Effect of Moisture Content, Grinding, and Extraction ...

Chapter 7

Effect of Moisture Content, Grinding, and ExtractionTechnologies on Crude Fat Assay

Devanand L. Luthriaa,*, Kirk Noelb, and Dutt Vinjamooric

aUSDA/ARS/FCL, Beltsville, MD 20705; bMonsanto Company, Ankeny, IA 50021; cMonsanto,St. Louis, MO 63167

Abstract

Conventional breeding as well as transgenic approaches constantly strives to makeimprovements to quality traits such as increasing the percentage of oil and/or modify-ing the oil composition. One of the key challenges faced by the industry is obtainingaccurate, cost-effective, and rapid analysis of oilseeds/grains with enhanced qualitytraits such as total crude fat (oil) content or a modified oil composition. Reliable crudefat analysis is of paramount importance to oilseed businesses because monetaryassessment in the trade of such seeds is based on total oil values. Although several dif-ferent primary and secondary technologies are available to determine crude fat contentin oilseeds, there are significant variations in the results reported by different proce-dures. A comparative evaluation of different grinders (Mega-grinder, Knifetec,Cyclotec, Cemotec, Mikro mill, UDY grinder, Brinkmann-Retsch grinding mill) andcommonly performed crude fat extraction methodologies [accelerated solvent extrac-tor (ASE), supercritical fluid extraction (SFE), Ankom batch extraction, automatedSoxtec extraction and classical Butt-tube] on the determination of total crude fat con-tent in soybean seeds is presented. The results clearly suggest a need for harmoniza-tion of official primary reference methods across different organizations (e.g., AOCS,AOAC, AACC, ISO, DGF). This is vital for the development of rugged calibrationsfor nondestructive, high-throughput secondary procedures involving near infraredtransmittance, near infrared reflectance/imaging, and nuclear magnetic resonancespectroscopy. Strategies aimed at harmonization of methods will aid in the develop-ment of successful business opportunities and obtaining fair trade value for the quali-ty-enhanced traits in the global market. Recommendations for developing secondarycalibrations and performing interlaboratory studies are also presented.

Introduction

Accurate and precise analysis of crude fat (oil) in corn, canola, and soybeans isimportant for different research and commercial programs such as seeds, animal

*Research work was done at Monsanto in Ankeny, IA.

Copyright © 2004 AOCS Press

feed trade, nutritional labeling, and biotechnology research. Reliable assays of qualityand quantity of crude fat content in oilseeds/grains are essential for determination ofthe fair trade value of enhanced traits/products in the global market. To facilitate thisprocess, several international societies such as the American Oil Chemists’ Society(AOCS) (1), the Federation of Oil Seeds and Fat Association Ltd. (FOSFA) (2), theGerman Fat Science Society (Deutsche Gesellschaft fur Fettwissenschaft, DGF) (3),the International Organization of Standardization (ISO) (4), the American Associationof Cereal Chemists (AACC) (5), and the Association of Official Analytical Chemists(AOAC) (6) have developed standard reference methods for the assay of quality andquantity of crude fat in a variety of matrices. Nevertheless, problems exist because ofinherent variations in the method practiced.

The common approach for total crude fat determination is based on the solu-bility of lipids in nonpolar organic solvents such as hexanes, petroleum ether, orsupercritical liquid carbon dioxide with or without a solvent modifier. The volatilesolvents are removed by evaporation and the nonvolatile residue (crude fat) is mea-sured gravimetrically. The nonvolatile fraction consists of triacylglycerols andtrace amounts of other components such as free fatty acids and their alkyl esters,sterols, sterol esters, long-chain aldehydes and alcohols, fat-soluble vitamins, andother nonpolar natural products. Alternatively, two high-throughput nondestructivemethods that utilize nuclear magnetic resonance and near infrared spectroscopictechniques have also been developed. These methods are secondary in naturebecause they require calibration support by primary reference methods (7–9). Inresponse to the U.S. 1990 Food Labeling Act, another reference method was devel-oped to measure crude fat content (10). In this approach, fat was defined as thesum of all fatty acids present in a food material expressed as triacylglycerol equiv-alents of all analytically measured fatty acids.

Three factors that affect crude fat analysis are moisture content, sample prepa-ration, and extraction methodologies. Accurate moisture content determination inoilseeds is essential because it plays a crucial role in determining the monetaryvalue of the oilseeds and appropriate storage conditions. There are several primary(Karl Fischer, Brown-Duvel, gas chromatography, air-oven drying, and phospho-rous pentoxide), and secondary technologies (near infrared, nuclear magnetic reso-nance, and conductance- and capacitance-based moisture meters) used for analysesof moisture content in grains. A variety of methods exists that have been approvedby different official societies. These methods differ in variables such as the dryingtemperatures, drying time, or sample quantity used for moisture determination. Allof these factors have differing effects on moisture content determination, and thisindirectly affects assays of crude fat, protein, fiber, and other constituents. Detailsof factors affecting the precision of moisture measurement (grinding, sample size,moisture dishes, ovens, relative humidity of the laboratory, desiccant) and differentmoisture reference methods were summarized in two publications (11,12).

We chose to evaluate two factors that affect the crude fat assay, i.e., the influenceof sample preparation (the grinder study) and crude fat extraction methodologies.


There have also been a number of publications comparing different extraction proce-dures on crude fat assay (10,13–15); however, all of the earlier studies except ourpreliminary report were carried out with one sample from each grain variety andcomparison with the Ankom batch extraction process was not evaluated earlier.

Several soy samples with varying crude fat content were ground on differentgrinders under varying grinding conditions to examine the effect of sample prepa-ration on crude fat assay. The comparative performance of five currently used oilextraction technologies was also evaluated. This study was conducted to identifythe most accurate and precise method for bulk sample (sample size >500 mg) andsingle-seed (sample size <150 mg) analysis. Sample extraction was conductedusing two different sample sizes. To determine the effect of different drying condi-tions, the fatty acid composition of the oil extracted and dried at different temperaturesand conditions was also evaluated.

Experimental

Effect of Sample Preparation (Grinder Study)

The principle of operation of the seven grinders used for sample preparation issummarized below.

Mikro Mill (Hosokawa). This is a high-capacity hammer mill designed for grind-ing a comprehensive range of materials. Grinding is caused by impact between therotating hammers, particles, and deflector liner mounted in the mill housing cover.The particle size of the ground material depends on the type of hammer, the rotorspeed, and the size of the screen opening. In the present study, a 1-mm screen sizewas used (16).

Knifetec Grinder (Foss). This grinder utilizes a high-speed (20,000 rpm) rotorblade for grinding samples. The particle size of the ground material depends on thegrind time and the number of cycles. The Knifetec grinding chamber is equippedwith a cooling feature that enables it to be connected to cold tap water or other lab-oratory chilling devices. Samples containing high levels of fat have a tendency tostick to the wall of the chamber because the fat softens during grinding, thus pre-venting adequate homogenization. In addition, fibrous samples may generate heatdue to friction. Utilizing the cooling option, the Knifetec grinder overcomes bothof these problems to ensure satisfactory sample preparation (17).

Cemotec Grinder (Foss). This is a disc-type grinder that uses two discs, one sta-tionary and one rotating. This is a fast grinder, allowing sample to be ground in <1min. Although it is a fast grind, the particle size is very coarse even with the finestsetting on the instrument. Grinder settings range from 1 (finest grind) through 7(coarsest grind) (17).


Cyclotec Grinder (Foss). This is a cyclone-type grinder that utilizes a high-speedturbine that spins seeds against a coarse chamber ring. This action causes the seedsto form a fine powder, which is pushed through a screen (1 or 2 mm). Samplescontaining high levels of fat have a tendency to stick to the screen and eventuallyblock the passage of the sample through the screen. This grinder requires frequentcleaning, thereby increasing the sample preparation time (17).

Mega-Grinder (built in-house at Monsanto). This is a ball-type grinder that con-sists of a 2-horse power electric motor that drives a crankshaft via a belt. Thecrankshaft drives a piston that holds the sample trays. The piston is moved in anup-down motion. The sample is loaded into a sealed Delrin tube with a steel ball.This grinder utilizes extremely rapid shaking (up to 3000 strokes/min) to drive thesteel balls into intact seeds causing seeds to become pulverized in 0.5–2 min. Thisgrinder has the capacity to grind 96 single-seed samples at one time in 2 min withno cross-contamination.

UDY Grinder (UDY). This is a cyclone sample mill designed for rapid grindingof a wide variety of soft to medium-hard materials. The grinding process involvesthe high-speed rotation of the impeller and air currents that throw particles into thegrinding ring and rolls them around. Particles remain in the grinding chamber untilimpact shattering and abrasion make them small enough to flow out of the exit screen(2 mm) with the air current. The airflow removes essentially all material and makesclean-out unnecessary. The airflow also minimizes heating and therefore eliminatesthermal degradation (18).

Brinkmann-Retsch Mill (Brinkmann). This grinder consists of a two-speedmotor, removable lid, rotors, and sieves. It is a centrifugal grinder that operatessimilarly to the Cyclotec and UDY grinders (19).

Experimental Details for the Grinder Study

Sample Preparation. Seven soybean samples with varying oil content were used tocompare the effect of grinding and particle size on crude fat assay from ground soy-beans. Grinding with Cemotec, Cyclotec, and the Mega-grinder was done atMonsanto’s Crop Analytics Laboratory in Ankeny, IA. Samples were ground with theMega-grinder at two Monsanto sites (St. Louis and Ankeny) to evaluate instrument-to-instrument variation. Samples for the Cyclotec were ground with 1- and 2-mmscreens. Samples were submitted to the contract laboratories for grinding on UDY,Knifetec, and Mikro-mill grinders. Samples ground on a Knifetec mill were groundfor either 30 or 90 s; ~150 ± 1 g of each sample was submitted for grinding.

Extraction. Blind replicates of all ground samples were extracted in triplicate foroil analysis by Soxtec extraction procedure. Moisture analysis on the ground sam-ples was performed by an oven-drying procedure (AOCS Ac 2-41).


To evaluate the effect of particle size on crude fat analysis, a single soybean sam-ple (~450 gm) was ground on a Cemotec grinder and Mega-grinder to provide a widerange of particle size distribution. The combined ground sample was sieved usingmultiple stacked sieves (1.4, 1.0, 0.6, and 0.3 mm). Approximately 30 g of groundsample was placed on the top 1.4 mm mesh size sieve. The sample was brushedthrough each sieve to eliminate/reduce the effect of sample caking. After brushing,fractions were collected from the top of each mesh sieve and the bottom tray belowthe finest mesh sieve. Each fraction was weighed separately to determine the particlesize distribution. Each of the fractions was analyzed for crude fat assay.

Experimental Section for Comparison of Five Extraction Procedures

Three soybean samples with varying oil content (19–23%) were used to comparethe extraction of crude fat by five different methods. To eliminate the effect of par-ticle size on crude fat extraction yield, all seeds were ground with the Mega-grinder, and these ground samples were used for analysis. Six replicate analyseswere carried out for each sample by all five methods. The results are reported on adry matter basis to eliminate the effect of varying moisture content. Extractionswith supercritical fluid extraction (SFE) were performed at ISCO Labs, and part ofthe extractions with the Ankom batch procedure was performed at the Ankomfacility.

Extraction was done with two sample sizes, 100 mg and 1 or 2 g. Extractionwith 100-mg sample size was conducted to evaluate the applicability of the methodto single-seed analysis, whereas gram quantity sample size extraction was per-formed with different samples as recommended by the instrument vendors [Soxtec(1 g), Butt-tube (2 g), accelerated solvent extractor (ASE; 1 and 2 g), Ankom (500mg), and SFE (2 g)]. Extraction conditions and sample processing for two samplesizes (100 mg and 1–2 g samples) were the same for all methods. The five extrac-tion procedures are summarized briefly below.

Butt-Tube Method. Extraction with the Butt tube is a single-step process thatinvolves continuous flow of distilled condensed solvent over the ground samplematrix (4–5 h). The amount of oil extracted is determined gravimetrically afterevaporating the extraction solvent (AOCS Ac 3-44) (1).

Soxtec Extraction (Foss). This method is a two-step process for the extraction ofcrude fat from ground samples. In the first step, the thimble containing the test por-tion is immersed in the boiling solvent. The intermixing of the matrix with the hotsolvent ensures rapid solubilization of extractables. Then the thimble is raisedabove the solvent and the test portion is further extracted by a continuous flow ofcondensed solvent. The resulting crude fat residue is determined gravimetricallyafter evaporation of the solvent.

The apparatus includes the following: (i) solvent extraction system (Soxtec 2050)with multiple extraction units for conducting a 2-stage Randall extraction process with


solvent recovery cycle, using Viton or TeflonTM seals compatible with hexanes; (ii)cellulose thimbles and a stand to hold the thimbles; (iii) extraction cups, which can bealuminum or glass. (Extraction temperature settings may differ; consult manufactur-er's operating instructions.) The reagents include hexanes and defatted cotton.

The determination is made as follows: Weigh 1 g of ground soy sample into taredcellulose thimbles. Record the weight to the nearest 0.1 mg (S) and the thimble num-ber. Place a defatted cotton plug (with solvent used for extraction) on top of the sam-ple to keep the material immersed during the boiling step and to prevent any loss ofthe sample from the top of the thimble. Prepare a cotton plug large enough to hold thematerials in place yet as small as possible to minimize the absorption of solvent.Extract the sample with the following instrument settings: hot plate temperature, 163± 5°C; boiling time/sample immersion cycle time, 20 min; sample rinse cycle time, 40min; solvent evaporation and recycling time, 8 min. Dry the sample cups at 105 ± 5°Cfor at least 30 min before transferring to a desiccator and cooling to room temperature.Weigh the empty sample cups (T) and the extraction cups and record the weights tothe nearest 0.1 mg.

Preheat the extractor and turn on the condenser cooling water. Attach thimblescontaining dried test portions to the extraction columns. Add a sufficient amount ofsolvent (80 ± 10 mL) to each extraction cup to cover the sample when thimbles are inboiling position. Place the cups under the extraction columns and secure in place.Make sure that the cups are matched to their corresponding thimble. Lower the thim-bles into the solvent and boil for 20 min. Verify proper reflux rate. Reflux rate is criti-cal to the complete extraction of fat. Adjust the reflux rate to ~3–5 drops/s.

Raise the thimbles out of the solvent and allow them to extract in this position for40 min. Then distill as much solvent as possible from the cups to reclaim solvent andto attain apparent dryness. Dry the extraction cups in a 105 ± 5°C oven for 30 min toremove moisture. Excessive drying may oxidize the fat and give high results. Cool ina desiccator to room temperature and weigh to the nearest 0.1 mg (F). The percentageof crude fat is calculated as follows:

% crude fat (as is) = (F – T)/S × 100 [1]

where F is the weight of cup + fat residue (g), T is the empty weight of cup (g),and S is the test portion weight (g).

Crude fat is calculated on a dry matter basis (DMB) as follows:

100 × average crude fat % (as is) = % crude fat DMB [2]

(100 – average % moisture loss)

Accelerated Solvent Extractor (Dionex). Accelerated solvent extraction (ASE 200)is achieved by extracting crude fat with an organic solvent (petroleum ether/hexanes)at elevated temperature and pressure. The solvent is pumped into an extraction cell


containing the sample, which is then brought to a specified temperature and pressure(Table 7.1). Increased temperature accelerates the extraction kinetics, whereas elevat-ed pressure prevents boiling at temperatures above the normal boiling point of the sol-vent. Crude fat is extracted and flushed into preweighed glass vials. The percentage ofcrude fat is determined gravimetrically after evaporation of the extracting solvent withnitrogen. Only small amounts of solvent are used and the extraction time is ~30 min.Time, solvent consumption, and worker exposure are significantly reduced throughthis technology compared with other techniques.

The Dionex ASE 200 system apparatus includes the following: a pump fordelivery of extraction solvent from the reservoir to extraction cell, a programmableoven in which the extraction takes place, and an autosampler tray. The sample cellsare made of stainless steel with interchangeable caps that screw onto each end ofthe cell bodies and are hand-tightened. Inside each cap is a stainless steel frit and aPEEK seal. For extraction of oil from corn and soy, 11-mL cells are used. Glassvials are used for rinse and collection glass; sand and filters are also used. Thereagents include petroleum ether (boiling point range 40–60°C) and sand (30–40mesh).

The determination is made as follows: Accurately weigh the ground sample ofcorn or soybean into the ASE cell. Add sand to the top of the cell and place two filtersat the top. Preweigh the 40 mL collection vials in milligrams to one decimal place,without the caps before loading on the ASE. The operating conditions of ASE-200 arelisted in Table 7.1. Before running the samples, blank cells (ASE cell with no sample)and a check sample (a previously run sample with a known crude fat content) are runas an instrument performance check. At least one check and one blank sample areanalyzed with each analytical run.

After the extraction process is completed, remove the collection vials, uncapthem and arrange them under a stream of nitrogen or house air (regulator pressure ≤5psi) at ~37 ± 5°C to evaporate the solvent. Dry the collection vials for 2 h ± 10 min.Allow vials to cool to ambient temperature before weighing; vials must be cappedwhile cooling. The oil amount is calculated from the difference between the finalweight and original vial weight as follows:

% crude fat (as is) = [(vial + oil) – vial weight] × 100 [3]

TABLE 7. 1Accelerated Solvent Extractor Operating Conditions

Preheat 0 min Pressure 1000 psiHeat 6 min Temperature 105°CStatic 5 min Solvent compartment AFlush 50% vol Petroleum ether 100%Purge 90 s Average run time/sample 30 minCycles 3





Ankom Fat Extractor (ANKOM). Extraction was performed using the Ankom XT20 Fat Analyzer. This extractor uses ~1500 mL solvent for a set of 20 samples. Thefirst step in this process involves the entrapment of the ground seed samples inspecial sealed polymer bags of controlled porosity. The samples are placed in aspinning basket and allowed to bathe in petroleum ether at 90°C for 40 min afterwhich the solvent is removed with a nitrogen flush. The dried bags are weighedand the difference in the weights before and after bathing with petroleum ether rep-resents the amount of total oil present in the samples.

The apparatus includes the Ankom XT 20 fat analyzer, fat extraction filterbags (Ankom ID # XT4), heat sealer (for Ankom #1915 or #1916), a moisture-stopweigh pouch (Ankom #F39), and a drying oven. The reagents include reagent-grade petroleum ether (boiling point 40–60°C).

The determination is made as follows: Weigh and record weight of an XT4 fil-ter bag and tare; accurately weigh ~1 g of sample into the bag and record weight tothe nearest 0.1 mg; heat-seal the filter bag to encapsulate the sample; extract thesample in Ankom XT20 according to the “operating instructions” for 40 min at90°C; oven-dry extracted samples at 100°C for 1 h; cool in moisture-stop weighpouch, weigh and record weight of the extracted sample in the filter bag.

Calculation of the % crude fat is as follows:

% crude fat =[(WPD

sample – Wbag) – (Wfinal – Wbag)] × 100[5]

Wsample

where Wsample is the original weight of the sample; WPDsample is the weight of the

predried sample; Wbag is the weight of the filter bag; and Wfinal is the weight of thesample after extraction.

Supercritical Fluid Extractor (ISCO). Extraction was carried out at ISCO Inc.,Lincoln, NE. Crude fat was extracted from ground corn and soybean samples fol-lowing ISCO’s standard operating procedures ISCO SOP # MSW-10–026 andISCO SOP # MSW-10–028, respectively. Crude fat is extracted from ground seedswith a supercritical fluid (liquid carbon dioxide) and ethanol (10%) as a modifier.The extract is trapped directly onto glass wool contained in a collection vial. Afterevaporation of moisture and any residual modifier from the glass wool and the col-lection vial, the weight of the oil extract is determined and used to calculate thepercentage of oil present in the sample.

The SFE apparatus (ISCO SFX 3560 or ISCO) includes the sample extractioncell, collection vial (glass or polypropylene tube), and microwave oven. The reagents


used include food-grade industrial liquid carbon dioxide, glass wool, ethanol(HPLC-grade), and granular diatomaceous earth.

The determination is made as follows: Accurately weigh 100 ± 5 mg or 2 ± 0.1g of ground seeds into a tared extraction cell. Record the weight to the nearest 0.1mg (S) and the extraction cell number. Fill the remaining void volume in the cellwith granular diatomaceous earth. Take a minimum of 1 g glass wool and pack itinto each collection vial. Accurately weigh the collection vial with the glass wooland record the weight (T). Load the extraction cell and collection vial. The extrac-tion parameters for corn and soybeans are given in Table 7.2. After the extractionis completed, the collection vials are transferred into a microwavable test tuberack. The tubes are kept in a microwave at 500 W for 10 min. The tubes are cooledto room temperature and weighed accurately (F).

Calculation of the % crude fat is as follows:

% crude fat (as is) = (F – T)/S × 100 [6]

where F is the weight of cup + fat residue (g), T is the empty weight of cup (g),and S is the test portion weight (g).




Analysis of Fatty Acid Methyl Esters (FAME) by GC

Fat was extracted from ground corn or soy samples using either an automatedDionex accelerated solvent extractor (ASE) with petroleum ether solvent or a FossTecator 2050 Soxtec extraction unit with hexane solvent. The solvent was evapo-rated by nitrogen stream. FAME are formed by transesterification of the extractedoil with acetyl chloride/methanol at ambient temperature with constant agitationovernight (16–24 h) or with constant stirring at 70 ± 5°C for ~2 h ± 15 min. TheFAME were extracted into hexane and analyzed by a capillary GC with a flameionization detector (FID) (20).

TABLE 7.2 Instrument Settings for the Extraction of Soybean Samples by Supercritical FluidExtraction

Soybeans

Extraction chamber pressure (psi) 7000 Extractor chamber temperature (°C) 100 Restrictor temperature (°C) 150 Modifier (ethanol) (%) 10 Dynamic Extraction Time (min) 45


Results and Discussion

Effect of Sample Preparation Step (Grinder Study)

Table 7.3 summarizes the differences among the various grinders with respect togrinding conditions, screen size, grinding time, sample preparation time, cross-conta-mination, and multiple grinding capabilities. The Monsanto-built and designedMega-grinder provides significant advantages over the commercially availablegrinders because it is the only grinder capable of grinding multiple samples (from1–96 samples) with no cross-contamination. The Mega-grinder is the best grinder forsmall size samples; there is minimal loss during the grinding process because thesample is ground in a single, closed container.

For very large sample sizes, the Mega-grinder is limited by the loading capaci-ty of each individual tube. The Mikro-mill and Knifetec grinders are bettergrinders for bulk samples because the grinding and sample preparation time aresignificantly lower with larger sample sizes. The grinding time was significantlyincreased with high-oil and high-moisture samples for the UDY, Cyclotec, andBrinkmann grinders due to frequent clogging of the 0.75-, 1-, and 2-mm screens.

Oil Extracted from Soy Samples. Table 7.4 summarizes the grand mean of oilextracted from seven soy samples ground with various grinders using the Soxtecextraction procedure. The grand mean of crude fat extracted from different samplesvaried from 11.0 to 21.4%. The Cemotec grinder provided the lowest crude fatyields with the highest SD due to inefficient extraction of oil from large particles.The highest crude fat yields were found in samples ground with the Mega-grinder(grand mean = 21.4%). The crude fat yields of samples ground with the othergrinders varied from 15.7 to 20.9%. Maximum and minimum oil yields were

TABLE 7.3 Comparison of Operating Performance of Different Grinders

Sample Multiple sampleGrinding preparation Cross- grinding

Grinder type Screen size time timea contamination possibility

Mega-grinder N/A 2 min 45 min No YesCyclotec 1 and 2 mm 30 min 75 min Yes NoKnifetec N/A 30/90 s 5 min Yes No

(3 × 2 × 5)(3 × 3 × 10)

Mikro-mill 1 mm 30 s 3 min Yes NoUDY 2 mm 15 min 30 min Yes NoBrinkmann- 0.75/1 mm 2 min 5 min Yes NoRetsch-millCemotec Setting #1 2 min 5 min Yes NoaSample preparation time is the average time required to grind 150 g of each sample.


obtained with the Monsanto Mega-grinder and Cemotec grinder, respectively. Thedifferences in crude fat yields with different grinders may be attributed to differ-ences in particle size distribution (Fig. 7.1).

Assessment utilizing four-screen particle size separation showed that the effi-ciency of crude fat extracted from ground soy samples increases with the decreasein particle size (Fig. 7.2). A similar effect was also noticed on the precision of theanalyses for these samples. High SD for coarse samples (>1.4 mm) stem from inef-ficient extraction of crude fat from large-size particles (Fig. 7.3). One cautionarynote that must be emphasized is that the very high oil recovery numbers for smallparticle samples could be due to the co-extraction of ultra fine particulates alongwith the crude total fat content or partial fractionation of oil- and protein-enrichedparts. A detailed study is currently underway to gain better insights into this issue.Similar studies are also planned for corn as well. Based on the current study, we

TABLE 7.4Grand Mean Average of Crude Fat Extracted by Soxtec Procedure from Seven SoySamples with Various Grinders

Grinder Grinding specifications Mean SD Range

Cemotec Grind Size #1 11.0 0.56 1.08Cyclotec Screen 1.0 mm 20.9 0.27 0.52Cyclotec Screen 2.0 mm 20.7 0.27 0.51Knifetec 3 × 2 × 5 s (30 s) 15.7 0.35 0.66Knifetec 3 × 3 × 10 s (90 s) 18.2 0.23 0.46Mega-grinder: ANKa SOP 21.4 0.23 0.44Brinkman-Retsch mill Screen 0.75 mm 19.6 0.22 0.42Mega-grinder: STLb SOP 21.3 0.22 0.42UDY Screen 2.0 mm 20.7 0.21 0.41Mikro-mill Screen 1.0 mm 20.6 0.18 0.33aFor the Mega-grinder: ANK, grinding was done at the Ankeny site.bFor the Mega-grinder: STL, grinding was done at the St. Louis site.

Fig. 7.1. Particle size distribution of soy samples ground on different grinders.

Camotec Cycolotec Cyclotec Knifetec Mega- Brinkman- UDY Micro Mill1 mm 2 mm grinder Retsch Mill 1 mm

Grinder

%D

istr

ibut

ion

2.4 mm

2.0 mm

1.0 mm

0.6 mm


recommend that a 1-µm particle screen be used with commercial grinders as muchas possible.

Grinding conditions can also influence the amount of crude fat extracted. Thegrand mean crude fat yields for soy for the Knifetec grinder varied with changes inthe grinding conditions. An increase of 2.5% in grand mean crude fat yield wasobtained when grinding time was changed from 30 to 90 s. A difference of 0.2% ingrand mean crude fat content was observed when samples were ground with twodifferent screen sizes (1 and 2 mm) on a Cyclotec grinder. These results confirmthat both grinder type and grinding conditions have an effect on crude fat extrac-tion yields. A minor difference (0.1%) in grand mean averages was observed withthe Monsanto Mega-grinder when two operators ground samples with differentinstruments at two different sites. Thus, one type of grinder can give consistent

Fig. 7.2. Effect of particle size on the percentage of crude fat extraction from a soybeansample.

Particle size (mm)

%C

rude

fat (

DM

B)

Fig. 7.3. Effect of particle size on the precision of crude fat extraction from a soybeansample.

Particle size (mm)

%C

rude

fat (

DM

B)


results regardless of operator or location when experiments are conducted with thesame standard operating procedure. A similar effect on crude fat content can beobserved with different grinders by modifying grinding time, tube sizes and grindingball.

Statistical analyses were carried out using JMP 5.0 (SAS) and SYSTAT (10)software. ANOVA showed that the P-values for the oil extracted with differentgrinders was <0.0001 for soy samples, suggesting that the crude fat extraction yieldswith at least one grinder were significantly different from the others. Tukey’s methodwas used for statistical comparison of crude fat extracted from samples ground withdifferent grinders. The results presented in Table 7.5 show that the crude fat contentfrom samples ground with a Cemotec, Knifetec (30 and 90 s), and Brinkmann Retschgrinders were clearly different from the samples ground with other grinders (Mega-grinder, Cyclotec, Mikro mill, and UDY). The crude fat extraction yields from sam-ples ground with Mega-grinder at two sites did not differ.

Comparison of the Five Extraction Procedures

A comparison of sample throughput and automation possibilities of the five meth-ods used for extraction of crude fat is presented in Table 7.6. The results in Tables7.7 and 7.8 show the comparison of the total percentage of crude fat extracted andthe precision of the five methods, Butt-tube, Soxtec, ASE, SFE, and Ankom FatExtractor. The results in Table 7.7 show that at the 100-mg sample size, the rangedifference in the grand mean of the percentage of crude fat extracted by three dif-ferent methods (ASE, Soxtec, and SFE) was 0.9% for soybean samples.

For single-seed analysis in which the sample size is limited, the highest preci-sion was obtained with Soxtec, which had the lowest SD (Table 7.8). The results

TABLE 7.5 Least Squares Means (LSM) Differences with Tukey’s Honestly Significant DifferenceTest for Soybeans (α = 0.050; Q = 3.29923)

Level LSM

Mikro-mill (1.0 mm) A 20.05Mega-grinder: ANK A B 19.55Cyclotec 1.0 mm A B 19.40UDY 2.0 mm A B 19.36Mega-grinder: STL A B 19.33Cyclotec 2.0 mm B 19.16Brinkman-Retsch mill (0.75 mm) C 17.87Knifetec-90 s D 16.51Knifetec-30 s E 14.32Cemotec F 10.02


presented in Table 7.7 show that the grand mean of the total percentage of crudefat extracted from soybean at the 100-mg sample size by ASE, SFE and Soxtecwere in good agreement with the grand mean crude fat extracted from bulk sam-ples by different methods. For soy samples, the SD for Soxtec and Ankom weresimilar and ~0.1% higher than those for ASE, SFE, and Butt-tube. Even thoughhigher precision was obtained with the 2-g sample size with ASE, we recommendthe 1-g sample size for soy due to lower maintenance associated with instrumentoperation. In the case of Soxtec, we used 1-g sample size for soy as recommendedby the instrument manufacturer (0–10% fat, 2–3 g sample size; >20–25% fat,0.5–1 g sample size).

In a comparison of the grand mean percentage of crude fat extracted by thevarious methods with 1- to 2-g sample sizes, the range of the means was 1.4%.All methods showed comparable accuracy for bulk crude fat analysis at 1–2 g ofground sample. The crude fat extracted by ASE was higher than that reported forthe other methods. This was either due to the extraction conditions and/or resid-ual moisture present in the sample because extractions were carried out on an as-is basis. The results obtained were converted to a dry matter basis after determin-ing the moisture content separately. The higher extraction yield may be due todifferences in extraction conditions or passage of very fine particles through thefrit, or passage of moisture during the flush cycle because analyses were carriedout on “as-is” basis and the results were converted to DMB after analyses.

For Butt-tube and Soxtec extraction procedures, the sample must be dried at105°C for 30 min. This drying process may cause oxidation of fatty acids, particu-larly for crude fat enriched in polyunsaturated fatty acids (PUFA). To evaluate theeffect of oxidation, two soy samples were extracted separately with ASE andSoxtec. The oil extracted by both of these procedures was derivatized and analyzedby GC. The results presented in Table 7.9 indicated no difference in the fatty acidcomposition obtained by the two methods. However, caution is advised with crudefat enriched in PUFA or if one is interested in minor constituents (PUFA); these

TABLE 7.6 Comparison of Sample Throughput and Automation Capabilities for Five Crude FatExtraction Methods

Method Sample throughput Automation

ASE 24 samples/12 h YesSFE-3560 24 samples/18 h YesSFE-FastFat-HT 4 samples/h NoSoxtec 6 samples/1.25 h PartialButt-tube Depends on set-up NoAnkom 20 samples/4 h Yes


TABLE 7.7 Total Percentage of Crude Fat Extracted from Ground Soybeans by Five Methodsa

ASE SOXTEC SFE SOXTECb ASE ASE SFE Ankom Butt-tubeAverage 100 mg 100 mg 100 mg 1 g 1 g 2 g 2 g 1 g 2 g

SOY 1 19.6 21.5 20.6 19.7 20.6 21.2 19.6 19.6 19.5SOY 2 21.4 20.8 22.3 21.9 22.5 22.7 22.2 21.9 21.2SOY 3 23.1 24.0 23.7 23.7 23.7 24.6 23.1 23.9 23.3

Grand mean 21.4 22.1 22.3 21.8 22.3 22.8 21.6 21.8 21.4aResults are averages of six replicate analyses.bSelection of sample size used for extraction of crude fat from ground oilseeds was based on instrument vendor’s recommendations.


TABLE 7.8 Standard Deviations of the Total Percentage of Crude Fat Extracted from Ground Corn and Soybeans by Five Methodsa

ASE SOXTEC SFE SOXTECb ASE ASE SFE Ankom Butt-tubeAverage 100 mg 100 mg 100 mg 1 g 1 g 2 g 2 g 1 g 2 g

SOY 1 0.55 0.35 0.46 0.20 0.29 0.07 0.05 0.32 0.08SOY 2 0.33 0.28 0.43 0.23 0.14 0.05 0.21 0.21 0.11SOY 3 0.44 0.34 0.72 0.18 0.29 0.12 0.12 0.09 0.11

Grand mean 0.44 0.32 0.54 0.20 0.24 0.08 0.13 0.21 0.10aSix replicate analyses were carried out with each sample.bSelection of sample size used for extraction of crude fat from ground oilseeds was based on instrument vendor’s recommendation.


are found in lower quantities because of the greater possibility of oxidation ofPUFA at a higher temperature (105°C) as used in the case of drying after Soxtecextraction.

Conclusions

The results presented in this study indicate that both sample preparation andextraction conditions have a direct effect on total crude fat extracted from oilseeds.For single-seed analysis, ASE and SFE can be considered to be primary methodsof choice for crude fat extraction and Mega-grinder for sample preparation. Forbulk samples, all five methods could be used for the determination of crude fatcontent. The method of choice would depend on the precision required, instrumentavailability, and any further analysis of the extracted crude fat (fatty acid composi-tion or microconstituent determination). Ankom technology, a batch extractionprocess, provides the highest sample throughput. The major limitation with Ankomtechnology is that it does not allow any further analyses of the oil extracted fromindividual samples because oil extracted from individual samples is present in thesame solvent pool. SFE is an environmentally friendly technique because it usespressurized CO2, and there is no solvent waste as associated with other techniques.

Two approaches for developing secondary calibrations are described briefly.Approach I: To develop the most precise secondary calibration, one can select a singleprimary reference method. However, there are disadvantages to developing a sec-ondary calibration on a single primary method. A secondary calibration based on asingle primary method will provide consistent results, but the results may notmatch primary chemistry results from other laboratories performing analyses usingdifferent preparation and analytical methodologies.

Approach II: Alternatively, one can can develop more rugged secondary cali-brations that contain more than a single source of primary data. The problem with

TABLE 7.9 Fatty Acid Methyl Ester Analysis by GC of Oil Extracted from Two Soy Samples by ASEand Soxtec Methods

16:0 18:0 18:1(n-7) 18:1(n-9) 18:2(n-6) 18:3(n-3) 20:0 22:0

Sample ID Area (%)

Soy3-ASEa 11.0 6.3 1.3 21.5 51.9 7.2 0.1 0.2Soy3-Soxb 10.9 6.3 1.3 21.6 51.6 7.1 0.5 0.3Difference 0.1 –0.0 0.0 –0.1 0.3 0.1 –0.4 –0.1

Soy1-ASE 11.1 6.1 1.3 21.4 52.4 6.97 0.1 0.2Soy1-Sox 11.0 6.0 1.3 21.4 52.1 6.97 0.3 0.4Difference 0.1 0.1 0.0 0.0 0.3 0.00 –0.2 –0.2aASE: Extraction carried out with accelerated solvent extractor.bSox: Extraction carried out with Soxtec.


this approach is that the analytical precision of the secondary method will decreasebecause primary data from multiple techniques will not match precisely.

We recommend approach I for research projects in which precision is critical.This will enable laboratories to monitor projects on a regular basis. For commercialpurposes, we recommend approach II because this will provide a more rugged calibra-tion. In developing secondary calibrations, one should document the source of everyreference value for each calibration sample in the secondary calibration model. Inaddition, replicate measurements on each sample at the selected primary laboratoryshould be determined. The number of replicates required is determined by the level ofprecision expected in the calibration vs. the precision of the primary data. If data frommultiple laboratories are to be used, the interlaboratory validation studies must be per-formed in advance. Any systematic differences among laboratories must be defined.

The following recommendations are made for round-robin studies to comparedifferent methods or interlaboratory variations: (i) Submit a minimum of 8–10 sam-ples with a range of concentrations for the analyte of interest. (ii) Sample preparationshould be done by identical processes by each participant. (iii) Always perform amoisture analysis and report the results on a dry matter basis; this will reduce theeffect of changes in the environmental conditions such as drying of sample or humid-ity. (iv) Moisture content analysis should be done using identical procedures by alllaboratories. (v) Perform all analyses during the same time frame to reduce the possi-bility of changes in the analyte concentration due to degradation, for example. (vi)Always use identical instrument conditions and chemicals for analysis. (vii) Alwayssubmit standard samples with known analyte concentrations to each laboratory toevaluate variations and results obtained from each laboratory.

Acknowledgments

The authors acknowledge Tracy Doane Weideman and Josh Tomczk, Isco, Inc., Lincoln,NE for their help in the evaluation of SFE.

References

1. Official Methods and Recommended Practices of the American Oil Chemist’s Society(1998) 5th edn. (Firestone, D., ed.) AOCS Press, Champaign, IL.

2. OSFA International Method: Determination of Oil Content in Oil Seeds-Solvent Extract(1998) Reference Method.

3. DGF, Deutsche Einheitsmethoden zur Untersuchung von Fetten, Fettprodukten (1998)Tensiden und verwandren Stoffen, Wissenschaftliche, Stuttgart.

4. International Organization for Standardization (1998) Oilseeds: Determination of HexaneExtract (or Light Petroleum Extract), Called Oil Content, ISO, Standard No. 659, Geneva.

5. Approved Methods of the American Association of Cereal Chemists (2000) 10th edn., St.Paul, MN.

6. Official Methods of Analysis of AOAC International (1999) 16th edn. (Cunniff, P., ed.)Gaithersburg, MD.

7. Brown, J.S. (1995) NMR Method to Determine Oil Content, INFORM 5: 320–321.


8. Daun, J.K., Clear, K.M., and Williams, P. (1994) Comparison of Three Whole Seed Near-Infrared Analyzers for Measuring Quality Components of Canola Seed, J. Am. Oil Chem.Soc. 71: 1063–1068.

9. Orman, B.A., and Schumann, R.A. (1992) Nondestructive Single-Kernel Oil Determinationof Maize by Near-Infrared Transmission Spectroscopy, J. Am. Oil Chem. Soc. 69: 1036–1038.

10. Barthet, V.J., Chornick, T., and Daun, J.K. (2002) Comparison of Methods to Measurethe Oil Contents in Oilseeds, J. Oleo Sci. 51: 589–597.

11. Hill, L.D., and Blender, K.L. (1994) Evaluation of Changing the Moisture ReferenceMethod, Cereal Foods World 39: 19–27.

12. Hunt, W.H., and Neustadt (1966) Factors Affecting the Precision of Moisture Measurementin Grain and Related Crops, J. Assoc. Off. Anal. Chem. 49: 757–763.

13. Matthaus, B., and Brü, L. (2001) Comparison of Different Methods for the Determinationof the Oil Content in Oilseeds, J. Am. Oil Chem. Soc. 78: 95–102.

14. Brü, L., Matthaus, B., and Fresenius, J. (1999) Extraction of Oilseeds by SFE. AComparison with Other Methods for the Determination of Oil Content, Anal. Chem. 363:631–634.

15. Luthria, D.L., and Cantrill, R. (2002) Evaluation of Five Different Methodologies forDetermination of Oil Content in Ground Corn and Soybean Seeds, INFORM 13: 893–894.

16. Hosokawa Micron Ltd., Hammer Mill, http://www.hosokawa.co.uk/flash/mikropulverizer.html

17. Foss, Solutions, Analytical Laboratories, Sample Preparation Mill, http://www.foss.dk 18. Seedburo Equipment Company, Lab Mills, http://www.seedburo.com/19. Brinkmann Instruments, Inc., http://www.foodonline.com/Content20. Luthria, D.L., and Sprecher, H. (1997) Studies to Determine If Rat Liver Contains

Multiple Chain Elongating Enzymes, Biochim. Biophys. Acta 1346: 221–230.


http://www.hosokawa.co.uk

http://www.hosokawa.co.uk

http://www.foss.dk/

http://www.seedburo.com/

http://www.foodonline.com

Effect of Moisture Content, Grinding, and Extraction ...

Documents

Transcript of Effect of Moisture Content, Grinding, and Extraction ...