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Evaluation and comparison of the CEM rapid
extraction method with official standard methods
for the determination of lipid content in fillets of
ž
/
farmed Atlantic salmon Salmo salar
P. Katikou
1, D.H.F. Robb
)Department of Clinical Veterinary Science, DiÕision of Food Animal Science, UniÕersity of Bristol,
Langford House, Langford, Bristol BS40 5DY, UK
Received 24 May 2000; received in revised form 16 August 2000; accepted 16 August 2000
Abstract
The CEM rapid extraction method for lipid content determination was compared with official standard methods and was found to give accurate results for individual samples, taking approxi-mately 8 min per sample. The study involved the lipid analysis of 47 white muscle samples from six fillets of farmed Atlantic salmon, using both the CEM and the Soxhlet method, in order to evaluate the performance of the former. Nine samples from each fillet with variable lipid content, ranging from 2.0% to 25.4%, were analysed. The comparison of the CEM with the Soxhlet
Ž 2 method showed that the CEM lipid values were significantly linearly correlated r s0.993,
.
P-0.001 , but were consistently lower than those of the standard by a factor of 0.37%. This difference was found to be more distinct in samples with lipid content below 10%. It is concluded that the CEM method is an effective rapid analysis method for the determination of lipid content in salmon fillets under commercial conditions for the routine monitoring of muscle lipid. For
Ž
research purposes, it may be necessary to add a correction factor 0.59% for levels below 10%
)Corresponding author. Tel.:q44-1179289312; fax:q44-1179289324.
Ž .
E-mail address: david.robb@bristol.ac.uk D.H.F. Robb . 1
Present address: Department of Technology of Animal Origin Products, School of Veterinary Medicine, Aristotle University, 540 06 Thessaloniki, Greece.
0044-8486r01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.
Ž .
.
lipid and 0.27% above 10% if direct comparisons with Soxhlet results are required. q2001
Elsevier Science B.V. All rights reserved.
Keywords: Salmo salar; Lipid content; CEM; Rapid extraction method; Soxhlet
1. Introduction
There is a need, both in industry and in research to be able to accurately measure the
Ž .
lipid content of fish muscle and several methods are available Fjellanger et al., 2000 . Ž .
Some of the extraction methods, such as those of Folch et al. 1957 , Bligh and Dyer Ž1959 and the Soxhlet, which is also the standard official AOAC-recommended. Ž .
Ž .
method Association of Official Analytical Chemists, 1990 , provide reliable results, but have the disadvantages of requiring skilled operators for their completion and are very time-consuming. Numerous automated appliances have been developed in order to reduce the time required for the Soxhlet method but still, the time required is too long to be acceptable by the industry. Consequently, there is a trend within the industry for new rapid lipid determination methods, as such methods allow on-line process control in order to maintain product uniformity and quality as well as regulation compliance ŽBostian et al., 1985 . One such method is the Torry fatness meter Sinnott, 2000 ,. Ž .
Ž
although there are concerns for its accuracy for individual measurements Douirin et al., .
1998 .
The CEM automatic analyser is one of the recently available rapid extraction methods, capable of simultaneously determining both lipid and moisture. The system utilises microwave drying for the determination of moisture, combined with an enclosed
Ž
solvent extraction system, using dichloromethane, for lipid extraction Bostian et al., 1985; Crosland and Bratchell, 1988; Association of Official Analytical Chemists, 1990;
.
Mann et al., 1991 . The performance of the CEM analyser, in comparison with different
Ž .
methods, such as the Soxhlet Bostian et al., 1985; Crosland and Bratchell, 1988 and
Ž .
the Folch Mann et al., 1991 , has been already studied in meat and meat products, where it has been commonly used for the rapid measurement of the moisture and lipid
Ž .
content. The results of Bostian et al. 1985 revealed good correlation between the CEM
Ž .
and the official Soxhlet method for both moisture and lipid contents. Crosland and Ž .
Bratchell 1988 , however, concluded that the CEM consistently underestimated the lipid content, in both raw meat and sausages, by values of 0.37% and 0.98%, respectively, compared to the Soxhlet method. The comparison between the CEM and Folch method showed that there was good agreement between the two methods for samples with a lipid content greater than 10%, on a wet weight basis. However, a
Ž
constant bias existed between the two methods in low lipid samples 0.72% for lamb and .
0.78% for beef , with the CEM consistently giving lower results. This was explained by Ž the fact that the CEM method does not quantitatively extract phospholipids polar
.
lipids , which constitute a significant proportion of the total lipids in low lipid meats ŽMann et al., 1991 ..
the analysis of salmon white muscle lipid content by the CEM rapid extraction method with the official Soxhlet method in order to evaluate the new methodology.
2. Materials and methods
2.1. Fish
Ž .
A large population of Atlantic salmon Salmo salar , with an average weight of approximately 2.3 kg, was raised on a standard commercial diet for 6 months. From this
Ž .
population, 120 fish were randomly selected and placed in a trials cage 5=5=5 m on the Marine Harvest McConnell Feed Trials Unit in Loch Eil, Scotland. After 6 months, on reaching approximately 3.5 kg liveweight, the fish were slaughtered, eviscerated, and filleted. The fillets were vacuum-packed and frozen aty208C. Six fillets were chosen at
Ž .
random, transferred to a refrigerator 2–48C and left for 16 h to defrost prior to sampling.
2.2. Sampling
The fillets were sectioned nine samples per fillet. The sampling pattern was selected to provide samples with variable lipid content, as different sections within the salmon
Ž
fillet have been shown to have significantly different lipid content Bell et al., 1998; .
Katikou and Robb, unpublished data . The white muscle of each sample was dissected from the dark muscle, skin, bones, visible connective tissue, and the fat attached to the skin.
2.3. Lipid content analysis
2.3.1. Soxhlet method
All the Soxhlet analyses were performed in duplicate according to Association of Ž .
Official Analytical Chemists 1990 standards. A total of 47 samples were analysed in duplicate.
Each sample was blended to a smooth paste using either a Multi-Moulinette ŽMoulinex, UK or a CH100 Mini-Chopper Kenwood, UK mixer–blender and was. Ž . sub-sampled four times. Two of the sub-samples, weighing between 3 and 5 g, were
Ž accurately weighed to 0.001 g in pre-weighed coded plastic tubs, using a Mettler Delta
. Ž .
Range PC440 balance Gerrards Cross, UK and were then dried for 48 h in a Beta
Ž .
ChrisA vacuum freeze-dryer DamonrIEC, Dunstable, Bedfordshire, UK . Upon re-moval from the freeze-dryer, the tubs containing the dried samples were re-weighed to the nearest 0.001 g and the percent moisture was calculated using the measured weight difference and the initial weight of the sample.
Ž The dried samples were chopped and placed in cellulose extraction thimbles
What-.
man International, Maidstone, England . Any traces of the samples left in the tubs were
Ž ) .
removed using medical wipes Kimcare , Kimberly-Clark, UK and were also trans-ferred into the thimbles. The thimbles were then placed in pre-weighed dried and coded
Ž
. Ž Loughborough, UK . The flasks were filled with 160 ml of petroleum ether 40–608
.
Petroleum spirit, Rathburn Chemicals, Walkerburn, Scotland, UK and fitted in an
Ž .
automatic Soxhlet analyser Soxtherm 2000 Automatic, C. Gerhardt UK, Northants, UK for lipid extraction, for a total of 2 h. After the completion of the extraction program, the
Ž
flasks were removed from the apparatus and placed in a drying oven Gallenkamp 300 .
plus series for 1 h at 1038C, to evaporate remaining solvent. The flasks were allowed to cool in a dessicator and were then re-weighed to the nearest 0.001 g, using the Mettler AE166 balance. The difference between the initial and the final flask weight was the weight of lipid extracted. This information, along with the initial weight of the sample was used to calculate the percent lipid content. Finally, the mean values for percent moisture and lipid content were calculated using the values of the duplicate samples.
2.3.2. CEM method
The two remaining sub-samples of the paste resulting from the Soxhlet preparation each weighing 5"0.35 g, were analysed for lipid content using a CEM Meat Analysis
Ž .
System CEM, Indian Trail, NC, USA . The CEM analyser consisted of two
instru-Ž .
ments. The first was the automatic volatility computer AVC80 , which was a moisturersolids analyser, with a microwave drying system linked to an electronic balance, weighing to an accuracy of 0.2 mg, and a microprocessor digital computer. The second instrument was the lipid extraction unit, which comprised an automatic
extrac-Ž .
tion system AES and a solvent recovery system.
Ž .
One round and two rectangular glass fibre sample pads CEM, Matthews, NC, USA
Ž .
were placed on the electronic balance inside the microwave moisture analyser AVC80 and tared. The rectangular pads were removed and approximately 5 g of the sub-sample was thinly spread between the two pads which were then returned to the balance. The machine was run on 100% power for 3 min to determine the moisture: the percent moisture being calculated from the weight loss.
The rectangular pads, containing the dried sample, were removed from the AVC80, folded in half and placed in the chamber of the AES. The round pad was placed in the recessed area at the top of the extractor chamber and the lid was closed and latched. The
Ž .
sample was blended with dichloromethane Rathburn Chemicals to extract the lipid. The dry, defatted residue was collected automatically on the round pad and was placed again on the balance in the AVC80. The microwave was run for 2 min on 95% power, to evaporate any remaining solvent and the percentage lipid content calculated from weight loss. The time required for the analysis of each sample was approximately 8 min. All analyses were performed in duplicate. However, when the difference between the duplicates was more than 1.0% for moisture or 0.5% for lipid, a third sample from the original paste was analysed to reduce the possibility of errors. The values obtained were then used to calculate the mean percent moisture and lipid of wet weight for each cut.
2.4. Statistical analysis
The differences between the lipid content determined by the two methods were first
Ž .
compared by the use of linear regression analysis. This analysis was used in order to investigate the existence of either a proportional andror a constant bias, and resulted in an equation of the form ysaxqb, where asslope and bsconstant term. A non-unity slope would indicate a proportional bias and a non-zero constant would imply
Ž .
a constant bias Caulcutt and Boddy, 1983 .
3. Results
3.1. Comparison of the two methods
The lipid values of the samples were found to range from 2.0% to 25.4% by the
Ž . Ž
Soxhlet method mean 10.5"1.02% , compared with 1.5% to 24.9% by CEM mean
. Ž . Ž .
9.9"1.00% , with no significant difference between the means P)0.05 Fig. 1 . Three separate comparisons were performed to evaluate the performance of the CEM automatic analyser in determining the lipid content of salmon fillets. The first compari-son included all the analysed samples, while the second and the third comparicompari-sons Ž . included the samples with lipid contents below and above 10%, respectively Fig. 1 .
Table 1
Performance comparison of the CEM and Soxhlet methods in evaluating the lipid content of salmon fillets,
Ž . Ž .
showing the standard error S.E. of the slope and the constant con. term in the resulting equations 2
x y Equation S.E. slope S.E. con. term r P value n
All samples Soxhlet CEM ys0.98 xy0.37 0.01 0.15 0.993 -0.001 47
0–10% Soxhlet CEM ys1.03 xy0.59 0.04 0.21 0.967 -0.001 27
10%q Soxhlet CEM ys0.98 xy0.27 0.04 0.73 0.970 -0.001 20
The level of 10% was selected to provide comparable results with the study of Mann et Ž .
al. 1991 , which compared the CEM with the Folch method. The results are sum-marised in Table 1.
4. Discussion
The CEM offered a very rapid method for the analysis of lipid and moisture content. On average, a sample took 8 min to analyse for both of these parameters, following the sample preparation. In contrast, the Soxhlet method required the sample to be dried for 48 h and then approximately 4 h for the extraction. This meant that using the CEM, small numbers of samples could be rapidly analysed, a useful feature for commercial use when results are often required very rapidly. Under research conditions, it is possible in a normal working day to analyse at least 30 samples in duplicate, using just one set of equipment and one operator.
The mean values obtained for lipid content using the CEM method were lower,
Ž .
although not significantly P)0.05 , than those measured by the Soxhlet. When all the lipid content values, measured by both methods, were compared using regression analysis, it was found that the CEM method underestimated the lipid content by a factor of 0.37%, but this was not a significant difference. The samples were then separated in
Ž .
two categories, according to Mann et al. 1991 . Samples with lipid content below 10% were considered of low lipid content, while samples above 10% of high lipid content. The regression analysis of the low lipid samples found that the CEM values were consistently lower by a factor of 0.59%, while in high lipid samples, the CEM values were lower only by 0.27%. This implied that the CEM was not as efficient as the Soxhlet at removing the lipid from the sample, a finding which is consistent with
Ž .
Crosland and Bratchell 1988 . However, the accuracy is greater than that of the Torry Ž .
meter as found by Douirin et al. 1998 , who showed that a calibration correction of 1.2% was required and stated that measurements on individuals varied widely even though the overall mean may be related to the Soxhlet measurement.
From the results of this study, it is not absolutely necessary to add a correction factor to the CEM lipid values in order to compare them to Soxhlet results. Thus, it is suitable
Ž
for research such as nutrition trials and sensory research Robb and Nute, unpublished .
data where comparisons between groups and individual fish may be required. However, for the sake of absolute accuracy, we would suggest the use of the following formulae:
where y is the percent lipid by wet weight of the sample measured by Soxhlet and x is the percent lipid measured by the CEM.
Ž . Ž .
The previous studies of Bostian et al. 1985 , Crosland and Bratchell 1988 , and Ž .
Mann et al. 1991 agreed that the CEM method is an effective rapid screening procedure for the determination of lipid content in meat and meat products. This included the proviso that suitable correction factors were established for bias adjustment in certain product categories.
The results of the present study for lipid content of Atlantic salmon are in agreement with the lipid content results of previous studies on red meat and poultry. The ANOVA showed that the observed differences between the lipid contents of the two methods were not statistically significant and it is therefore concluded that the CEM rapid extraction method can be reliably used commercially for lipid determination in salmon fillets, whether or not correction factors are used, as such minor differences would not be of significance in industrial or commercial situations. For research, the corrections need not be applied as the differences are constant, unless direct comparisons with Soxhlet results from other works are required.
Acknowledgements
The authors would like to thank Mrs. Sue Hughes for her assistance with the statistical analyses of the results and Mr. Steve Kestin for his comments on the paper.
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