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5-1993
Formation of Polycyclic Aromatic Hydrocarbons On the Surfaces of Ultra-High Temperature Treated Meat
Michelle T. Foote Utah State University
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Recommended Citation Foote, Michelle T., "Formation of Polycyclic Aromatic Hydrocarbons On the Surfaces of Ultra-High Temperature Treated Meat" (1993). All Graduate Theses and Dissertations. 5397. https://digitalcommons.usu.edu/etd/5397
This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Copyright © Michelle T. Foote All Rights Reserved FORMATION OF POLYCYCLIC AROMATIC HYDROCARBONS
ON THE SURFACES OF ULTRA-HIGH TEMPERATURE
TREATED MEAT
By
Michelle T. Foote
A thesis submitted in partial fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Nutrition and Food Sciences
Approved:
UTAH STATE UNIVERSITY Logan, Utah
1993 ii ACKNOWLEDGMENTS
I thank the Center of Excellence for funding this project. My special
thanks is given to Dr. Von T. Mendenhall for making it possible for me to
complete this degree and for his endless counsel and advice. I also thank my
other committee members, Dr. Charles Carpenter, Dr. Donald Sission, and Dr.
Jeff Broadbent, for their assistance in completing this research. A special thanks
is extended to Dr. Charlotte Brennand for her assistance in using her equipment.
I thank my parents for their inspiration and unfailing support. Most of all I thank my husband, David, for his love, patience, encouragement, and support in helping me reach this point.
Michelle T. Foote iii CONTENTS
ACKNOWLEDGMENTS ...... ii
LIST OF TABLES ...... iv
LIST OF FIGURES ...... v
ABSTRACT ...... vi
INTRODUCTION ...... 1
LITERATURE REVIEW ...... 3
MATERIALS AND METHODS ...... 8
Sample Preparation ...... 8 PAH Extraction ...... 8 PAH Purification ...... 9 PAH Analysis ...... 1O Total Solid Determination ...... 10 Statistical Analysis ...... 1O
Sample Size Estimation ...... 10 Analysis of Variance ...... 11
RESULTS AND DISCUSSION ...... 12
Benzo[a]pyrene ...... 15 Benzofluoranthenes ...... 15 Benz[a]anthracene ...... 17 Standard Recovery ...... 17
CONCLUSIONS ...... 20
REFERENCES ...... 21
APPENDIX ...... 24 iv LIST OF TABLES
Table Page
1 Comparison of adjusted mean values of benzo[a]pyrene (B[a]P) found in treated steak samples using LSD ...... 16
2 Comparison of adjusted mean values of benzofluoranthenes (B[b]F+B[k]F) found in treated steak samples using LSD ...... 18
3 Comparison of adjusted mean values of benz[a]anthracene (B[a]A) found in treated steak samples using LSD ...... 19
4 Comparison of mean values of benzo[a]pyrene, benzofluoranthenes, and benz[a]anthracene found in treated steak samples ...... 25
5 Analysis of variance table used to determine treatment effects on the production of benzo[a]pyrene ...... 26
6 Analysis of variance table used to determine treatment effects on the production of the sum of benzo[b]fluoranthene and benzo[k]fluoranthene ...... 27
7 Analysis of variance table used to determine treatment effects on the production of benzo[a]anthracene ...... 28
8 Standard recoveries of benzo[a]pyrene, benzofluoranthenes, and benzo[a]anthracene ...... 29 v LIST OF FIGURES
Figure Page
1 Structure of parent hydrocarbon benz[a]anthracene 3
2 Example of initial calibration of the HPLC instrument for collection of PAH fraction ...... 13
3 Structures of polycyclic aromatic hydrocarbons quantified in this study ...... 14
4 Example of gas chromatograph of a PAH extract from a UHT-treated steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard ...... 30
5 Example of gas chromatograph of a PAH extract from a UHT/Marks-treated steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard ...... 31
6 Example of gas chromatograph of a PAH extract from a UHT/Marks/Micro-treated steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard ...... 32
7 Example of gas chromatograph of a PAH extract from a charcoal grilled steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard ...... 33 vi ABSTRACT
Formation of Polycyclic Aromatic Hydrocarbons on
the Surfaces of Ultra-High Temperature-Treated Meat
by
Michelle T. Foote, Master of Science
Utah State University, 1993
Major Professor: Dr. Von T. Mendenhall Department: Nutrition and Food Sciences
The effect of ultra-high temperature (UHT) on production of polycyclic aromatic hydrocarbons (PAHs) on the surface of beef steaks was determined.
Beef steaks were treated with five treatments, raw, UHT, UHT/grill marks,
UHT/grill marks/microwave, and charcoal grilled. Four PAHs, benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, and benzo[k]fluoranthene, were quantified. High performance liquid chromatography (HPLC) and gas chromatography (GC) were used to purify and analyze the PAH extracts, respectively. Levels of PAH found on charcoal-grilled steaks were higher than those observed in the literature. A balanced incomplete block design was used to analyze the data. There were no significant differences among the treatments in the production of the benzofluoranthenes. There were significant increases in production of benzo[a]pyrene and benz[a]anthracene when grill marks were applied to the UHT steak. Microwaving significantly decreased the levels of benzo[a]pyrene and benz[a]anthracene. The production of these PAHs on vii UHT/grill mark/microwave steak did not differ significantly from the charcoal grilled steak in the levels of PAH quantified.
(40 pages) INTRODUCTION
Ultra-high temperature (UHT) pasteurization of the surfaces of meat is a
new concept in processing technology. The objectives in developing this
processing method were first, to destroy vegetative cells of pathogenic bacteria;
second, to extend the shelf-life of the product; and third, to create a
microwaveable meat product with a more desirable appearance and flavor than
traditional microwave meat.
In the UHT process, meat surfaces are exposed to an air temperature of
11 oo0 c in an electric furnace for 20 seconds. The temperature and length of exposure were selected based on the appearance of the treated steak. This process does not cook the meat completely. It only denatures the proteins one half to one millimeter into the meat surface. After high temperature treatment the meat is seared with a grill to create marks giving the appearance of a charcoal broiled steak. The steak is cooled to 4.4°C, vacuum packaged, and then stored at 4.4°C.
One of the concerns with UHT pasteurization is the formation of polycyclic aromatic hydrocarbons (PAHs) on the surface of the meat due to the extremely high temperature of the treatment and the burning of the meat to create the grill marks. PAHs are potent carcinogenic compounds that are widely distributed in the human environment (Suess, 1976). PAHs form when organic matter comes in contact with a high temperature heat source. PAHs may form on UHT pasteurized meat in two ways: when fat comes in contact with the heat source during UHT treatment, or when the grill marks are made (Larsson et al., 1983;
Hotchkiss and Parker, 1990). The levels of PAH found on meat surfaces depend on many factors, such as the fat content of the meat, distance from the heat source, and type of fuel used in cooking. 2 The presence of polycyclic aromatic hydrocarbons is an important concern
in UHT pasteurization of the surface of the meat. The extreme temperature used
in the UHT process and the searing of the grill marks into the meat may
contribute to PAH formation. The purposes of this study were to measure the
effect of the UHT process on the formation of polycyclic aromatic hydrocarbons on beef steaks and to compare the levels found on UHT-treated steaks to PAH
levels found on charcoal-broiled steaks. 3 LITERATURE REVIEW
Polycyclic aromatic hydrocarbon or polynuclear hydrocarbon (PAH)
formation on grilled meat has been studied extensively (Fretheim, 1983; Larsson,
1986; Hotchkiss and Parker, 1990; Lijinsky and Ross, 1967; Lijinsky and Shubik,
1964). At least 100 structurally distinct PAHs have been found in food and
environmental samples. Benz(a)anthracene (Fig. 1) is the basis of nearly all
other PAH (Hotchkiss and Parker, 1990). PAHs are formed from the incomplete
combustion of organic matter; they are found in the residue of tar, soot, tobacco smoke, petroleum, and combustion effluent. PAHs are reported in foods that are smoked, treated with smoke condensate, and cooked or grilled. Although environmental contamination of air, water, and soil is also a source of PAH in foods, smoking and grilling over an open flame generally determine the PAH content of foods (Dennis et al., 1984; Hotchkiss and Parker, 1990; Lawrence and
Weber, 1984) Large organic molecules, such as carbohydrates and fat, crack into smaller free radicals at high temperatures. These radicals recombine to form more stable polycyclic hydrocarbons (Larsson, 1986; Hotchkiss and Parker,
1990)
Fig. 1 - Structure of parent hydrocarbon benz[a]anthracene 4 There are three routes by which PAHs enter meats and other foods. They
may be deposited on the food surface due to incomplete combustion of fuel used
as the heat source; they may form directly on the food due to very high
temperatures; or they may be transferred to the food surface as melted fat drips
onto the heat source and is pyrolyzed to form PAHs (Larsson, 1986; Hotchkiss
and Parker, 1990). Since PAHs generally do not diffuse into the food, meat with
a large surface-to-weight ratio has higher levels of PAH than meat with a small
surface-to-weight ratio (Larsson, 1986).
Many factors contribute to the level of PAHs formed on cooked meat,
including fat content of the meat, type of fuel, closeness to the heat source, and cooking time. As a result, levels of PAHs on meat reported in the literature vary greatly. Lijinsky and Shubik (1964) reported values of 8 µg benzo[a]pyrene/kg meat and 4.5 µg benz[a]anthracene/kg meat on charcoal-broiled steaks.
Panalaks (1976) found similar results of 0-2 µg benzo[a]pyrene/kg meat and 1-8
µg benz[a]anthracene/kgmeat; however, much higher values were found by
Lijinsky and Ross (1967). They found 11.1 µg benzo[a]pyrene/kg meat and 10.3
µg benz[a]anthracene/kg meat on sirloin steaks and 50.4 µg benzo[a]pyrene/kg meat and 31.0 µg benz[a]anthracene/kg meat on T-bone steaks (Lijinsky and
Ross, 1967). Although there are no PAH regulations currently in place for food products in the United States, there is a 1 µg benzo[a]pyrene/kg meat limit in smoked meat products in the Federal Republic of Germany (Larsson et al. 1983).
Humans are exposed to PAH in car exhaust, tobacco smoke, industrial pollution and environmental contamination in addition to PAH found in smoked and grilled foods. Although PAHs are known to be carcinogenic, they seem to be less powerful by inhalation or ingestion than by skin contact (Lijinsky, 1991 ). 5 Very few studies have been done on ingestion of PAH. Chu and Malmgren
(1965) administered 0.05% benzo[a]pyrene mixed with food 4 days per week to
Syrian hamsters. Of 13 animals treated, 61% had invasive cancer of the forestomach and 30% had either intestinal cancer or papillomas in the esophagus. Three animals died at the age of 6.5 months. Huggins and Yang
(1962) found that a single large dose (100 mg) of benzo[a]pyrene induced mammary cancer in 89% of 7-week-old female Sprague-Dawley rats. However, a single dose (200 mg) of benz[a]anthracene did not induce mammary cancer in the same strain of rats.
It has been determined that the primary source of PAH in grilled meat is the pyrolysis of fat. In 1964 Lijinsky and Shubik stated, "The most likely source of the polynuclear hydrocarbons is the melted fat which drips on the hot coals and is pyrolysed at the prevailing high temperature. The polynuclear hydrocarbons in the smoke are then deposited on the meat as the smoke rises" (p. 54). They found no nitrogen-containing compounds; therefore, pyrolysis of compounds that contain only carbon, hydrogen, and oxygen were the source of PAH (Lijinsky and
Shubik, 1964). Several studies have examined the effect of fat content on the formation of benzo[a]pyrene. By adjusting the fat content of ground meat samples before grilling, Doremire et al. (1979) found lesser amounts of benzo(a)pyrene in grilled samples with a low fat content than in high fat samples.
Lijinsky and Ross (1967) also found less benzo(a)pyrene in grilled lean hamburger than in hamburger with higher fat. The absence of benzo(a)pyrene on meat cooked on a grill with a no-drip pan that prevented contact between the fat and the flame also showed that PAHs are derived from pyrolyzed fat.
Other determinants of PAH formation are the type of fuel and the closeness of the meat to the fuel source. It is well known that grilling of food over 6 an open flame or hot coals can lead to increased levels of PAH. Extremely high
levels were found in meat cooked in the flames of an open log or cone fire (54.2
µg Benzo[a]pyrene/kg meat). Charcoal, because it is prepyrolyzed, produced a
much cleaner smoke (Larsson et al., 1983) which contained lower PAH levels
(1µg Benzo[a]pyrene/kg meat). When meat is cooked by electrical broiling or
frying, small or undetectable amounts of PAH are found (Larsson, 1986). Lijinsky
and Ross (1967) determined that a thick T-bone steak cooked close to the coals
for an extended length of time resulted in a higher concentration of PAH (50 µg
Benzo[a]pyrene/kg meat) than T-bone steaks cooked in a gas flame (4.4 µg
Benzo[a]pyrene/kg meat).
There are a variety of methods for quantification of PAHs. Most, however, are tedious and very lengthy. Paper and thin-layer chromatography were the first methods used. These methods included initial extraction, solvent-solvent partitioning, lengthy chromatography, and ultraviolet spectroscopy and spectrophotofluorometry quantification. Also, because there are so many different PAHs, it may be necessary to use several different thin-layer systems to separate all PAH (White and Howard, 1967). Gas, capillary, and high performance liquid chromatography (HPLC) have greatly advanced quantification of PAH (Dennis et al., 1984). Performance of the packed column for gas chromatography (GC) has fallen short of the improvements in resolution and decreased analysis time of the capillary column for gas chromatography (Lee and
Wright, 1980).
The use of HPLC for PAH separation and quantification is gaining favor because it can be used to perform crucial isomer separations that may be difficult by capillary GC. Peak capacity of a capillary GC column, however, is superior to a HPLC column, and capillary GC displays a better resolving power in terms of 7 plate number than HPLC. Therefore, a greater number of compounds can be separated by capillary GC than by HPLC. In a comparison study Dennis et al.
(1984) found that the means and standard deviations of the analysis from HPLC and GC were in agreement, and that 25 out of 35 pairs of analyses were not significantly different within 95% confidence limits . 8 MATERIALS AND METHODS
Sample Preparation
Raw and treated steaks 20 mm thick were cut from USDA choice beef top
loin boneless with fat limitations of 6 mm. Charcoal-broiled samples were cooked
to a well-done state (71.1°C internal) 15 cm above the coals. UHT samples were
treated in a 11 oo°C electric oven for 20 seconds. UHT/grill-mark samples were
UHT treated as described above and grill marks were placed on them using a
flame-heated stainless steel grill. UHT/grill-marks/micro samples were treated as
UHT/grill-marks samples and microwaved on high for 1 minute 20 seconds.
Treated samples were cooled to 4.4°C, packaged, and refrigerated at 4.4°C (not
more than 3 days), and analyzed. Six steaks were given each treatment. There
was one repetition on each steak and one measurement on each repetition.
PAH Extraction
PAHs were extracted by the method of Macleod et al. (1985). Each meat
sample (100 g) was homogenized in an Osterizer food chopper. Three grams of
sample were placed in a 200 ml centrifuge bottle with 35 ml methylene chloride
(CH2Cl2), 25 g sodium sulfate (Na2S04), and 100 µI aromatic hydrocarbon
internal standard (Perkin Elmer, Norwalk, CT). The sample was macerated for 1
min using a Polytron homogenizer (Brinkman Instruments, Westbury, NY). The
probe was washed with CH2Cl2 and the washings were collected in the
centrifuge bottle. The sample was centrifuged at QOOO rpm for 1O min. The extract was decanted into a labeled flask. An additional 35 ml CH2C12 were added to the original 3 g sample. The sample was macerated, centrifuged, and the extract decanted into the flask as before. The Na 2 SO~sample mass was 9 washed with 10 ml CH2Cl2 and swirled to mix, and the extract was decanted into
the flask.
PAH Purification
The tissue extract was filtered through a CH2Cl2 washed silica-alumina
column followed by 25 ml CH2Cl2 to remove some of the lipids. The extract was
collected in another labeled flask containing glass beads. A snyder column was
attached to the flask and the extract concentrated in a 60°C water bath to 1O to
15 m I. The concentrated extract and the glass beads were transferred to a
concentrator tube. The flask was washed three times with 3-4 ml CH2Cl2, and
the washings were added to the tube. The extract was concentrated on a tube
heater to"~ 0.9 ml,< 1.0 ml. HPLC recovery standards were added. HPLC clean-up was performed using two model 11 OA solvent delivery systems, a 21 OA
injector, a model 420 system controller, a model 164 variable wavelength detector, and a model 427 integrator from Beckman Instruments, Inc. (Palo Alto,
CA). A stainless steel column 250 x 22.5 mm 1.0. containing Phenogel 100-A size exclusion packing (Phenomenex, Rancho Palos Verdes, CA) was used with pre-column, 75 x 22.5 mm 1.0., with identical packing. An inline filter (2 µm,
Rheodyne, Model 7302) was placed before the pre-column. The flow program was isocratic, 100% methylene chloride at a flow-rate of 5 ml/min for 20 min at room temperature; 500 µI of sample extract were injected onto the HPLC column.
A fraction was coliected according to previous calibration. Calibration of the
HPLC was performed using biphenyl and perylene to adjust the times for collecting PAH fractions. The extract was exchanged into hexane as the volume was reduced to approximately 1 ml. Gas chromatography standards were added to the extract and the extract was placed in the freezer (-18°C) until analysis
(Krahn et al., 1988). 10 PAH Analysis
The frozen tissue extracts were thawed and analyzed by capillary gas
chromatography. A 30 m x 0.25 mm l.D. fused silica capillary column coated with
0.50 µm of 95% dimethyl-5% diphenylpolysiloxane (Restek, Bellefonte, PA) was
used in a Varian Aerograph Model 2740-30 (Palo Alto, CA) capillary gas
chromatograph with flame ionization detection (GC-FID) and a Hewlett Packard
3390A Integrator (Avondale, PA). A sample size of 1 µI was injected onto the
capillary column. The GC-FID was programmed from 1OOOC to 3000C
(40C/min.). The total run time was 75 min (Krahn et al., 1988). Concentrations
of PAH in the samples were determined using the area of an internal standard.
Standard blank samples containing no tissue extracts were prepared and
analyzed to determine percent recovery of PAH.
Total -Solid Determination
Total solid determinations of each sample were performed in duplicate.
Approximately 3 g of sample was placed in an aluminum dish. The sample was dried to a constant weight (ca. 5 h) at 95-1 oo0 c under ::;100 mm Hg (AOAC, 1990).
Statistical Analysis
Sample Size Estimation
The sample size was defined as the number of steaks that would be treated with each of five treatments. Coefficients of variance of 2.8% and 5.8% were given by Grimmer and Bohnke (1975). The sample size (n) was then calculated by the following equation (Ott, 1988): 11 n = z2s2 z =standard normal variable at a=0.05 E2 s = 5.8 =Coefficient of Variation E = 5% = amount error allowed
n = (1.962) {5 .82) = 5.17 = 6.0 (52)
Therefore, six steaks were subjected to each of five treatments.
Analysis of Variance
A balanced incomplete block design was used to compare the five
treatments. All possible comparisons were made to show treatment effects on
the formation of PAH. A comparison of the raw treatment to all other treatments
showed the effect of heating. Comparison of charcoal-grilled sample to all UHT treated samples showed the effect of UHT cooking on the formation of PAH.
Comparison of UHT-treated samples to UHT/grill-mark samples showed the effect of applying the grill marks on the formation of PAH . Comparison of
UHT/grill-mark samples to UHT/grill-mark/micro samples showed the effect of microwave cooking on the formation of PAH . 12 RESULTS AND DISCUSSION
The HPLC clean-up procedure proved sufficient to separate the polycyclic
aromatic hydrocarbons from interfering compounds. The column was initially
calibrated using biphenyl, the earliest eluting PAH, and perylene, the latest
eluting PAH. A collection time from about 14.05 to 18.37 min was established
(Fig. 2). Standards were chromatographed each day to ensure collection of the
entire PAH fraction of the sample. Four polycyclic aromatic hydrocarbons were quantified in this experiment (Fig. 3).
The different volitilities and affinities to the column allow for separation and quantitative measurement of Benzo[a]pyrene, Benz[a]anthracene, and the
Benzofluoranthenes. PAH standards (Sigma Chemical, St. Louis, MO) were used to identify individual hydrocarbons. The separation of the compounds was increased when the gas chromatograph program began at a higher initial temperature. Levels of the four PAHs found on charcoal-grilled steaks were much higher than those found in the literature. This may be due to another compound in the meat eluting at the same retention time from the GC column.
There was a large amount of variability among the levels of PAH detected on samples within the same treatment. This variability may be due to the small sample size and variability in the instrumentation. Another source of variation was the preparation of the steaks. Each steak was prepared independently, and there could be fluctuations in the temperatures of the UHT oven and charcoal. Fig. 2 - Example of initial calibration of the HPLC instrument for collection of PAH fraction. t1 =Retention time of Biphenyl, t2=Earliest detection of Biphenyl, t3=t2+0.2 min, t4=Retention time of Perylene, ts=Latest detection of Perylene, te=ts+0.5 min, t1=PAH collection time. 13
.£ E
Q) c -· LO Q) ...... ~ :::r·n Q) ------a_ T ------~, __ : J__
...... Q) E 0t"c:T ...... L:'. -rfl6 "T E C\J c ci ...... 0 c ...... Q) Q) a: A B
c D
Fig. 3 - Structures of polycyclic aromatic hydrocarbons quantified in this study. A--Benzo[a]pyrene, 8- Benz[ a]anth racene, C--Benzo[k]f luoranthene, D--Benzo[b ]fluoranthene
...... ~ 15 For analysis of variance an incomplete block design was used. An
apprmimate F value was computed; however, due to the nature of the
incomplete block design, the F test may not show significance that could exist.
Therebre, Fisher's least significant difference (LSD) test at a= .05 was applied
to the idjusted means of each of the three compounds. Treatment means were
adjushd for the day effect (Cochran and Cox, 1957). This resulted in some
negati1e values.
Benzc[a]pyrene
The LSD test showed there were significant differences among the
treatrrents. The production of benzo[a]pyrene on the UHT treatment was not
significantly different from any other treatment (Table 1 ). Therefore, searing of grill mirks into the meat did not cause a significant increase in the benzoa]pyrene found. UHT/grill-mark treatment was significantly higher than
raw, c1arcoal-grilled, and UHT/grill-mark/micro treatments. This was due to the
combimtion of the extreme high temperature of UHT treatment and the grill mark
process. UHT/grill-marks/micro treatment did not differ significantly from the raw
or chcrcoal-grilled treatment (Table 1 ). The microwave seemed to destroy some
of the benzo[a]pyrene formed by the UHT and grill mark processes.
Benzofl uoranthenes
Benzo[b]fluoranthene and benzo[k]fluoranthene could not be completely separated by the GC column. Therefore, the peaks were summed, and the 16
TABLE 1 - Comparison of adjusted mean values of benzo[a]pyrene (B[a]P) found in treated steak samples using LSD
Treatment µg B[a]P I kg meat
Raw
Grill
UHT 499 ab
UHT/grill-marks 1281 a
UHT/grill-marks/micro a-b Means not sharing a common superscript are significantly different (p ~ 0.05) 17 results were reported as one. An LSD test indicated there were no significant
differences found among the treatments in the production of the
benzofluoranthenes {Table 2).
Benz[A]Anthracene
The LSD test showed there were significant differences among some
treatments. UHT/grill-marks treatment differed significantly from all treatments.
UHT/grill-marks treatment was significantly higher than the UHT treatment {Table
3). The grill mark process appeared to increase the amount of
benz[a]anthracene found. This may be due to the burning of the fat when the
meat is seared with the hot grill. The UHT/grill-marks/micro treatment was significantly lower than the UHT/grill-marks treatment. Again, the microwave destroyed some of the benz[a]anthracene that was formed by the UHT and grill mark processes. There was no significant difference among the other treatments in the production of benz[a]anthracene.
Standard Recovery
Standard samples containing no tissue were analyzed to determine percent recoveries of benzo[a]pyrene, benz[a]anthracene, and the benzofluoranthenes. The recovery of benzo[a]pyrene ranged from 31% to 106% with a mean of 74.5%. Recoveries of benz[a]anthracene and the benzofluoranthenes ranged from 3% to 130% and 70% to 112%, respectively.
The means were 92.0% and 88.8%, respectively. This reflects the variability of the entire procedure. 18
TABLE 2 - Comparison of adjusted mean values of benzofluoranthenes (B[b]F+B[k]F) found in treated steak samples using LSD
Treatment µg B[b]F+B[k]F I kg meat
Raw 1245 a
Grill 1390 a
UHT 1340 a
UHT/Marks 2288 a
UHT/Marks/Micro 1015 a a-b Means not sharing a common superscript are significantly different (p ~ 0.05) 19
TABLE 3 - Comparison of adjusted mean values of benz[a]anthracene (B[a]A) found in treated steak samples using LSD
Treatment µg B[a]A I kg meat
Raw
Grill
UHT
UHT/Marks
UHT/Marks/Micro 80 b a-b Means not sharing a common superscript are significantly different (p ~ 0.05) 20 CONCLUSIONS
The combination of UHT and grill marks and the grill marks alone
increased the amount of benzo[a]pyrene and benz[a]anthracene, respectively. At
the high temperature of the UHT treatment, melted fat drips onto the pedestal of
the oven and is pyrolyzed. PAHs are deposited on the meat when the smoke
rises. The grill marks are made with a red-hot stainless steel iron. This burns the
fat and forms PAH directly on the meat. However, microwave cooking seemed to
decrease the amounts of these polycyclic aromatic hydrocarbons. Additional
work needs to be performed to determine if microwave radiation could be
breaking down or altering the structure of the PAH, or if the PAHs are being volatilized at the temperature achieved by the microwave during cooking. The
UHT-treated steak (high temperature treatment, grill marks, and microwave) did not have an increase in polycyclic aromatic hydrocarbons over that of the charcoal-grilled steak. 21 REFERENCES
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APPENDIX 25
TABLE 4 - Comparison of mean values of benzo[a]pyrene (B[a]P), benzofluoranthenes (B[b]F+B[k]F), and benz[a]anthracene (B[a]A) found in treated steak samples
Treatment B[a]P (µg/kg) B[b]F+B[k]F (µg/kg) B[a]A (µg/kg)
Raw 89 1348 3
Grill 45 1203 21
UHT 603 1733 57
UHT/Marks 1260 2723 626
UHT/Marks/Micro 89 270 10 26
TABLE 5 - Analysis of variance table used to determine treatment effects on the production of benzo[a]pyrene.
Mean Source df Squares
Treatment (Unadj) 4 1649222.49
Day {adj) 9 690129.59
Error 16 453467.18
Treatment (adj)* 4 1426985.78
* Adjusted according to Cochran and Cox, 1957 27
Table 6 - Analysis of variance table used to determine treatment effects on the production of the sum of benzo[b]fluoranthene and benzo[k]fluoranthene
Mean Source df Squares
Treatment (unadj) 4 4747521.77
Day (adj) 9 7140960.72
Error 16 1572327.14
Treatment (adj)* 4 1186233.05
* Adjusted according to Cochran and Cox, 1957 28
TABLE 7 - Analysis of variance table used to determine treatment effects on the production of benz[a]anthracene
Mean Source df Squares
Treatment (unadj) 4 438921.76
Day (adj) 9 112602.92
Error 16 127809.90
Treatment (adj)* 4 387759.37
* Adjusted according to Cochran and Cox, 1957 29
TABLE 8 - Standard recoveries of benzo[a]pyrene, benzofluoranthenes, and benz[ a]anth racene
PAH 1 (%) 2 (%) 3 (%) 4 (%) Mean(%)
B[a]P 95 31 66 106 75
BF 81 70 92 112 89
B[a]A 130 3 129 106 92 4
·:i:· --· o:· •::Cl ~'I -.::- · 1:r·. ~r:- er;. ~ :-. .. •:C ·~""'' 1..1·;.. '-.C.• l;")_J r""':• t,;r., (,"l..J ,...., ·=ii:; •:"U ~u cu c r-':• r-, .,.,..,., C) - ""'Lr:• -u~ '·.t:-0 en i ~ 2 ... ~ 0 I ..r.;.. ~ (..) I ~?" "·'"'' ~.; - 1.•;= <1> - 00•::Ii) -<1> 0 ...... 3 ,...~ "'O .. .., <1> ir,. ~ c: 0 <1> 1 E ,..., ...,. ~ ~ LL ,_,..::•
'~
Retention Time
Fig. 4 - Example of gas chromatograph of a PAH extract from a UHT-treated steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard. Numbers indicate where peak is or would be.
w 0 ~· - ...,- 1._ci r .. ~, ...,. "tr':• •:"W •::r"> = ·~ r .. ~ nrs:.. 0'"1 c ..- .. ,..-, 0.J 1 co """" ("U 0.J c:: ; :J':f:1 !J£D 1"'"-.. u··0i:: •:7·, ~~ · 1:'1'.:i .-~~~~- ~ u ""' •..c ... •:.'r"l ;:'.r'\ O> 1::tJ:'I ,..._,("I..! u-... en uO'-,. IJ'":• LI '") u-) 4 ~ I 0 2 ("U= (.) <3:J....c: ~ ..i::· •'."U - '.J.") •-. 1.":• CD ...... Q) ...,, u-:. 0- · ~. 3 ~ ""''!!~~~ Q) l 1 1.(J: c::~ 0 I - I Q) I ~j ::: .u
/------Retention Time
Fig. 5 - Example of gas chromatograph of a PAH extract from a UHT/Marks-treated steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard. Numbers indicate where peak is or would be.
w...... 4
0 , _r .. -:• r.:?::• r~ .. ...,.. '"-' '-.C• '·.Cl •. .c• •..() ct1 ·:r·1 °"'~ · =r...-: 1 ""' •;"U .,..._, 1;"'t,J c cu u:rc r-.. ::-w t;'"i../ u··:ict;• ...:.r.1 - •...:> O> en i.... 0 -(.) CD Ci) 0- "O CD 2 N ·("U- c: co 0 I":....,.• !.(") CD -•:O ~ E iJ"") ct1 u.. 1
Hetention Time
Fig. 6 - Example of gas chromatograph of a PAH extract from a UHT/Marks/Micro-treated steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene, (4) Internal Standard. Numbers indicate where peak is or would be.
w I\) •'."1.J ...... IC1LI ,, , .S.• _..., co ..-:i:. ""-' ,...., ""i:t"' l:'\.,i 4 c !""°'? ,...... o:u i.."\J r..-:. 0) u··:i t.r:o ,...., 1:c CJ) ,...,.,, "- o-. '° 0 ,,....,~-=· u··:. (.) -Q) ,...-, ..., .. Q) '"" ...,. 0- "'O Q) 2 N ;--.. .. c ..,.. :""'":• LJ-:• - 0 .::S:• 1 'D 3 Q) E g~ co l.J'. u..
Retention Time
Fig. 7 - Example of gas chromatograph of a PAH extract from a charcoal grilled steak. (1) Benz[a]anthracene, (2) Benzo[b]fluoranthene+Benzo[k]fluoranthene, (3) Benzo[a]pyrene (4) Internal Standard. Numbers indicate where peak is or would be.
c.u c.u