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Detection of Foreign Fat Adulteration of Milk Fat by Near Infrared Spectroscopic Method
Detection of Foreign Fat Adulteration of Milk Fat by Near Infrared Spectroscopic Method TETSUO SATO and SUMIO KAWANO Food Analysis and Nutrition Division MUTSUO IWAMOTO Food engineering Division National Food Research Institute 2-1-2 Kannondal Tsukuba-c1ty IbarakI-ken Japan 305 ABSTRACT
INTRODUCTION
A near infrared spectroscopic method for the detection of foreign fat adulteration in dairy products was examined using the extracted fats from samples of butter and margarine mixtures and from milk. and soymilk mixtures. Butter and margarine mixtures were also analyzed. The near infrared absorptions at 1164, 1660, 2144, and 2176 om are due to cis unsaturation of fatty acid moieties, the ratio of which is intrinsic to each oil. We suggest that the near infrared spectra around these wavelengths could be used as a simple index for fatty acid profiles in oil. As for the original near infrared spectra, the difference of spectral data at log 1164, 1660,2144, or 2176 om from 2124 om correlated very well with the mixing ratios. As for their second derivative spectra, spectral data at log 1164, 1660, 2144, or 2176 om correlated with the mixing ratios very well, and further, the correlation proved to be better by employing the difference between these values and those at 2124 om. The application of near infrared method to dairy products indicated that the adulteration of the fat with as little as 3% foreign fat could be detected simply, rapidly, and "nondestructively" for butter and margarine mixtures. (Key words: fat adulteration, near infrared, spectroscopy)
In order to reduce production cost, less expensive vegetable fats are used as substitutes for milk. fat in dairy products. Such products should be properly labeled for consumers, and it is important to develop a potential method for detection of vegetable fat to ensure correct labeling. Many methods have been developed to detect fat adulteration, and some of them have been officially recognized (3, 7, 8, 11, 17). However, they require time-consuming and sophisticated analytical procedures. This paper describes feasibility of near infrared (NIR) method for the rapid and simple detection of foreign fat adulteration in dairy products.
Received February 28, 1990. Accepted June 18, 1990. 1990 J Dairy Sci 73:3408-3413
MATERIALS AND METHODS Preparation 01 Samples
Commercially available margarine and butter were mixed in ratios of 0, 2.5, 5.0, 7.5, 10.0, 20.0, 25.0, 50.0, 100.0% (wt/wt) and milk: and soymilk. mixture ratios were 0, 2.5, 5.0, 7.7, 10.1, 24.7, 49.3, 100.0% (wt/wt). From each mixture, fat was extracted by Rose-Gottlieb method (6) and used for the following analyses. Moisture and fat contents in butter and margarine were measured by modified Kohman's method (13). Moisture and fat were 16.80 and 80.04% in butter 15.04 and 81.39% in margarine, respectively. The total solids and fat contents in milk. and soymilk. were measured by the standard gravimetric method (14) and RoseGottlieb method. They were 12.01 and 3.52% in milk, and 11.36 and 4.30% in soymilk., respectively. 3408
3409
DETEcrION OF FAT ADULTERATION
Analysis of Samples by Near Infrared Method
An InfraAlyzer 500 (Bran & Luebbe Co., West Gennany) was used to measure NIR transflectance in the wavelengths from 1100 to 2500 run. The extracted fat was warmed at 35·C to become liquid, cooled to room temperature, applied on a sample holder, a British cup (Bran & Luebbe), covered with a slide glass (microslide glass S2226, Matsunami Glass Ind., Ltd., Japan), and was measured. In case of butter and margarine mixtures, NIR measurement was conducted by using an Italian cup (Bran & Luebbe) as described in a previous paper (16). The conditions to obtain second derivative spectra were as follows: 4 om between output points, 4 om in moving average, 12 nm/derivative segments, and 12 om between derivative segments.
2.50 2.00
~
0::
-
'--
1. 50
~
'Oll
.Q
1.00
Gas Chromatographic Analysis
The extracted fats were analyzed with gas chromatography (GC) after methyl-esterification according to a conventional method (1). The GC conditions for determining the fatty acid methyl esters were as follows: 2 m X 3 mm Ld. Pyrex column packed with 10% Sitar 7CP coated on 80/100 mesh chromosorb W (AW, DMCS); 250·C injection temperature; 30 mlImin carrier gas flow; column temperature programmed from 60 to 200·C by 3·C/min, then holding for 14 min at 200·C. Each sample was analyzed twice, the average of duplicate measurements was calculated, and samples were normalized to 90% for major fatty acid components (9). RESULTS AND DISCUSSION Near Infrared Spectra and Fatty Acid Profiles
Figure I shows NIR spectra of butter and margarine (Figure lA) and their second derivative spectra (Figure IB and C). As shown in Figure lA, butter and margarine show almost similar NIR spectral patterns except those at 1164, 2144, and 2176 om, at which margarine spectra have sharper peaks than butter. Holman et al. (4) measured NIR spectra of carbon tetrachloride solution of various fatty
-
~-
.30 t200 1400 1600 1800 2000 2200 2400 nm wavelength ().) Figure 1. Original near infrared spectra of butter and lIUU'glIl'ine (A) and second derivative ones of butter (8) and lIUU'glIl'ine (C). Arrows indicate 1164, 1660. 2144. and 2176 nm, respectively. A dot indicates 2124 nm.
acids and their related compounds. They concluded that C-H bond, which is associated with cis double bonds, had a fundamental absorption at 3300 om, moderation combination absorptions at 2190 and 2150 nm, a weak first overtone at 1680 om, and a second overtone at 1180 om. Experimental conditions of Holman et al. (4) were different from ours: solution of acids versus extracted fat itself, pure substances versus mixtures of triglycerides (crude fat), and so on. However, our wavelengths (1164, 1660, 2144,2176 om) might be from C-H vibrations associated with cis double bonds due to cis Journal of Dairy Science Vol. 73.
No. 12, 1990
3410
SATO BT AL.
Wlsaturated fatty acid moieties, as assigned by Law et al. (12). Near infrared spectrum gives the infonnation of cis isomer, whereas infrared (IR) spectrum gives that of transisomer. Absorption intensity in log (11R) for the spectra of extracted fats was rather weak, and in addition, peaks due to water, i.e., peaks at 1450 and 1940 om, WeIe not observed. However, general trend of the spectra showed similarity to Figure 1. Conversely, as for their second derivative spectra, as in the original, the spectra of margarine were sharper than those of butter arowd these wavelengths, but in the opposite direction. According to Osborne et al. (15), second derivative spectra have a trough corresponding to each peak. in the original spectra. Further, with its second derivative spectra, a linear background can be cancelled, and peaks discriminated that overlap substantially in the original NIR spectrum. In addition, a new trough at 1660 and 2124 om appeared in the second derivative spectra. In the butter spectra, the trough at 2144 om was a little more shallow than that at 2124 om. However, in the margarine spectra, the trough at 2144 om was much deeper than at 2124 om. According to the result of measuring an NIR spectrum of 11-hydroxy-lD-eicosanone, Holman et aI. (4) reported that neighboring ~H and ~ groups showed absorptions in a Wlique couplet at 2050 and 2120 om, respectively. H the absorption at 2124 om might be due to the carbonyl groups close to the polar groups, this absorption might be contributed by the ester carbonyl groups of triglycerides. We
will adopt this wavelength, 2124 om, as a comparative standard, because it was close to 2144 or 2176 om. Using the 2150 or 2140 om wavelength, the estimation of iodine value, the degree of cisunsaturation, and the degree of hydrogenation of oil were determined (2, 5). However, in the attempts of Fenton and Crisler (2) and Holman et al. (5) corrections by IR methods were necessary because of the presence of trans tDlsaturation. Because these spectral data give information about cis Wlsaturation as mentioned, they can be used in another way. Each oil has intrinsic fatty acid profiles, i.e., each has a characteristic ratio of cisunsaturation. Figure 1 shows that butter has different fatty acid profiles from margarine. We suggest that the NIR spectral data near 1164, 1660,2124,2144, and 2176 om could be used as a simple index to describe fatty acid profiles with no particular correction. The NIR spectral patterns are like fingerprints of each oil H the spectral pattern in these wavelength regions changed, we can assume that some mixing practice, e.g., foreign fat adulteration, might have been conducted. The feasibility of the NIR method for the detection of foreign fat adulteration in dairy products was confirmed in the following section, based on this information. Fetty Acid Proftle by Gas Chrometographlc Method
Table 1 shows fatty acid profiles (Wi %) of butter, margarine, milk, and soymilk fats as
TABLE 1. Fatty acids profiles ofboltel' fat, margarine fat, milk fat, and soymiIk fat as methyl eslerS normalized to 90% for major fatty acid coIllpODCllts. Patty acid
B~
(wt'l» C4:O
Gi:o C8:O ClO:O C12:0 Cl4:O Cl6:O C16:1 C18:O C18:1 C18:2 C18:3
.67 1:1.7 .81 2.93 3.46 11.89 31.36 2.87 10.36 22:1.2 1.84
.33
Journal of DaIry Scimce VoL 73,
MargariDe SD
(wt'l»
Milk SD
.OS .71 .63 .52
.05 .40 1.52 .00 .51
.15 .42 .16 No. 12, 1990
.10 .48 17.35 .15 6.25 33.51 28.62 3.54
.01 .07 .54 .03 .10 .40 .08 .28
(wt %)
Soyml.Ik SD
.97 2.34 1.45 2.88 2.99 10.48 28.52
.85 2.23
2.06
.11
11.32 23.74 3.24
.48
.11
(wt'l»
SD
:1.7 .13
.11
.11
.32
.08 .02 .17 11.23 .12 5.47 20.67 48.36 3.77
.05
.77 .58 .00
0 .02 0 0 .01 0 .01 .06 .04 .01
3411
DE'mCIlON OF FAT ADULTERATION TABLE 2. Correlation coefficienls between mixing ratio and
DC8l'
iDfrared (NIR) spectral data.
L(1164) L(1660) L(2144) L(2176) L(1164)3 L(1660) L(2124) L(2144) L(2176) -1.(2124) -L(2124) -1.(2124) -1.(2124) Original spectra Bulter and margarinel Butter and ~ Milk and soy IDinc2
Second derivative ~tra Butter and margarioel Butter and mar~2 Milk and soy IDinc2
.0892 .4064
.0799
.074S
.0ISS
.OS27
.OSSS
.4414 -.3788
.7104
-.2191
.s62O
.slOB
.s38O
.8S30
.6010 .8549
-.6860
-.8704 -.9800 -.9794
.7707 .9662 .9623
-.9883
-.911S -.99S3 -.9900
-.9704 -.9801 -.9847
-.9732 -.9799
-.9789
-.9099
-.9848
.1811 .6223 -.3722
.9104 .9983
.8260
.99S9
.948S
-.83S7 -.9125
-.9843
-.9701
-.9967
-.9639 -.9899 -.9917
-.9992
.9688
IThe NIR measurement Cor butter and margarine mixtures (UDeXtneted). ~e NIR measurement for extracted faL 3Spectral value of log(llR) at 1164 nm or spectral value of d 2 (log(llR» at 1164 nm.
methyl esters. which were nonnalized to 90% for major fatty acid components in order to compare with the data recalculated by Iverson and Sheppard (9). In this experimental condition. the values of C4:0 to C8:0. C18:1 and C18:3 were underestimated. Those of ClO:O to C 18:0 were the same level as the data recalculated by Iverson and Sheppard (9). The value of C 18:2 in milk was slightly overestimated; that in butter was the same level as in the data of Iverson and Sheppard (9. 10). The value of C16:1 in butter was overestimated but that in milk was underestimated (9, 10). Table 1 shows that the fatty acid profile of dairy products was different from that of margarine or soymilk. Correctly speaking. these values for unsaturated fats are not the degree of cis unsaturation but are a reflection of it. As the mixing ratio increased. the ratios of C18:2 and C18:3 increased. i.e., the cis unsaturation might increase and the intensity of the spectral value due to them might be expected to become high, as described in the previous section. Detection of Foreign Fat Adulteration by Near Infrared Method
In the following. the spectral value at 2144 DIn is abbreviated as 1..(2144). which means log (1/R) value at 2144 DIn in the original NIR spectra. and d2 (log (1/R» value at 2144 DIn in the case of its second derivative spectra. Using the spectral values. the mixing ratios (wt/wt). and the fat contents of each product. the c0rrelation coefficients and standard deviation (SD) from the regression line were calculated.
Table 2 describes correlation coefficients between 1..(1164). 1..(1660). L(2144). or 1..(2176) and mixing ratios. Original NIR spectra. L(l164). L(166O), 1..(2144) and L(2176) showed no significant correlation with the mixing ratios because of the presence of a linear background (bias) in the spectrum. However. by employing differences between these values and 1..(2124). the correlation coefficient was much improved. However in the second derivative spectra. 1..(1164). 1..(1660). 1..(2144) and L(2176) showed a good correlation with the mixing
2108
2144 2152nm
2124
wavelength (A)
Ci '-
.:=. 'OlJ
.2 -
!b -
.002,.-p;;-----------, .000 t--r---------+-I .002 .004 .006 .008 2108 2124 2144 2152nm -~~~~~,.__,...._,____,,____,,..--,-J
wavelength (,l.)
PiguIe 2. Original near iDfrared spectra (2108 to 21S2 nm) of extracted fat from butter and margarine mixtures (A) aod Iheir secood derivative spectra (B). The values in the figure mean the ratio of margariDe to butter in the b1eDds: 0 (-)• .OS (....)•• 10 (- •••) and .20 (---). Journal of Dairy SciCDCe Vol.
73, No. 12. 1990
3412
SATO ET AL. . 002
A
.002
't (\J (\J
8
C
o
0
...J I
't <::t
.002
o
(\J
...J -
.004 '-;-'-'~~~-,:":,-o---~---:--':"7'
.50
1.00
-
. 002 '-:-'-~~~~~~~...,.,.. 0 .50 1.00
ratio of margarine in mixture
ratio of margarine in mixture
. 002'-:;-'-~~'----'--::~~~-':7' 0 .50 1.00 ratio of soymik in mixture
Figure 3. Correlation between the ratios (wt/wt) of margarine or soy milk in mixtures and the differences in spectral dara: (2144 minus 2124 urn.). Larger circles (0) are for the original near infrared spectra case, smaller circle (.) are for the second derivative ones. A) butter and margarine mixtures, (8) extracted fats from butter and margarine mixtures, C) extracted fats from milk and soymilk mixtures.
ratios. Correlations improved appreciably by employing the differences between these values and L(2124). Figure 2 shows a limited wavelength range of NIR spectra obtained for extracted fat from butter and margarine mixtures. In Figure 2A (original spectra), as the mixing ratio increased, the slope of the straight line connecting two peaks at 2124 and 2144 DID became steeper. In Figure 2B (second derivative spectra), the slope of straight line connecting two troughs at 2124 and 2144 DID changed from a positive value, through zero, to a minus value as the mixing ratio increased. The adulteration could be more easily detected visually by the second derivative NIR. spectra than by the original one. Figure 3 shows a relationship between L(2144) minus L(2124) and the practical mixing ratio. This set gave the best correlations, as shown in Table 2. Figure 3A shows measuring butter and margarine mixtures directly by NIR method. The standard deviation from the regression line was very bad (.0055) for the original NIR spectra (larger circles), but the NIR method could not be used for a quantitative detection of adulteration. However, the standard deviations of the second derivative spectra (smaller circles), improved (.0027), indicating that NIR. method could be used for this purpose. As for the extracted fat (Figures 3B and C), both original and second derivative showed good correlation. The standard deviation from the regression line was .0001 to .0004. The slope of the milk and soymilk mixJournal of Dairy Science Vol. 73,
No. 12, 1990
tures (Figure 3C) was steeper than that of the butter-margarine mixtures (Figure 3B), because of the amount of cis unsaturation (fable 1). In the second derivative spectra, L(2144) minus L(2124) changed from positive value, through zero, to a minus value as the mixing ratio increased (Figure 3). The practical mixing ratio of .025, where L(2144) minus L(2124) equals zero, means the adulteration of the butter or milk fat with 2.54% (= 81.39 x .025 x 100/ (80.10 x .975 + 81.39 x .025» margarine fat or 3.04% (= 4.30 x .025 x 100/(3.52 x .975 + 4.30 x .025}) soymilk fat, respectively (see fat content of each product). The application of NIR method to dairy products indicated that fat adulteration, with as little as 3% foreign fat, could be detected simply and rapidly. The NIR method usually depends on sample-specific calibrations. However, there was no need to change the wavelengths for measuring these various raw materials. CONCLUSIONS
In GC analysis, it takes about 1 h to detect fat adulteration. However, it takes only 90 s to obtain a final result, including scanning from 1100 to 2500 DID in NIR analysis. Extraction of fats without further esterification is necessary for the preparation of the sample. Moreover, for butter and margarine mixtures, foreign fat adulteration could be detected "nondestructively" when using second derivative spectra. In the present study, 700 wavelengths were used Design of a testing instrument that would detect
DETECTION OF FAT ADULTERATION
foreign fat adulteration, would limit the wavelength region to be scanned from 2110 to 2160 om, or only the two wavelengths 2124 and 2144 om. In order to use this method in a practical situation, it might be necessary to check nonnal variation of L(2144) minus L(2122) of milk fat due to feed, season, stage of lactation, and other factors. However, this NIR method could be adopted as a simple screening test to search suspicious samples being submitted for official testing. REFERENCES 1 Chikuni, K., S. Ozawa, T. Mitsuhashi, M. Mitsumoto, T. Koisikawa, S. Kato, and K. Ozutsumi. 1989. Effects of oonadrenaline injections on the composition of plasma free fatty acids in beef cattle. Ipn. 1. Zootech. Sci. 60:29. 2 Fenton, A. 1. lr., and R. O. Crisler. 1959. Determination of cis unsaturation in oils by near infrared spectroscopy. 1. Am. Oil Chem. Soc. 36:620. 3 Fox, 1. R., A. H. Duthie, and S. Wulff. 1988. Precision and sensitivity of a test for vegetable fat adulteration of milk fat 1. Dairy Sci. 71:574. 4 Holman, R. T., and P. R. Edmondson. 1956. Ncarinfrared spectra of fatty acids and some related substanccs. Anal. Cbcm. 28:1533. 5 Holman, R. T., S. Eller, and P. R. Edmondson, 1959. Detection and measurement of cis unsaturation in fatty acids. Arch. Biocbem. Biopbys. 80:72. 6 International Dairy Federation. 1983. Determination of fat content-gravimetric method. IDF Standard lB. Int. Dairy Fed., Brussels, Belgium.
3413
7 International Dairy Federation. 1965. Detection of vegetable fat in milk fat by the phytosteryl acetate test IDF Standard 32. Int Dairy Fed., Brussels, Belgium. 8 International Dairy Federation. 1970. Detection of vegetable fat in milk fat by gas-liqnid chromatography of sterols. IDF Standard 54. Int. Dairy Fed., Brussels, Belgium. 9 Iverson, 1. L., and A. 1. Sheppard. 1986. Determination of fatty acids in butter fat using temperature-programmcd gas chromatography of the butyl esters. Food Chem. 21:223. 10 Iverson, 1. L., and A. 1. Sheppard. 1989. Detection of adulteration in cow, goal, and sheep cheeses utilizing gas-liqnid chromatographic fatty acid data. 1. Dairy Sci. 72:1707. 11 Keeney, M., K. C. Bachman, H. H. Tikriti, and R. L. King. 1971. Rapid vitamin E method for detecting adulteration of dairy products with non-coconut vegetable oils. 1. Dairy Sci. 54:1702. 12 Law, D. P., and R. Tkachuk. 1977. Ncar infrared diffuse reflectance spectra of wheat and wheat components. Cereal Chem. 54:256. 13 Marth, E. H. ed. 1978. Page 376 in Standard methods for the examination of dairy products. 14th ed. Am. Pub!. Health Assoc., Washington, DC. 14 Ministry of Health and Welfare of lapan. 1983. Ministerial ordinance concerning compositional standards, etc. for milk and milk products. Revision Ordinance 37. Tokyo, Ipn. 15 Osborne, B. G., and T. Fearn. 1986. Page 117 in Ncar infrared spectroscopy in food analysis. Longman Scientific & Technical, Harlow, Essex, Engl. 16 Sato, T., M. Yoshino, R. K. Cho, and M. Iwamoto. 1988. Ncar-infrared reflectancc spectroscopic analysis for butter constituents. Ipn. 1. Zootech. Sci. 59:806. 17 Sheppard, A. I., C.SJ. SheD, and T. S. Rudolf. 1985. Detection of vegetable oil adulteration in ice cream. 1. Dairy Sci. 68:1103.
lournal of Dairy Science Vol. 73,
No. 12, 1990
INTRODUCTION
A near infrared spectroscopic method for the detection of foreign fat adulteration in dairy products was examined using the extracted fats from samples of butter and margarine mixtures and from milk. and soymilk mixtures. Butter and margarine mixtures were also analyzed. The near infrared absorptions at 1164, 1660, 2144, and 2176 om are due to cis unsaturation of fatty acid moieties, the ratio of which is intrinsic to each oil. We suggest that the near infrared spectra around these wavelengths could be used as a simple index for fatty acid profiles in oil. As for the original near infrared spectra, the difference of spectral data at log 1164, 1660,2144, or 2176 om from 2124 om correlated very well with the mixing ratios. As for their second derivative spectra, spectral data at log 1164, 1660, 2144, or 2176 om correlated with the mixing ratios very well, and further, the correlation proved to be better by employing the difference between these values and those at 2124 om. The application of near infrared method to dairy products indicated that the adulteration of the fat with as little as 3% foreign fat could be detected simply, rapidly, and "nondestructively" for butter and margarine mixtures. (Key words: fat adulteration, near infrared, spectroscopy)
In order to reduce production cost, less expensive vegetable fats are used as substitutes for milk. fat in dairy products. Such products should be properly labeled for consumers, and it is important to develop a potential method for detection of vegetable fat to ensure correct labeling. Many methods have been developed to detect fat adulteration, and some of them have been officially recognized (3, 7, 8, 11, 17). However, they require time-consuming and sophisticated analytical procedures. This paper describes feasibility of near infrared (NIR) method for the rapid and simple detection of foreign fat adulteration in dairy products.
Received February 28, 1990. Accepted June 18, 1990. 1990 J Dairy Sci 73:3408-3413
MATERIALS AND METHODS Preparation 01 Samples
Commercially available margarine and butter were mixed in ratios of 0, 2.5, 5.0, 7.5, 10.0, 20.0, 25.0, 50.0, 100.0% (wt/wt) and milk: and soymilk. mixture ratios were 0, 2.5, 5.0, 7.7, 10.1, 24.7, 49.3, 100.0% (wt/wt). From each mixture, fat was extracted by Rose-Gottlieb method (6) and used for the following analyses. Moisture and fat contents in butter and margarine were measured by modified Kohman's method (13). Moisture and fat were 16.80 and 80.04% in butter 15.04 and 81.39% in margarine, respectively. The total solids and fat contents in milk. and soymilk. were measured by the standard gravimetric method (14) and RoseGottlieb method. They were 12.01 and 3.52% in milk, and 11.36 and 4.30% in soymilk., respectively. 3408
3409
DETEcrION OF FAT ADULTERATION
Analysis of Samples by Near Infrared Method
An InfraAlyzer 500 (Bran & Luebbe Co., West Gennany) was used to measure NIR transflectance in the wavelengths from 1100 to 2500 run. The extracted fat was warmed at 35·C to become liquid, cooled to room temperature, applied on a sample holder, a British cup (Bran & Luebbe), covered with a slide glass (microslide glass S2226, Matsunami Glass Ind., Ltd., Japan), and was measured. In case of butter and margarine mixtures, NIR measurement was conducted by using an Italian cup (Bran & Luebbe) as described in a previous paper (16). The conditions to obtain second derivative spectra were as follows: 4 om between output points, 4 om in moving average, 12 nm/derivative segments, and 12 om between derivative segments.
2.50 2.00
~
0::
-
'--
1. 50
~
'Oll
.Q
1.00
Gas Chromatographic Analysis
The extracted fats were analyzed with gas chromatography (GC) after methyl-esterification according to a conventional method (1). The GC conditions for determining the fatty acid methyl esters were as follows: 2 m X 3 mm Ld. Pyrex column packed with 10% Sitar 7CP coated on 80/100 mesh chromosorb W (AW, DMCS); 250·C injection temperature; 30 mlImin carrier gas flow; column temperature programmed from 60 to 200·C by 3·C/min, then holding for 14 min at 200·C. Each sample was analyzed twice, the average of duplicate measurements was calculated, and samples were normalized to 90% for major fatty acid components (9). RESULTS AND DISCUSSION Near Infrared Spectra and Fatty Acid Profiles
Figure I shows NIR spectra of butter and margarine (Figure lA) and their second derivative spectra (Figure IB and C). As shown in Figure lA, butter and margarine show almost similar NIR spectral patterns except those at 1164, 2144, and 2176 om, at which margarine spectra have sharper peaks than butter. Holman et al. (4) measured NIR spectra of carbon tetrachloride solution of various fatty
-
~-
.30 t200 1400 1600 1800 2000 2200 2400 nm wavelength ().) Figure 1. Original near infrared spectra of butter and lIUU'glIl'ine (A) and second derivative ones of butter (8) and lIUU'glIl'ine (C). Arrows indicate 1164, 1660. 2144. and 2176 nm, respectively. A dot indicates 2124 nm.
acids and their related compounds. They concluded that C-H bond, which is associated with cis double bonds, had a fundamental absorption at 3300 om, moderation combination absorptions at 2190 and 2150 nm, a weak first overtone at 1680 om, and a second overtone at 1180 om. Experimental conditions of Holman et al. (4) were different from ours: solution of acids versus extracted fat itself, pure substances versus mixtures of triglycerides (crude fat), and so on. However, our wavelengths (1164, 1660, 2144,2176 om) might be from C-H vibrations associated with cis double bonds due to cis Journal of Dairy Science Vol. 73.
No. 12, 1990
3410
SATO BT AL.
Wlsaturated fatty acid moieties, as assigned by Law et al. (12). Near infrared spectrum gives the infonnation of cis isomer, whereas infrared (IR) spectrum gives that of transisomer. Absorption intensity in log (11R) for the spectra of extracted fats was rather weak, and in addition, peaks due to water, i.e., peaks at 1450 and 1940 om, WeIe not observed. However, general trend of the spectra showed similarity to Figure 1. Conversely, as for their second derivative spectra, as in the original, the spectra of margarine were sharper than those of butter arowd these wavelengths, but in the opposite direction. According to Osborne et al. (15), second derivative spectra have a trough corresponding to each peak. in the original spectra. Further, with its second derivative spectra, a linear background can be cancelled, and peaks discriminated that overlap substantially in the original NIR spectrum. In addition, a new trough at 1660 and 2124 om appeared in the second derivative spectra. In the butter spectra, the trough at 2144 om was a little more shallow than that at 2124 om. However, in the margarine spectra, the trough at 2144 om was much deeper than at 2124 om. According to the result of measuring an NIR spectrum of 11-hydroxy-lD-eicosanone, Holman et aI. (4) reported that neighboring ~H and ~ groups showed absorptions in a Wlique couplet at 2050 and 2120 om, respectively. H the absorption at 2124 om might be due to the carbonyl groups close to the polar groups, this absorption might be contributed by the ester carbonyl groups of triglycerides. We
will adopt this wavelength, 2124 om, as a comparative standard, because it was close to 2144 or 2176 om. Using the 2150 or 2140 om wavelength, the estimation of iodine value, the degree of cisunsaturation, and the degree of hydrogenation of oil were determined (2, 5). However, in the attempts of Fenton and Crisler (2) and Holman et al. (5) corrections by IR methods were necessary because of the presence of trans tDlsaturation. Because these spectral data give information about cis Wlsaturation as mentioned, they can be used in another way. Each oil has intrinsic fatty acid profiles, i.e., each has a characteristic ratio of cisunsaturation. Figure 1 shows that butter has different fatty acid profiles from margarine. We suggest that the NIR spectral data near 1164, 1660,2124,2144, and 2176 om could be used as a simple index to describe fatty acid profiles with no particular correction. The NIR spectral patterns are like fingerprints of each oil H the spectral pattern in these wavelength regions changed, we can assume that some mixing practice, e.g., foreign fat adulteration, might have been conducted. The feasibility of the NIR method for the detection of foreign fat adulteration in dairy products was confirmed in the following section, based on this information. Fetty Acid Proftle by Gas Chrometographlc Method
Table 1 shows fatty acid profiles (Wi %) of butter, margarine, milk, and soymilk fats as
TABLE 1. Fatty acids profiles ofboltel' fat, margarine fat, milk fat, and soymiIk fat as methyl eslerS normalized to 90% for major fatty acid coIllpODCllts. Patty acid
B~
(wt'l» C4:O
Gi:o C8:O ClO:O C12:0 Cl4:O Cl6:O C16:1 C18:O C18:1 C18:2 C18:3
.67 1:1.7 .81 2.93 3.46 11.89 31.36 2.87 10.36 22:1.2 1.84
.33
Journal of DaIry Scimce VoL 73,
MargariDe SD
(wt'l»
Milk SD
.OS .71 .63 .52
.05 .40 1.52 .00 .51
.15 .42 .16 No. 12, 1990
.10 .48 17.35 .15 6.25 33.51 28.62 3.54
.01 .07 .54 .03 .10 .40 .08 .28
(wt %)
Soyml.Ik SD
.97 2.34 1.45 2.88 2.99 10.48 28.52
.85 2.23
2.06
.11
11.32 23.74 3.24
.48
.11
(wt'l»
SD
:1.7 .13
.11
.11
.32
.08 .02 .17 11.23 .12 5.47 20.67 48.36 3.77
.05
.77 .58 .00
0 .02 0 0 .01 0 .01 .06 .04 .01
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DE'mCIlON OF FAT ADULTERATION TABLE 2. Correlation coefficienls between mixing ratio and
DC8l'
iDfrared (NIR) spectral data.
L(1164) L(1660) L(2144) L(2176) L(1164)3 L(1660) L(2124) L(2144) L(2176) -1.(2124) -L(2124) -1.(2124) -1.(2124) Original spectra Bulter and margarinel Butter and ~ Milk and soy IDinc2
Second derivative ~tra Butter and margarioel Butter and mar~2 Milk and soy IDinc2
.0892 .4064
.0799
.074S
.0ISS
.OS27
.OSSS
.4414 -.3788
.7104
-.2191
.s62O
.slOB
.s38O
.8S30
.6010 .8549
-.6860
-.8704 -.9800 -.9794
.7707 .9662 .9623
-.9883
-.911S -.99S3 -.9900
-.9704 -.9801 -.9847
-.9732 -.9799
-.9789
-.9099
-.9848
.1811 .6223 -.3722
.9104 .9983
.8260
.99S9
.948S
-.83S7 -.9125
-.9843
-.9701
-.9967
-.9639 -.9899 -.9917
-.9992
.9688
IThe NIR measurement Cor butter and margarine mixtures (UDeXtneted). ~e NIR measurement for extracted faL 3Spectral value of log(llR) at 1164 nm or spectral value of d 2 (log(llR» at 1164 nm.
methyl esters. which were nonnalized to 90% for major fatty acid components in order to compare with the data recalculated by Iverson and Sheppard (9). In this experimental condition. the values of C4:0 to C8:0. C18:1 and C18:3 were underestimated. Those of ClO:O to C 18:0 were the same level as the data recalculated by Iverson and Sheppard (9). The value of C 18:2 in milk was slightly overestimated; that in butter was the same level as in the data of Iverson and Sheppard (9. 10). The value of C16:1 in butter was overestimated but that in milk was underestimated (9, 10). Table 1 shows that the fatty acid profile of dairy products was different from that of margarine or soymilk. Correctly speaking. these values for unsaturated fats are not the degree of cis unsaturation but are a reflection of it. As the mixing ratio increased. the ratios of C18:2 and C18:3 increased. i.e., the cis unsaturation might increase and the intensity of the spectral value due to them might be expected to become high, as described in the previous section. Detection of Foreign Fat Adulteration by Near Infrared Method
In the following. the spectral value at 2144 DIn is abbreviated as 1..(2144). which means log (1/R) value at 2144 DIn in the original NIR spectra. and d2 (log (1/R» value at 2144 DIn in the case of its second derivative spectra. Using the spectral values. the mixing ratios (wt/wt). and the fat contents of each product. the c0rrelation coefficients and standard deviation (SD) from the regression line were calculated.
Table 2 describes correlation coefficients between 1..(1164). 1..(1660). L(2144). or 1..(2176) and mixing ratios. Original NIR spectra. L(l164). L(166O), 1..(2144) and L(2176) showed no significant correlation with the mixing ratios because of the presence of a linear background (bias) in the spectrum. However. by employing differences between these values and 1..(2124). the correlation coefficient was much improved. However in the second derivative spectra. 1..(1164). 1..(1660). 1..(2144) and L(2176) showed a good correlation with the mixing
2108
2144 2152nm
2124
wavelength (A)
Ci '-
.:=. 'OlJ
.2 -
!b -
.002,.-p;;-----------, .000 t--r---------+-I .002 .004 .006 .008 2108 2124 2144 2152nm -~~~~~,.__,...._,____,,____,,..--,-J
wavelength (,l.)
PiguIe 2. Original near iDfrared spectra (2108 to 21S2 nm) of extracted fat from butter and margarine mixtures (A) aod Iheir secood derivative spectra (B). The values in the figure mean the ratio of margariDe to butter in the b1eDds: 0 (-)• .OS (....)•• 10 (- •••) and .20 (---). Journal of Dairy SciCDCe Vol.
73, No. 12. 1990
3412
SATO ET AL. . 002
A
.002
't (\J (\J
8
C
o
0
...J I
't <::t
.002
o
(\J
...J -
.004 '-;-'-'~~~-,:":,-o---~---:--':"7'
.50
1.00
-
. 002 '-:-'-~~~~~~~...,.,.. 0 .50 1.00
ratio of margarine in mixture
ratio of margarine in mixture
. 002'-:;-'-~~'----'--::~~~-':7' 0 .50 1.00 ratio of soymik in mixture
Figure 3. Correlation between the ratios (wt/wt) of margarine or soy milk in mixtures and the differences in spectral dara: (2144 minus 2124 urn.). Larger circles (0) are for the original near infrared spectra case, smaller circle (.) are for the second derivative ones. A) butter and margarine mixtures, (8) extracted fats from butter and margarine mixtures, C) extracted fats from milk and soymilk mixtures.
ratios. Correlations improved appreciably by employing the differences between these values and L(2124). Figure 2 shows a limited wavelength range of NIR spectra obtained for extracted fat from butter and margarine mixtures. In Figure 2A (original spectra), as the mixing ratio increased, the slope of the straight line connecting two peaks at 2124 and 2144 DID became steeper. In Figure 2B (second derivative spectra), the slope of straight line connecting two troughs at 2124 and 2144 DID changed from a positive value, through zero, to a minus value as the mixing ratio increased. The adulteration could be more easily detected visually by the second derivative NIR. spectra than by the original one. Figure 3 shows a relationship between L(2144) minus L(2124) and the practical mixing ratio. This set gave the best correlations, as shown in Table 2. Figure 3A shows measuring butter and margarine mixtures directly by NIR method. The standard deviation from the regression line was very bad (.0055) for the original NIR spectra (larger circles), but the NIR method could not be used for a quantitative detection of adulteration. However, the standard deviations of the second derivative spectra (smaller circles), improved (.0027), indicating that NIR. method could be used for this purpose. As for the extracted fat (Figures 3B and C), both original and second derivative showed good correlation. The standard deviation from the regression line was .0001 to .0004. The slope of the milk and soymilk mixJournal of Dairy Science Vol. 73,
No. 12, 1990
tures (Figure 3C) was steeper than that of the butter-margarine mixtures (Figure 3B), because of the amount of cis unsaturation (fable 1). In the second derivative spectra, L(2144) minus L(2124) changed from positive value, through zero, to a minus value as the mixing ratio increased (Figure 3). The practical mixing ratio of .025, where L(2144) minus L(2124) equals zero, means the adulteration of the butter or milk fat with 2.54% (= 81.39 x .025 x 100/ (80.10 x .975 + 81.39 x .025» margarine fat or 3.04% (= 4.30 x .025 x 100/(3.52 x .975 + 4.30 x .025}) soymilk fat, respectively (see fat content of each product). The application of NIR method to dairy products indicated that fat adulteration, with as little as 3% foreign fat, could be detected simply and rapidly. The NIR method usually depends on sample-specific calibrations. However, there was no need to change the wavelengths for measuring these various raw materials. CONCLUSIONS
In GC analysis, it takes about 1 h to detect fat adulteration. However, it takes only 90 s to obtain a final result, including scanning from 1100 to 2500 DID in NIR analysis. Extraction of fats without further esterification is necessary for the preparation of the sample. Moreover, for butter and margarine mixtures, foreign fat adulteration could be detected "nondestructively" when using second derivative spectra. In the present study, 700 wavelengths were used Design of a testing instrument that would detect
DETECTION OF FAT ADULTERATION
foreign fat adulteration, would limit the wavelength region to be scanned from 2110 to 2160 om, or only the two wavelengths 2124 and 2144 om. In order to use this method in a practical situation, it might be necessary to check nonnal variation of L(2144) minus L(2122) of milk fat due to feed, season, stage of lactation, and other factors. However, this NIR method could be adopted as a simple screening test to search suspicious samples being submitted for official testing. REFERENCES 1 Chikuni, K., S. Ozawa, T. Mitsuhashi, M. Mitsumoto, T. Koisikawa, S. Kato, and K. Ozutsumi. 1989. Effects of oonadrenaline injections on the composition of plasma free fatty acids in beef cattle. Ipn. 1. Zootech. Sci. 60:29. 2 Fenton, A. 1. lr., and R. O. Crisler. 1959. Determination of cis unsaturation in oils by near infrared spectroscopy. 1. Am. Oil Chem. Soc. 36:620. 3 Fox, 1. R., A. H. Duthie, and S. Wulff. 1988. Precision and sensitivity of a test for vegetable fat adulteration of milk fat 1. Dairy Sci. 71:574. 4 Holman, R. T., and P. R. Edmondson. 1956. Ncarinfrared spectra of fatty acids and some related substanccs. Anal. Cbcm. 28:1533. 5 Holman, R. T., S. Eller, and P. R. Edmondson, 1959. Detection and measurement of cis unsaturation in fatty acids. Arch. Biocbem. Biopbys. 80:72. 6 International Dairy Federation. 1983. Determination of fat content-gravimetric method. IDF Standard lB. Int. Dairy Fed., Brussels, Belgium.
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7 International Dairy Federation. 1965. Detection of vegetable fat in milk fat by the phytosteryl acetate test IDF Standard 32. Int Dairy Fed., Brussels, Belgium. 8 International Dairy Federation. 1970. Detection of vegetable fat in milk fat by gas-liqnid chromatography of sterols. IDF Standard 54. Int. Dairy Fed., Brussels, Belgium. 9 Iverson, 1. L., and A. 1. Sheppard. 1986. Determination of fatty acids in butter fat using temperature-programmcd gas chromatography of the butyl esters. Food Chem. 21:223. 10 Iverson, 1. L., and A. 1. Sheppard. 1989. Detection of adulteration in cow, goal, and sheep cheeses utilizing gas-liqnid chromatographic fatty acid data. 1. Dairy Sci. 72:1707. 11 Keeney, M., K. C. Bachman, H. H. Tikriti, and R. L. King. 1971. Rapid vitamin E method for detecting adulteration of dairy products with non-coconut vegetable oils. 1. Dairy Sci. 54:1702. 12 Law, D. P., and R. Tkachuk. 1977. Ncar infrared diffuse reflectance spectra of wheat and wheat components. Cereal Chem. 54:256. 13 Marth, E. H. ed. 1978. Page 376 in Standard methods for the examination of dairy products. 14th ed. Am. Pub!. Health Assoc., Washington, DC. 14 Ministry of Health and Welfare of lapan. 1983. Ministerial ordinance concerning compositional standards, etc. for milk and milk products. Revision Ordinance 37. Tokyo, Ipn. 15 Osborne, B. G., and T. Fearn. 1986. Page 117 in Ncar infrared spectroscopy in food analysis. Longman Scientific & Technical, Harlow, Essex, Engl. 16 Sato, T., M. Yoshino, R. K. Cho, and M. Iwamoto. 1988. Ncar-infrared reflectancc spectroscopic analysis for butter constituents. Ipn. 1. Zootech. Sci. 59:806. 17 Sheppard, A. I., C.SJ. SheD, and T. S. Rudolf. 1985. Detection of vegetable oil adulteration in ice cream. 1. Dairy Sci. 68:1103.
lournal of Dairy Science Vol. 73,
No. 12, 1990