oil extraction and analysis phần 4 doc

The amount of oil is determinedeither by directly weighing the extracted oil Direct Method, AOAC Method920.39a or by measuring the loss of weight from the sample Indirect Method,AOAC Met

Chapter 4

Evaluation of the Rapid, High-Temperature Extraction

Analyzer to Determine Crude Fat Content

R.J Komarek, A.R Komarek, and B Layton

ANKOM Technology Corporation, Macedon, NY 14502

Abstract

The process of extraction for the quantitative separation of fat/oil is the basis forthe majority of official methods The extraction process, which separates the sam-ple into two fractions, permits two approaches to quantitative measurement Theanalysis can be performed by either weighing the fat/oil fraction directly, or indi-rectly by measuring the loss of weight due to extraction Acceleration of theextraction process has been achieved by elevating the temperature of the solvent.This chapter discusses a recently developed primary method called the Filter BagTechnique (FBT) This technique utilizes temperatures of up to twice the boilingpoint of petroleum ether to accelerate extraction High sample throughputs areaccomplished by batch processing of samples encapsulated in filter media formed

in the shape of a bag The extraction is performed automatically in an ANKOMXT20Fat Analyzer, an instrument that can process 20 samples in 20–60 min The fat/oilpercentage is calculated indirectly from the loss of weight from the sample in thefilter bag Various studies related to the extraction and gravimetric measurements

of these fractions are discussed in this chapter for both the conventional methodand the FBT The accuracy of the FBT depends on effective predrying and properweighing of the sample Studies of the conventional method suggest that samplescontaining polyunsaturated fatty acids are sensitive to oxidation particularly duringthe solvent evaporation step when the oil is heated in the presence of oxygen.Various studies of the ruggedness of the FBT indicate that the method is not sensi-tive to small changes in analytical conditions The ruggedness of the method wasconfirmed in an experiment utilizing Youden’s Ruggedness Test When the accu-racy of the FBT was compared to that of the conventional method with a widevariety of samples (n = 22) in a regression analysis, the two methods were highly

correlated (R2= 0.9996) There was essentially no bias (–0.046 intercept) and nodistortion over the range of the samples (slope 1.001) Two collaborative studieswith laboratories from five countries provided similar evidence of the accuracy ofthe FBT The second collaborative study, designed to evaluate the FBT as anAOCS official method, was conducted with 28 samples presented as 56 blind

duplicates Twelve international collaborating laboratories used the FBT for theanalysis, whereas three AOCS certified laboratories utilized the official methods.

This study resulted in a similar highly significant R2 of 0.9990 compared with theofficial methods, with an intercept of 0.046 and a slope of 1.005 The averagerepeatability within laboratories was Sr= 0.31 and reproducibility among laborato-ries was SR = 0.46 These studies indicate that the FBT is an accurate and precisemethod capable of analyzing large quantities of samples in an efficient and auto-mated fashion

Introduction

Knowledge of the fat content of food and feed, or the oil content in oilseeds is ofcritical importance when evaluating the value of these materials The oil content ofoilseeds determines their commercial value, whereas the fat content is important ingaining an understanding of the nutritional value and energy metabolism of a diet.Both fat and oil represent the fraction of lipids generally associated with triacyl-glycerides and compounds of similar solubility in nonpolar solvents In this chap-ter, the terms “fat” and “oil” will be used interchangeably

The quantitative analysis of “Oil” as it is termed by American Oil Chemists’Society (AOCS) (1) or “Crude Fat,” as designated by Association of OfficialAnalytical Chemists (AOAC) (2), is based on separating the fat/oil from the sam-ple matrix by extraction with nonpolar solvents The amount of oil is determinedeither by directly weighing the extracted oil (Direct Method, AOAC Method920.39a) or by measuring the loss of weight from the sample (Indirect Method,AOAC Method 920.39b, 948.22a) This process is described in the flow diagram inFigure 4.1 Each step in the process affects the accuracy and precision of the analy-sis There are several critical drying, weighing, extraction, and evaporation steps.The process terminates with two fractions, i.e., the residue extracted by the solvent,for which the percentage can be calculated directly, and that portion of the samplenot soluble in the solvent for which the percentage can be calculated indirectly.Because both values can be determined on the same sample, their agreement veri-fies the accuracy of the analysis

Nonpolar solvents such as diethyl ether, petroleum ether, and hexane dissolve fatsand oils and leave behind proteins, carbohydrates, and other compounds insoluble inthese solvents This fractionation is the basis for most of the “Official” analyticalmethods established by AOCS, AOAC, International Organization for Standardization(ISO) (3), German Fat Science Society (DGF) (4), and Federation of Oils, Seeds andFats Associations (FOSFA) (5) These methods utilize either the Soxhlet extractionapparatus, developed by Franz Von Soxhlet (6) in 1939, the Butt-type apparatus (2),

or the Goldfisch apparatus (7) All of these methods boil the solvent and utilize thecondensed solvent to extract the sample The Soxhlet apparatus allows the samplechamber to fill and periodically siphon off into the boiling flask; the others simplyallow the condensed solvent to pass through the sample as the solvent is refluxed The

sample is therefore extracted with solvent at a temperature below the boiling point ofthe liquid, requiring extraction times from 4 to 16 h.

The rate of extraction has been increased by immersing the sample in the ing solvent (8), thereby extracting the fat/oil at a higher temperature and reducingthe extraction time Further improvements in the kinetics of extraction have beenachieved by performing the extraction in a sealed chamber at elevated pressuresthat permit extraction to be performed at temperatures well above the boiling point

boil-of the solvent [ANKOM (9) Dionex (10) and supercritical fluid extraction (11)].This results in a further reduction in the extraction time

A recently developed method that utilizes high solvent temperatures in an mated batch process is being evaluated as an Official AOCS Method This techniqueresponds to the need for a rapid, efficient, high-volume process for the analysis of fats/oils that is equivalent to a primary method using petroleum ether The method is enti-tled, “Rapid Determination of Oil/Fat Utilizing High Temperature Solvent Extraction.”

auto-Fig 4.1 A diagrammatic representation of the analy- sis of fat/oil by solvent extraction.

This method is performed by the ANKOM Fat Analyzer (XT20) and can also

accomplished by encapsulating each sample in a special filter medium, preservingits quantitative identity while performing the high temperature extraction of multi-ple samples in a common extraction chamber The filter media is made in theshape of a bag and is heat sealed after the introduction of the sample This method

of analysis will be referred to as the Filter Bag Technique (FBT) and has the bility of high sample throughput (>200 sample/d) This chapter will discuss thebackground of the extraction process and the evaluation of the precision (repro-ducibility among different laboratories in a collaborative study), accuracy (compar-ison with standard methods), and ruggedness of the FBT in laboratory and interlab-oratory collaborative studies

capa-Materials and Methods

Conventional Method The Goldfisch Method, conducted on a Labconco

Goldfisch Fat Extraction Apparatus, was used in a number of studies as the ventional standard for comparison with the FBT (7) The apparatus functionsessentially the same as the Butt-type apparatus, continually refluxing solvent overthe sample during the extraction The method can follow both paths, i.e., directanalysis and indirect analysis of fat/oil (Fig 4.1) Extractions were performed over

con-a 4- to 5-h period con-and the solvent wcon-as pcon-articon-ally evcon-aporcon-ated con-and recovered in con-a glcon-assbeaker In earlier studies, the residual solvent (~10 mL) was evaporated above thehot plate on a holder in the apparatus In subsequent studies, with sensitive sam-ples, the residual solvent was evaporated on a steam bath under nitrogen Theanalysis was conducted by weighing the sample in a tared thimble, drying the sam-ple at 100°C for 3 h, and weighing it at ambient temperature from a desiccantpouch The thimbles in these studies were made from the hydrophobic filter medi-

um used for the filter bags Typical cellulose thimbles are very hydroscopic and aredifficult to weigh The thimbles containing the samples were inserted into theapparatus and a tared glass beaker with 50 mL of petroleum ether was attached toeach reflux unit The cycle was started by turning on the hot plate When theextraction was completed and the solvent evaporated, both the residual sample inthe thimble and the fat/oil in the beaker were dried at 100ºC for 30 min, cooled toroom temperature in a desiccator, and weighed Both direct and indirect analyseswere performed on the same sample as a check for accuracy

Filter Bag Technique The FBT follows the path in Figure 4.1 of the indirect

analysis and was performed in the XT20 (9) The sample was weighed in the filterbag, heat sealed, dried at 100ºC for ~3 h, cooled in a desiccant pouch, andweighed Samples (n = 20) were placed in a carousel in the extraction chamber.The temperature (90ºC) and time of extraction, usually from 10 to 60 min, wereselected and the instrument was started The XT20 automatically processed the

samples in the following fashion: sealed and purged the chamber, inserted andheated the solvent, rotated the bag carousel, and emptied when the extraction timewas complete Solvent was then added for the first rinse, emptied after 3 min andrefilled with fresh solvent for a second rinse After the solvent was emptied, theresidual solvent was evaporated and the chamber was purged with nitrogen Whenattached to an ANKOMXTRecovery System, the instrument automatically distills

Extractor The samples were then dried at 100ºC for 30 min, cooled to room perature in a desiccant pouch, and weighed

tem-The desiccant pouch was developed to more conveniently handle the filter bagsduring the weighing process The pouches were made from resealable polyethylenebags containing desiccant and were used in all of the FBT studies Filter bags wereremoved from the oven and placed directly in the desiccant pouch The air waspressed out and the pouch was sealed The samples rapidly equilibrated to room tem-perature and were effectively protected from ambient moisture by the limited headspace in the pouch The introduction of moist air during the removal of each bag wasreduced by minimizing the size of the opening and pressing the pouch flat

Solvents Although other solvents can be used, petroleum ether is the preferred

solvent for the FBT because of its safety, cost, and ease of recycling Petroleumether was used in all the studies reported in this chapter The boiling point range ofcommercial petroleum ether is specified by the supplier as 35–65ºC (12) The dis-tribution of the solvent components over the temperature range was investigated in

a fractional distillation study of both new and recycled petroleum ether (distilled toremove fat) Fractions were collected within 5°C increments from 36 to 80ºC

Sample Preparation The objective of sample preparation is to provide a sample

that accurately represents the “population” being studied and sufficiently disruptsthe matrix to permit more efficient extraction Meat samples were ground to a uni-form consistency with a food processor and mixed thoroughly For shipping conve-nience and sample uniformity, the meats in the international collaborative studieswere dried for 3 h at 100°C and then ground in a cyclone mill to pass through a 2-

mm screen The feed samples were ground in a cyclone mill to pass through a 1-mmscreen and mixed thoroughly The food samples were processed with a food proces-sor or cyclone mill to produce a representative sample of uniform consistency.Soybean samples were first dried at 130°C for 30 min and then ground in a cyclonemill to pass through a 1-mm screen Other oilseeds were ground in a cyclone mill topass through a 1- or 2-mm screen, depending on the level of screen occlusion.The effects of grinding were demonstrated in a study with soybeans by pro-cessing them three ways In the first treatment, soybeans were ground through a 2-

mm screen and extracted In the second treatment they were processed according tothe AOCS procedure (13) by first heating the soybeans in a 130°C oven for 30 minand then grinding through a 1-mm screen followed by an extraction The third

treatment involved regrinding the soybean samples from the second treatmentthrough a 1-mm screen and then extracting a second time.

Conventional Method Weighing Procedures The weighing procedure is critical

to the gravimetric analysis of fats/oils Accuracy of the analytical balance was fied and checked each day that weighing was performed Accurate weighing of driedsamples requires rapid processing directly from a desiccating environment, limitingexposure to moist ambient air The glass beakers used in the conventional methodwere hydroscopic and can, under certain circumstances, carry a significant staticcharge The effect of static charge was investigated in an experiment with samples of

veri-a pig diet Sveri-amples were extrveri-acted for 4 h with petroleum ether, veri-and the residuveri-al oil inbeakers from six replicates was dried at 100°C for 30 min After equilibration toroom temperature in a desiccator, the beakers were weighed The oil was then trans-ferred with a small amount of petroleum ether to tared aluminum pans because they

do not retain a static charge After evaporation of the solvent, the samples in the minum pans were dried in the oven, equilibrated in a desiccator, and weighed

alu-Oxidation A study designed to evaluate the relative accuracy of the direct and

indirect measurements was conducted on duplicate samples of ground beef, hotdogs, potato chips, high-energy horse diet, pig diet, corn, oats, and soybeans Bothdirect and indirect determinations were performed on the same sample using theconventional method The oil was evaporated using the holder on the Labconcoapparatus, which holds the beaker above the hot plate

Due to the lack of agreement of the direct and indirect measurements with tain samples, studies were conducted to evaluate the role of oxidation in the elevatedvalues of samples containing unsaturated lipids An experiment was conducted with acorn sample that in previous studies had shown elevated direct values relative to theindirect values A series of treatments were designed to first limit oxidation and thenincrementally increase the opportunity for oxidation It was observed that the bulk ofthe oil/fat was extracted at the beginning of the extraction period and that the oil/fatdissolved in the solvent was subjected to the boiling temperatures for hours during therefluxing of the solvent Because the system was not anaerobic, there was a possibili-

cer-ty that these conditions could present an opportunicer-ty for oxidation In this experiment,extracted oil was removed from the apparatus during the extraction process in the firsttwo treatments at 1.5 and 3.0 h, and continued with fresh solvent to complete the 5-hextraction The remaining treatments were refluxed for 5 h without the removal of thefirst fraction The last 10 mL of solvent was evaporated in several ways For treat-ments 1, 2, and 3, solvent was evaporated on a steam bath with a nitrogen streamdirected on the surface In treatment 4, the solvent was evaporated on a steam bathwithout nitrogen In treatment 5, solvent was evaporated above the hot plate in theLabconco holder In treatment 6, the solvent was evaporated directly on the hot plateuntil all the solvent was observed to have been removed After extraction, the samplesfor treatments 1 and 2 were dried in a desiccator and purged with nitrogen for 4 h

The remaining treatments were dried in the oven at 100°C for 30 min When sampleswere removed from the oven they were equilibrated to room temperature in a desicca-tor purged with nitrogen The vacuum in the desiccator was returned to atmosphericpressure with nitrogen.

Oil recovered from treatments 1, 5, and 6 was analyzed by thin-layer raphy (TLC) Samples were chromatographed on silica gel plates with methylene chlo-ride and visualized with bromo thymol blue (14) This procedure separates the sterols,triacylglycerides, and the less polar fractions

chromatog-FBT Predrying Before extraction, all samples were dried at 100°C for 3 h for

both the conventional and the FBT methods It is particularly important to removethe residual moisture from samples analyzed by the FBT because the moisture isremoved during the extraction process, causing erroneously inflated values Astudy was made of the effects of predrying on ground beef, a high-energy horsediet, corn, soybeans, and a pig diet for different periods of time and at differenttemperatures Samples were weighed in a filter bag and dried at 100, 105, and110°C The samples were analyzed at intervals of 30 min up to 180 min and eachtreatment was replicated three times

FBT Sample Size The effect of sample size (1.00, 0.50, and 0.25 g) on the precision

of the analysis of six corn and three soybean samples was investigated in a study withthe FBT The samples, analyzed in triplicate, were finely ground and had a uniformconsistency Because of the sensitivity of the analytical balance (capable of weighing

to 0.1 mg) and the relatively small tare weight of the filter bags (0.5 g), it was

expect-ed that weighing errors would be minimizexpect-ed and the variance associatexpect-ed with thisstudy would be related to sample handling and sample homogeneity

FBT Extraction Temperature Because elevated solvent temperatures enhance the

extraction kinetics, the effects of extractions at three temperatures, 85, 90, and 95°Cwere studied Samples were extracted in 15-min intervals over a 60-min period TheFBT analyses were conducted in triplicate on ground beef, soybeans, potato chips, and

a high-energy horse diet

FBT Postextraction Drying After extraction and solvent evaporation in the

XT20, samples can absorb weight from exposure to ambient moisture and can tain traces of solvent that must be removed Postdrying periods of 10 and 20 minwere studied Samples were weighed directly upon removal from the XT20 andthen placed in an oven at 100°C for two consecutive 10-min periods and weighedafter cooling in a desiccant pouch Samples (n = 10) were analyzed in duplicate(oat meal, brownie mix, crackers, dog food, pig diet, ham, turkey, corn, soybeans,and canola) A second study was conducted to determine the effect of drying at100°C for intervals of 20, 40, 60, and 80 min The FBT analyses were conducted intriplicate on soybeans, canola, potato chips, and horse feed

con-FBT Youden’s Ruggedness Test Youden’s Ruggedness Test (15) was performed

to evaluate seven variables in the method and the effect of modest changes in thesevariables The variables were sample size (0.8–0.9 g vs 1.2–1.3 g), predry time(2.5 vs 3.0 h), predry temperature (98 vs 102°C), extraction time (25 vs 35 min),extraction temperature (89 vs 94°C), postdry time (25 vs 35 min), and postdrytemperature (98 vs 102°C) Nine sample types were analyzed in triplicate, includ-ing ground beef, chicken thighs, hot dogs, corn, soybeans, potato chips, cattle feed,poultry feed, and dog food

Comparison of the FBT with the Conventional Method The relative accuracy

of the FBT was evaluated by comparing the results of this method with those of theconventional method Samples (n = 22) were analyzed by both methods; each wasreplicated five times to compare the relative precision The samples included arange of samples encompassing meats, grains and oilseeds, feeds and foods Thedata were analyzed by Regression Analysis

Multilaboratory FBT Study A study was designed to evaluate the precision and

accuracy of the FBT by analyzing five samples in quadruplicate using the sameprotocol in 13 laboratories and completing the analysis within a 3-wk period Thisstudy provided an opportunity for the laboratories to familiarize themselves withthe FBT protocol to be used in the more extensive collaborative study The labora-tories were located in the United States, Canada, and Europe The samples usedwere ground beef, cheese curls, soybeans, corn, and a horse diet The conventionalanalysis was performed by ANKOM Technology

FBT Collaborative Study A collaborative study, performed in conjunction with

AOCS, was designed to evaluate the precision and accuracy of the FBT with a widevariety of samples that represented foods, feeds, meats, and oilseeds Samples (n = 28)were sent to 12 laboratories in the United States, Canada, and Europe in the form of 56blind duplicates Each laboratory was given a detailed protocol and had an opportunity

to become familiar with the method in a preliminary study These samples were alsoanalyzed by three AOCS Certified Laboratories using the relevant official methods

Results and Discussion

Reusing Solvent The results of the solvent fractionation study of petroleum ether

(Fig 4.2) indicated that the majority of the solvent (~70%) distilled in the range of36–40°C with no other fraction >8% The distributions of all of the fractions were sim-ilar for both recycled and purchased solvent This study indicates that petroleum ethercan be recycled without significantly changing the distribution of the solvent compo-nents

Sample Matrix Disruption Fats and oils that are not hindered by the sample

matrix or by various types of binding rapidly dissolve in fat solvents Oils trapped

in plant cell matrices are particularly difficult to extract due to the cell wall Thismicrostructure can act as a semipermeable membrane where larger molecules havelimited access to exit the structure even though the smaller solvent molecule canpenetrate the structure Plant matrices are difficult to disrupt on a cellular basis,and this has led to the development of extensive grinding procedures The grindingand regrinding procedures required in the AOCS and FOSFA methods for certainoilseed samples attest to the difficulty of preparing these samples for analysis Thegrinding study with soybeans illustrates the problem of sample preparation forcomplete extraction of the oil (Fig 4.3) The drying of the whole soybean at 130°Cfor 60 min before grinding improved the yield by ~3%, whereas regrinding afterextraction improved the recovery by another 2% In both treatments, it would beexpected that more extensive fracturing of the cell wall had occurred, enablinggreater extraction of oil Unfortunately, the oven treatment and extensive grinding

Boiling point fractions (°C)

Fig 4.2 The boiling point distribution of new reagent grade and recycled pretroleum ether The recycled petroleum ether was recovered by distilling waste solvent from fat extractions.

increase the chances of oxidation of the unsaturated fatty acids in the soy lipids,potentially increasing the weight of the oil extracted However, there may be suffi-cient protection within the matrix afforded by tocopherols and other antioxidants toretard this oxidation

Weighing Errors It is necessary in all gravimetric procedures to pay particular

attention to factors that affect the weighing process When samples are oven dried,water molecules are driven off binding sites on the sample and on the sample con-tainer These active sites are rapidly refilled by ambient moisture if given theopportunity Desiccators provide protection but care must be taken not to compro-mise this protection and to limit the exposure time during weighing When glassvessels are dried, they can hold a static charge that can interfere with the weighingprocess This phenomenon is illustrated in Figure 4.4 with the conventional analy-sis of a pig diet The erratic weights of five glass beakers containing the residualoil from replicate extractions were greatly improved by eliminating the staticcharge This was accomplished by transferring the oil sample to aluminum weigh-ing pans and reweighing The SD of the oil value was reduced from 0.33 to 0.05.This effect can also be controlled by using an ionizing source to dissipate the staticcharge on the glass beakers

Fig 4.3 The effect of three grinding treatments on the quantity of oil extracted from soybeans Soybeans were ground through a cyclone mill before extraction (Treatment 1), ground after drying at 130°C for 1 h before extraction (Treatment 2), and ground after drying at 130°C, extracted, ground again, and reextracted (Treatment 3).

Treatment 1 Treatment 2 Treatment 3

Oxidation During a series of experiments with the conventional method, it was

found that for certain samples, such as hot dogs, ground beef, and potato chips, thedirect measurements of fat (the weights of the fat recovered) were in good agree-ment with the indirect measurements (weight lost due to extraction) (Fig 4.5) Bycontrast, Figure 4.5 shows that the direct measurements of fat/oil were consider-ably higher than the indirect measurements in oats, corn, soybeans, and a pig diet.The distinguishing characteristics of this group include their plant origin and higherconcentrations of polyunsaturated fatty acids compared with the meat and potato chipsgroup Similar studies with corn and oats also showed higher values for the directcompared with the indirect analysis when solvent was evaporated on the Labconcoholder Oxidation increases the weight of the oil (16), thereby increasing the directmeasurement of the oil The extracted sample is not subject to the same effect, and nodistortion of the indirect measurement would be expected due to oxidation

In the experiment designed to investigate variables in the method that wouldenhance or avoid oxidation (Fig 4.6), the indirect measurements of the oil contentwere in excellent agreement across all six treatments This was not true for thedirect measurement of the oil Incremental changes in the time the oil was boiled in

Fig 4.4 The effect of static charge on glass beakers was examined in five samples of

a pig diet by first weighing the fat in the beaker and then transferring the fat to minum pans and weighing the fat again.

alu-the solvent during alu-the reflux resulted in slight but inconclusive increases in alu-thedirect value (Treatments 1–3) In Treatment 5, the solvent was evaporated usingthe Labconco holder, which positions the beaker above the heater and allows thetemperature of the oil to rise above 100°C The direct measurement of oil yielded avalue that was 4% higher that the indirect value In Treatment 6, in which the sol-vent was evaporated on the hot plate in the Labconco, the oil was subjected to tem-peratures of 200°C for ~1 min This resulted in a direct value that was lower thanthe indirect value In the TLC chromatogram of Treatments 1, 5, and 6 (Fig 4.7),

the triacylglyceride spot (R f 0.45) was the dominant spot for Treatment 1 wherethe direct value closely agreed with the indirect value In Treatment 5, with an

increase in oil weight, a large spot (R f 0.67) developed above the triacylglyceride

spot (R f 0.45) Only a trace of that spot (R f 0.67) was detected in Treatment 6 inwhich the oil received the highest heat treatment and had the lowest weight.Presumably, hydroperoxides were formed in both Treatments 5 and 6; some oftheir breakdown products (aldehydes and carbonyls) were observed on the chro-

Fig 4.5 Comparison of the direct and indirect analysis of samples containing rated oils (a pig diet, corn, oats, and soybeans) with samples containing predominantly saturated fat (ground beef, hot dogs, and potato chips) (n = 2).

unsatu-Direct Indirect

matogram for Treatment 5 but were essentially absent for Treatment 6 Althoughthe results of this experiment may represent a special case, they support the conclu-sion that when the samples were exposed to air at elevated temperatures, oxidativeformation of hydroperoxides occurred In Treatment 5, the hydroperoxides decom-posed but were not volatilized, whereas in Treatment 6, the breakdown productswere volatilized (16) by the higher temperatures These experiments indicate that

Fig 4.6 Effect of a progressive increase in oxidative conditions on the weight of oil recovered from a corn sample.

Solvent Evap.

Reflux Time

Treatment

care has to be taken to avoid oxidation when measuring the oil fraction directly,particularly with plant samples containing significant quantities of polyunsaturates

Sample Predrying During the refinement of the FBT, critical steps in the protocol

were investigated and optimized The requirements of predrying were investigated for

a variety of sample types A ground beef sample provides an example (Fig 4.8) of therelationship of moisture removal and the fat percentage The percentage of dry matterdecreased for the first 120 min and then leveled off The percentage of fat followedthe same pattern starting off high and leveling off after 120 min The moisture thatwas not removed in the oven was removed during the extraction and resulted in ele-vated fat values The three oven temperatures (100, 105, and 110°C) produced similarresults with ground beef The same experiment with a high-energy horse diet (Fig.4.9) indicated that the lipids in this diet were sensitive to temperature and that time inthe oven increased the effect The horse diet, starting with <10% moisture, reached aplateau in fat percentage in 60 min and maintained that plateau up to 150 min ofpredrying at 100 and 105ºC When the horse diet was heated at 110°C, a plateau inthe fat values was reached at 30 min and then declined exponentially after 60 min

Fig 4.7 TLC chromatogram showing the separation of oil samples with different heat treatments Sample 1 was analyzed under the mildest conditions; Sample 5 was heated on the Labconco holder and Sample 6 was heated directly on the hot

plate Triacylglycerides migrated to an R f

of 0.45 and suspected oxidation

degrada-tion products migrated to an R fof 0.67 Samples were separated on silica gel G TLC plates with methylene chloride.

Solvent front

Origin

The sources of fats and oils in the horse diet were rice bran, flaxseed, and vegetableoil These are excellent sources of polyunsaturated fatty acids, which are sensitive tooxidation, particularly in the presence of minerals that could act as catalysts The loss

of weight could be explained by the formation of hydroperoxides, their degradation,and subsequent volatilization of the resulting end products In both the 100 and 105°Ccurves, there was an indication of a depression in the fat values after 150 min In mostsamples, such as corn, soybeans, and a pig diet, the fat percentage stabilized within2–3 h of predrying at 100°C (Fig 4.10) Provided that the temperature is accuratelycontrolled at ~100 ± 3°C, the period between 2 and 3 h is a relatively “rugged” step inthe procedure

Extraction Rate The majority of the fats and oils in samples properly prepared

are rapidly removed from the matrix by petroleum ether These are the fat/oil ecules that are completely exposed and not hindered by the sample matrix The

mol-Time (min)

Fig 4.8 Effect of the predrying temperature on dry matter (DM) and the FBT ment of the fat in ground beef over a period of 30–180 min (n = 3).