oil extraction and analysis phần 7 ppt

A comparative evaluation of different grinders Mega-grinder, Knifetec, Cyclotec, Cemotec, Mikro mill, UDY grinder, Brinkmann-Retsch grinding mill and commonly performed crude fat extract

Chapter 7

Effect of Moisture Content, Grinding, and Extraction Technologies 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 make improvements 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 obtainmodify-ing accurate, cost-effective, and rapid analysis of oilseeds/grains with enhanced quality traits such as total crude fat (oil) content or a modified oil composition Reliable crude fat analysis is of paramount importance to oilseed businesses because monetary assessment 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 content

in 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) and commonly performed crude fat extraction methodologies [accelerated solvent extrac-tor (ASE), supercritical fluid extraction (SFE), Ankom batch extraction, automated Soxtec 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 organizaharmoniza-tions (e.g., AOCS, AOAC, AACC, ISO, DGF) This is vital for the development of rugged calibrations for nondestructive, high-throughput secondary procedures involving near infrared transmittance, near infrared reflectance/imaging, and nuclear magnetic resonance spectroscopy 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 secondary calibrations and performing interlaboratory studies are also presented

Introduction

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

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

feed trade, nutritional labeling, and biotechnology research Reliable assays of quality and quantity of crude fat content in oilseeds/grains are essential for determination of the fair trade value of enhanced traits/products in the global market To facilitate this process, several international societies such as the American Oil Chemists’ Society (AOCS) (1), the Federation of Oil Seeds and Fat Association Ltd (FOSFA) (2), the German Fat Science Society (Deutsche Gesellschaft fur Fettwissenschaft, DGF) (3), the International Organization of Standardization (ISO) (4), the American Association

of Cereal Chemists (AACC) (5), and the Association of Official Analytical Chemists (AOAC) (6) have developed standard reference methods for the assay of quality and quantity of crude fat in a variety of matrices Nevertheless, problems exist because of inherent 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, or supercritical liquid carbon dioxide with or without a solvent modifier The volatile solvents are removed by evaporation and the nonvolatile residue (crude fat) is mea-sured gravimetrically The nonvolatile fraction consists of triacylglycerols and trace amounts of other components such as free fatty acids and their alkyl esters, sterols, sterol esters, long-chain aldehydes and alcohols, fat-soluble vitamins, and other nonpolar natural products Alternatively, two high-throughput nondestructive methods that utilize nuclear magnetic resonance and near infrared spectroscopic techniques have also been developed These methods are secondary in nature because they require calibration support by primary reference methods (7–9) In response 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 the sum 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 in oilseeds is essential because it plays a crucial role in determining the monetary value 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 analyses

of moisture content in grains A variety of methods exists that have been approved

by different official societies These methods differ in variables such as the drying temperatures, drying time, or sample quantity used for moisture determination All

of these factors have differing effects on moisture content determination, and this indirectly affects assays of crude fat, protein, fiber, and other constituents Details

of factors affecting the precision of moisture measurement (grinding, sample size, moisture dishes, ovens, relative humidity of the laboratory, desiccant) and different moisture reference methods were summarized in two publications (11,12)

We chose to evaluate two factors that affect the crude fat assay, i.e., the influence

of 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 our preliminary report were carried out with one sample from each grain variety and comparison with the Ankom batch extraction process was not evaluated earlier Several soy samples with varying crude fat content were ground on different grinders under varying grinding conditions to examine the effect of sample prepa-ration on crude fat assay The comparative performance of five currently used oil extraction technologies was also evaluated This study was conducted to identify the most accurate and precise method for bulk sample (sample size >500 mg) and single-seed (sample size <150 mg) analysis Sample extraction was conducted using 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 temperatures and conditions was also evaluated

Experimental

Effect of Sample Preparation (Grinder Study)

The principle of operation of the seven grinders used for sample preparation is summarized below

Mikro Mill (Hosokawa) This is a high-capacity hammer mill designed for

grind-ing a comprehensive range of materials Grindgrind-ing is caused by impact between the rotating 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 rotor speed, and the size of the screen opening In the present study, a 1-mm screen size was used (16)

Knifetec Grinder (Foss) This grinder utilizes a high-speed (20,000 rpm) rotor

blade for grinding samples The particle size of the ground material depends on the grind time and the number of cycles The Knifetec grinding chamber is equipped with 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 to stick to the wall of the chamber because the fat softens during grinding, thus pre-venting adequate homogenization In addition, fibrous samples may generate heat due to friction Utilizing the cooling option, the Knifetec grinder overcomes both

of 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 <1 min Although it is a fast grind, the particle size is very coarse even with the finest setting 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-speed

turbine that spins seeds against a coarse chamber ring This action causes the seeds

to form a fine powder, which is pushed through a screen (1 or 2 mm) Samples containing high levels of fat have a tendency to stick to the screen and eventually block the passage of the sample through the screen This grinder requires frequent cleaning, 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 The

crankshaft drives a piston that holds the sample trays The piston is moved in an up-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 the steel balls into intact seeds causing seeds to become pulverized in 0.5–2 min This grinder has the capacity to grind 96 single-seed samples at one time in 2 min with

no cross-contamination

UDY Grinder (UDY) This is a cyclone sample mill designed for rapid grinding

of a wide variety of soft to medium-hard materials The grinding process involves the high-speed rotation of the impeller and air currents that throw particles into the grinding ring and rolls them around Particles remain in the grinding chamber until impact 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 makes clean-out unnecessary The airflow also minimizes heating and therefore eliminates thermal degradation (18)

Brinkmann-Retsch Mill (Brinkmann) This grinder consists of a two-speed

motor, removable lid, rotors, and sieves It is a centrifugal grinder that operates similarly to the Cyclotec and UDY grinders (19)

Experimental Details for the Grinder Study

Sample Preparation Seven soybean samples with varying oil content were used to

compare 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 at Monsanto’s Crop Analytics Laboratory in Ankeny, IA Samples were ground with the Mega-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-mm screens Samples were submitted to the contract laboratories for grinding on UDY, Knifetec, and Mikro-mill grinders Samples ground on a Knifetec mill were ground for 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 for

oil 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 wide range of particle size distribution The combined ground sample was sieved using multiple stacked sieves (1.4, 1.0, 0.6, and 0.3 mm) Approximately 30 g of ground sample was placed on the top 1.4 mm mesh size sieve The sample was brushed through 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 below the finest mesh sieve Each fraction was weighed separately to determine the particle size 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 compare the 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 analyses were carried out for each sample by all five methods The results are reported on a dry matter basis to eliminate the effect of varying moisture content Extractions with supercritical fluid extraction (SFE) were performed at ISCO Labs, and part of the extractions with the Ankom batch procedure was performed at the Ankom facility

Extraction was done with two sample sizes, 100 mg and 1 or 2 g Extraction with 100-mg sample size was conducted to evaluate the applicability of the method

to 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 (500 mg), and SFE (2 g)] Extraction conditions and sample processing for two sample sizes (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 that

involves continuous flow of distilled condensed solvent over the ground sample matrix (4–5 h) The amount of oil extracted is determined gravimetrically after evaporating the extraction solvent (AOCS Ac 3-44) (1)

Soxtec Extraction (Foss) This method is a two-step process for the extraction of

crude 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 hot solvent ensures rapid solubilization of extractables Then the thimble is raised above the solvent and the test portion is further extracted by a continuous flow of condensed solvent The resulting crude fat residue is determined gravimetrically after 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 Teflon seals compatible with hexanes; (ii) cellulose thimbles and a stand to hold the thimbles; (iii) extraction cups, which can be aluminum 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 tared

cellulose 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 of the sample from the top of the thimble Prepare a cotton plug large enough to hold the materials 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, 40 min; solvent evaporation and recycling time, 8 min Dry the sample cups at 105 ± 5°C for 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 to

the nearest 0.1 mg

Preheat the extractor and turn on the condenser cooling water Attach thimbles containing dried test portions to the extraction columns Add a sufficient amount of solvent (80 ± 10 mL) to each extraction cup to cover the sample when thimbles are in boiling 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 for

40 min Then distill as much solvent as possible from the cups to reclaim solvent and

to attain apparent dryness Dry the extraction cups in a 105 ± 5°C oven for 30 min to remove moisture Excessive drying may oxidize the fat and give high results Cool in

a desiccator to room temperature and weigh to the nearest 0.1 mg (F) The percentage

of crude fat is calculated as follows:

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)

(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 of crude fat is determined gravimetrically after evaporation of the extracting solvent with nitrogen Only small amounts of solvent are used and the extraction time is ~30 min Time, solvent consumption, and worker exposure are significantly reduced through this technology compared with other techniques

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

The determination is made as follows: Accurately weigh the ground sample of corn or soybean into the ASE cell Add sand to the top of the cell and place two filters

at 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 are listed 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 run

as an instrument performance check At least one check and one blank sample are analyzed with each analytical run

After the extraction process is completed, remove the collection vials, uncap them and arrange them under a stream of nitrogen or house air (regulator pressure ≤5 psi) 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 capped while cooling The oil amount is calculated from the difference between the final weight and original vial weight as follows:

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

TABLE 7 1

Accelerated Solvent Extractor Operating Conditions

Static 5 min Solvent compartment A

Purge 90 s Average run time/sample 30 min Cycles 3

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

100 × average crude fat % (as is)

(100 – average % moisture loss)

Ankom Fat Extractor (ANKOM) Extraction was performed using the Ankom XT

20 Fat Analyzer This extractor uses ~1500 mL solvent for a set of 20 samples The first step in this process involves the entrapment of the ground seed samples in special sealed polymer bags of controlled porosity The samples are placed in a spinning basket and allowed to bathe in petroleum ether at 90°C for 40 min after which the solvent is removed with a nitrogen flush The dried bags are weighed and 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 filter bags (Ankom ID # XT4), heat sealer (for Ankom #1915 or #1916), a moisture-stop weigh 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 to the nearest 0.1 mg; heat-seal the filter bag to encapsulate the sample; extract the sample in Ankom XT20 according to the “operating instructions” for 40 min at 90°C; oven-dry extracted samples at 100°C for 1 h; cool in moisture-stop weigh pouch, weigh and record weight of the extracted sample in the filter bag

Calculation of the % crude fat is as follows:

% crude fat =[(W

PD sample– Wbag) – (Wfinal– Wbag)] × 100

[5]

Wsample where Wsampleis the original weight of the sample; WPDsampleis the weight of the

predried sample; Wbagis the weight of the filter bag; and Wfinalis the weight of the sample 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 and ISCO SOP # MSW-10–028, respectively Crude fat is extracted from ground seeds with 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 After evaporation 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 the percentage of oil present in the sample

The SFE apparatus (ISCO SFX 3560 or ISCO) includes the sample extraction cell, 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 ± 0.1

g of ground seeds into a tared extraction cell Record the weight to the nearest 0.1

mg (S) and the extraction cell number Fill the remaining void volume in the cell

with granular diatomaceous earth Take a minimum of 1 g glass wool and pack it into each collection vial Accurately weigh the collection vial with the glass wool

and 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 extracextrac-tion

is completed, the collection vials are transferred into a microwavable test tube rack The tubes are kept in a microwave at 500 W for 10 min The tubes are cooled

to room temperature and weighed accurately (F)

Calculation of the % crude fat is as follows:

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)

(100 – average % moisture loss)

Analysis of Fatty Acid Methyl Esters (FAME) by GC

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

TABLE 7.2

Instrument Settings for the Extraction of Soybean Samples by Supercritical Fluid Extraction

Soybeans Extraction chamber pressure (psi) 7000 Extractor chamber temperature (°C) 100

Results and Discussion

Effect of Sample Preparation Step (Grinder Study)

Table 7.3 summarizes the differences among the various grinders with respect to

grinding conditions, screen size, grinding time, sample preparation time,

cross-conta-mination, and multiple grinding capabilities The Monsanto-built and designed

Mega-grinder provides significant advantages over the commercially available

grinders because it is the only grinder capable of grinding multiple samples (from

1–96 samples) with no cross-contamination The Mega-grinder is the best grinder for

small size samples; there is minimal loss during the grinding process because the

sample 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 better

grinders for bulk samples because the grinding and sample preparation time are

significantly lower with larger sample sizes The grinding time was significantly

increased with high-oil and high-moisture samples for the UDY, Cyclotec, and

Brinkmann 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 oil

extracted from seven soy samples ground with various grinders using the Soxtec

extraction procedure The grand mean of crude fat extracted from different samples

varied from 11.0 to 21.4% The Cemotec grinder provided the lowest crude fat

yields 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 other

grinders varied from 15.7 to 20.9% Maximum and minimum oil yields were

TABLE 7.3

Comparison of Operating Performance of Different Grinders

Grinding preparation Cross- grinding Grinder type Screen size time timea contamination possibility

(3 × 2 × 5) (3 × 3 × 10)

Retsch-mill

aSample preparation time is the average time required to grind 150 g of each sample.