J Food Sci Technol (August 2019) 56(8):3957–3961
https://doi.org/10.1007/s13197-019-03878-4
SHORT COMMUNICATION
Effect of modified Soxhlet (Soxtec) and Folch extraction method
selection on the total lipid determination of aged beef
Benjamin W. B. Holman1,2 • Kristy L. Bailes3 • Richard G. Meyer3
David L. Hopkins1,2
Revised: 4 June 2019 / Accepted: 13 June 2019 / Published online: 24 June 2019
Ó Crown 2019
Abstract This study compared the modified Soxhlet
(Soxtec) and Folch extraction methods for determining the
total lipid or intramuscular fat (IMF) content of aged beef
samples. Ageing periods tested were 0, 5, 8, 10 and
12 weeks during which samples were held in vacuo and at
* 1.0 °C. Prior to solvent extraction, all samples were
freeze-dried and ground. The Folch method was found to
result in higher IMF values and therefore higher extraction
efficiency when compared to the Soxtec. A linear rela-
tionship was evident between the two methods that descri-
bed 80.9% of the variation between IMF values. An
interaction between ageing period and extraction method
was identified, although ageing period did not indepen-
dently impact on averaged IMF findings. Based on these
observations and reagent toxicity not being a limiting fac-
tor, it is recommended that the Folch extraction method be
used to determine beef IMF, although it is acknowledged
that the Soxtec method IMF results were strongly aligned
with those found using the Folch extraction method and
tended towards comparability for beef aged \ 5 weeks.
Keywords Intramuscular fat
Beef
Total lipid
Ageing
period
Randall method
& Benjamin W. B. Holman
benjamin.holman@dpi.nsw.gov.au
1
Centre for Red Meat and Sheep Development, NSW
Department of Primary Industries, Cowra, NSW 2794,
Australia
2 solvent extraction; (3) purification of lipid fraction; and
Graham Centre for Agricultural Innovation, NSW
Department of Primary Industries, Charles Sturt University,
Wagga Wagga, NSW 2650, Australia
3
Wagga Wagga Agricultural Institute, NSW Department of
Primary Industries, Wagga Wagga, NSW 2650, Australia
•
Introduction
Intramuscular fat (IMF) refers to the total lipid or crude
fat content of trimmed beef, and is important to our
understanding of beef’s organoleptic and nutritional
properties. Research of this association includes findings
that IMF content is indicative of beef juiciness (Aldai
et al. 2006), as IMF was negatively associated with drip
loss; Serra et al. (2004) used trained sensory panellists to
show that IMF, odour and flavour have a positive rela-
tionship in 14 days aged beef samples; and Nishimura
et al. (1999) reported IMF to impact on beef tenderness as
a result of myofibril disruptions caused from adipose
tissue formation within endomysium structures—albeit
this was observed in heavily marbled muscles. From this
relationship, a 3–4% IMF lower limit for consumer
acceptability for beef has been defined (Savell and Cross
1988), which interestingly proved similar to the 5% IMF
limit later defined for sheep meat (Hopkins et al. 2006). It
is also noteworthy that IMF provides a major source of
essential fatty acids and a significant component of the
nutritional value for beef products which are desired by
consumers (Valsta et al. 2005). Apparent from these
observations and associations, is a requirement for con-
fidence in the results of any total lipid determination
method to reliably represent the IMF status of a beef
sample.
There are numerous approaches used to measure total
lipid content. These often adhere to a generalised
methodological pathway of (1) sample preparation; (2)
(4) data integration or calculation. Of these, solvent
extraction is considered a critical phase for IMF analysis
of meat samples (Sahasrabudhe and Smallbone 1983). To
this end, the Folch method (Folch et al. 1957) was
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developed to use a chloroform–methanol–water phase
system to extract lipids, but has somewhat lost favour in
the scientific community because of its comparative
expense (labour and reagents) and its use of more haz-
ardous chemicals (Reuber 1979). The Soxhlet method
(FOSS 2003) instead was designed to use a less toxic
hydrocarbon solvent, hexane and a Soxtec extractor to
isolate sample lipids, increasing sample throughput times
and resource use efficiencies (Thiex et al. 2003). Differ-
ences in the results of these two methods have been
reported. Indeed, Pérez-Palacios et al. (2008) reported
Folch extraction efficiencies to be better than Soxhlet
when testing whole muscle beef samples; Brooks et al.
(1998) found minor discrepancies when comparing chlo-
rinated and hydrocarbon solvents to determine total car-
cass lipid content of rats; and Segura and Lopez-Bote
(2014) used pork samples to conclude that method
extraction efficiency is dependent on the relative fattiness
of a sample. The basis for these discrepancies was
reported to be the variety of form, content and complexity
of the lipid profile. Overcoming these limitations has
prompted modification to these original methods and
consequently created an imperative; to compare the results
of these modified extraction methods when using them to
quantify the total lipid content of beef samples.
In response, this study aimed to test the effects of a
modified Soxhlet extraction method compared to the Folch
method when determining the IMF content of aged, trim-
med, freeze-dried and ground beef samples.
Materials and methods
Sample preparation
A total of eight beef striploins (M. longissimus lumborum)
were selected at random from the boning room of a com-
mercial Australian abattoir. All striploins were from grass-
fed assurance programs and processed as per industry
norm. Each loin was divided into five equal portions that
were individually vacuum-packaged and assigned to one of
five ageing periods (0, 5, 8, 10 or 12 weeks). Samples were
held, chilled (mean ± standard deviation: 1.49 ± 0.80 °C)
with allocation randomised, and balanced so that n = 4 per
ageing period per chiller and n = 40 in total. All samples
were sectioned at the completion of their ageing period and
held at - 80 °C until analysis so as to inhibit further
oxidation.
Prior to lipid extraction, approximately 25.0 g samples
were freeze-dried at - 50 °C (ScanVac CoolSafeTM,
LaboGene Ltd., Lynge, DEN) and ground using a sample
mill (model 1095, KnifetechTM, FOSS Pacific Ltd., New
South Wales, AUS) to generate a homogenous sample.
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Soxhlet extraction method
This Soxhlet extraction protocol followed the method of
Hopkins et al. (2014). This used an extraction unit (ST 255
SoxtecTM, FOSS Analytical Solutions Pty. Ltd., Victoria,
AUS) and 2.5 g freeze-dried sample placed in a porous
thimble and extracted in 85 mL hexane for 60 min within
individual extraction tins (FOSS 2003). The residue was
allowed to evaporate for a further for 20 min, before being
placed in a forced draft oven for 30 min at 105 °C so as to
further remove (vaporise) any residual solvent. The varia-
tion in sample weight before and after extraction was used
to calculate crude fat content as per Thiex et al. (2003), and
then expressed as a percentage of fresh (wet) sample
weight (IMF).
Folch extraction method
The method of Folch et al. (1957) was modified so that
0.5 g freeze-dried sample was combined with 10.0 mL
chloroform:methanol (2:1 v/v) in a glass screw top test
tube. Tubes were then vortexed for 30 s before being agi-
tated for 15 min in an orbital shaker at room temperature
(* 25 °C). Following this tubes were centrifuged at
15009g for 15 min before the majority of the chloro-
form:methanol solution was carefully decanted, ensuring
that all solid material remained in the bottom of the tube.
Tubes were then evaporated to dryness in a fume cupboard.
Once dry, 10.0 mL of hexane added to each tube and these
vortexed and allowed to settle before eluent was again
poured off and tubes permitted to once again dry. This
process was then repeated before dry tubes and residue
were weighed, IMF calculated and it expressed as a per-
centage of fresh (wet) sample weight.
Statistical analysis
Data were analysed using a simple linear regression model
(Genstat 19th Edition, VSN International Ltd., www.vsni.
co.uk) with Soxhlet extraction method as the response
variate and Folch extraction method as the fitted term.
Using this same software package, data were also analysed
using a linear mixed model with extraction method, ageing
period and their interaction fitted as fixed effects; and with
striploin, portion within striploin and their interaction as
random effects. Differences between predicted means were
considered to be significant when P \ 0.05.
Results and discussion
The summary data shown in Table 1 demonstrates the
range of IMF values observed and included in this study.
J Food Sci Technol (August 2019) 56(8):3957–3961
Table 1 Summary Soxhlet and Folch extraction method data of beef
M. longissimus lumborum samples aged for different chilled storage
periods, up to 12 weeks
n Mean Range Median SD
Soxhlet method 48 2.54 1.28–3.89 2.49 0.61
Folch method 47 2.84 1.72–4.01 2.90 0.59
Fig. 1 The intramuscular fat content (IMF) of aged beef M.
longissimus lumborum a the effect of extraction method (Soxhlet
and Folch) with predicted mean ± standard error (bars) plotted; and
b the relationship between IMF values determined using these same
extraction methods (Soxhlet and Folch) with a linear trend line
included
This study found beef sample IMF results to be highest
when the Folch extraction method was used, comparative
to the Soxhlet method (2.81 ± 0.04% vs 2.49 ± 0.04%,
respectively; P \ 0.001; Fig. 1a). Doing so, this observa-
tion has provided support to the conclusions of past
research (Silva et al. 2015). For example, Tanamati et al.
(2005) also reported the Soxhlet method to have a lower
extraction efficiency than the Folch method. A suggested
basis for this difference is the hexane solvent being less
effective at isolating the polar lipid fraction from beef or
tissue samples. The polar lipid fraction refers to lipopro-
teins, phospholipids and similar conjugated lipids—op-
posed to the neutral triglycerides, free fatty acids, mono-
and diglycerides, and sterols that belong to the non-polar
3959
category (Sahasrabudhe and Smallbone 1983). Character-
istic of polar lipids are their strong linkages between fats
and proteins that can inhibit their extraction, unless a sol-
vent is used with sufficient polarity to overcome these
bonds, such as a miscible mixture of polar and non-polar
solvents such as those found in the Folch method. That
said, caution is recommended when selecting an extraction
solvent as excessive polarity could suppress solubilisation
of non-polar lipids, such as triacylglycerol, to the detriment
of IMF representation (Iverson et al. 2001). Nonetheless,
the implications of a failure to capture the polar lipid
fraction on understanding the fatty acid composition of
beef—specifically health claimable long chain fatty acids
that are prolific within phospholipid structures—are a
potential concern. This has already been demonstrated with
Xiao et al. (2012) finding lower levels of eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA) fatty acids to
be extracted when a Soxhlet extraction method was used
comparative to the Bligh and Dyer method, another
extraction method that similarly to the Folch method uses a
chloroform based solvent.
An alternate basis for the difference between Soxhlet
and Folch extraction methods could be differences in the
amount and therefore the fattiness of the sample combined
with the solvent. As Iverson et al. (2001) suggests, it is
critical to maintain a lipid concentration to organic phase
ratio—that is, as sample fat content increases, so should the
volume of extraction solvent—because failure to do so will
increase the risk of organic phase saturation, incomplete
lipid isolation, and an underestimation of IMF values. The
findings of Thiex et al. (2003) illustrate this potential
masking of actual total lipid content by showing that the
extraction efficiency of low fat animal feed rations was
reduced when an additional 100% fat supplement was
included. In terms of a beef sample, this position lends
itself to the previous discussion of method difference in
their extraction efficiency of polar lipids, as samples with
lower total lipid content would have a higher ratio of polar
to non-polar lipids (Xiao et al. 2012).
It should be acknowledged that even if there was a
difference in polar lipid isolation efficiency between
methods, we could expect relatively comparable IMF
results because of their mutual extraction of non-polar lipid
fractions. This is illustrated by the linear relationship found
between Folch and Soxhlet extraction methods (P \ 0.001)
that accounted for 80.9 ± 0.27% of the variance (Fig. 1b).
Investigations with pork resulted in comparable results,
with Ragland et al. (1996) reporting a correlation coeffi-
cient of 0.84 observed between a Soxhlet method, albeit
using a petroleum ether solvent rather than hexane as per
this study, and a Bligh and Dyer extraction method.
The found similarity between extraction methods could
result from the freeze-drying and mechanical grinding
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Fig. 2 The effect of extraction method (Soxhlet and Folch) on the
intramuscular fat content (IMF) values for beef M. longissimus
lumborum samples aged for up to 12 weeks. Predicted mean ± stan-
dard error (bars) are plotted
when experimental samples were prepared, prior to solvent
extraction. These actions may have made sample fat stores
more assessable to both extraction solvents and in the case
of hexane, improved is efficiency by physically breaking
the protein-fat bonds of conjugated polar lipids that would
have remained intact if whole beef samples were tested
(Pérez-Palacios et al. 2008). Past research using freeze-
dried meat samples supports this position, having con-
cluded that both chlorinated and hydrocarbon solvents
could be used to effectively extract sample lipid fractions
(Sahasrabudhe and Smallbone 1983). Furthermore, it could
be inferred that a similar pathway would result from pro-
teolytic actions known to continue through a chilled stor-
age, ageing period (Coombs et al. 2017). This could
explain the interaction between ageing period and extrac-
tion method on sample IMF values (P = 0.003; Fig. 2)—
whereby IMF values determined using the Folch extraction
method were shown to increase with increased ageing. That
said, when extraction method data was averaged, ageing
period was shown not to impact on beef IMF (P = 0.257).
This outcome reflects the collective results of much past
research (Mahecha et al. 2009; Ba et al. 2014; Holman
et al. 2017) and doing so, provides credence to the repre-
sentativeness of this study.
Conclusion
From these results, the Folch extraction method was found
to provide a better representation of aged beef total lipid
(IMF) content and is therefore recommended. This state-
ment is constrained to analytics where reagent toxicity,
amount and throughput speed are not limiting factors. In
these instances, it would be advantageous to instead use the
Soxhlet method—especially as it provided comparable
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J Food Sci Technol (August 2019) 56(8):3957–3961
IMF results for beef aged \ 5 weeks and its results had a
strong linear relationship to Folch extraction method
results.
Acknowledgements The authors are grateful to the Graham Centre
for Agricultural Innovation for the Member Support Grant—Category
2 funds. The support of the NSW Department of Primary Industries
(NSW DPI) and our abattoir collaborators is also acknowledged.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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