vacuum-dried, and hot air-dried Arternia showed slightly higher T B A values than the freeze-dried sample. However, there was no significant difference among the four samples (cf. Dahle et al., 1962; Pryor etal., 1976). In Table 5, the iodine values of newly hatched, freeze-dried, and vacuum-dried Artemia were not significantly different. T h e hot air-dried had the lowest iodine value among the four samples.

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P. Sorgeloos. O. Roels & E. Jasper. Ecology, Culturing, use in Aquaculture. Universa Press. Wetteren, Belgium. pp. 277-90. Gabaudan, J., Pigott, G. M. & Halver, J. E. (i980). The effect of processing on protein in~edients for larval diets: Biolo~cal evaluation. Proc. World MaricuL Soc.. 11,424-32. Gallagher. M. & Brown, W. D. (1975). Composition of San Francisco Bay brine shrimp (Artemia salina ). J. A gr. Food Chem., 23,630-2. Grabner, M., Wieser, W. & Lackner, R. (198t]. The suitability of frozen and freeze-dried zooplankton as food for fish larvae: A biochemical test program. Aquaculture, 26, 85-94. Gray, J. I. (t978). Measurement of lipid oxidation: A review. J. Am. Oil Chem. Sot., 55,539-55. Gutteridge, J. M. C. (1977). The protective action of superoxide desmutase on metal-ion catalysed peroxidation of phospholipids. Biochem. Biophys. Res. Comm., 77,379-86. Harem, R. & Deatherage, F. E. (1960). Changes in hydration solubility, and charges of muscle proteins during heating of meat. Food Research, 25, 587-6 L0. Hardy, R. W., Mugrditchian, D. S. & lwaoka, W. T. (1983). Storage stability of lipids in a dry salmonid diet. Aquacult,~re, 34. 239-46. Horvath, C. (1966). Quantitative determination of cholesterol in autoxidation mixtures bv thin-laver chromatography. J. Chromatog., 22, 52-9. Hung, S. S. O., Cho, C. Y. & Slinger, S. J. (1980). Measurement of oxidation in fish oil and its effect on vitamin E nutrition of rainbow trout (SahTlo gairdneri). Can. J. Fish. Aquat. Sci., 37, 1248-53, Karel, M. (1975). Freeze dehydration of foods. In Principle oftZbod Science, Part ll. Physicul Principles ofkbod Preservation. ed. O. R. Fennema. Marcel Dekker, Inc., New '~%rk and Basel, pp. 359-95. Kates, M. (1972). Techniques of Lipidology: Isolation, Analysis and Identification of Lipids. North-Holland, New York, pp. 347-53; 358-61. Kayama, M., Tsuchiya, Y. & Meadm, J. F. (1963). A model experiment of aquatic food chain with special significance in fatty acid conversion. B,dl. Jap. Soc. Sci. Fish., 29,452-8. Klose. A. A. & Olcott, H. S. (1964). In Food Dehydration, Vol. I1. The AVI Publishing Company, Inc., Westport, Connecticut, pp. 563-90. Labuza, T. P. (1980). The effect of water activity on reaction kinetics of food deterioration. Food Technol., 34, 36-41, 59. Leger, Ph., Bengston, D. A., Simpson, K. L. & Sorgeloos, P. (1986). The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biol. Ann. Rev., 24, 521-623. Lemon, D. W. i 1975). An improved TBA test for rancidity. New Series Circular, No. 51. Environment Canada Fisheries and Marine Service, Halifax, Nova Scotia. Levine, D. M. & Sutkin, S. D, (1984). Nutritional significance of long-chain polyunsaturated fatty acids to the zoeal development of the Brachyuran crab, Eurs.'panopeus depressus [Smith]. J. Exp. Mar. Biol. Ecol., 81, 2l 1-23. Lovern, J. A. i 1965), Some analytical problems in the analysis of fish and fish products. J. Assoc. O]'fic. Agr. Chemists, 48, 60-8. Melton, S. L. (1983). Methodology for following lipid oxidation in muscle foods. Food Tech.. 37, I 05-11.

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