WO1992017766A1

WO1992017766A1 - Method for quantitative determination of fat in an emulsion which contains fat particles

Info

Publication number: WO1992017766A1
Application number: PCT/FI1992/000099
Authority: WO
Inventors: Lauri Jalkanen
Original assignee: Individual
Current assignee: Individual
Priority date: 1991-04-05
Filing date: 1992-04-03
Publication date: 1992-10-15
Anticipated expiration: 1993-10-05
Also published as: FI87837B; FI87837C; FI911654A0

Abstract

The invention relates to a method for quantitative determination of fat from an emulsion, such as milk or a milk product, containing fat particles. The method uses an IR absorption technique in which the determination is based on a specific absorption peak in the absorption spectrum of the emulsion sample, and the essential idea of the invention is that the absorption peak, at a wave number of approx. 1440 (1/cm), of the carbon-carbon bonds of the fat molecules is used in the determinations. When the determinations are carried out on milk or on a milk product, the effect, on the said peak, of the proteins and lactose present in the milk is taken into account by measuring the characteristic absorption peaks of the said substances and by carrying out, on the basis of the measurements, compensation in the value of the fat concentration yielded by the absorption peak of the carbon-carbon bond. The basis of the determination can in this case be a continuous IR absorption spectrum measured within a wave number range of approx. 1050-1550.

Description

Method for quantitative determination of fat in an emulsion which contains fat particles

The present invention relates to a method for quantitative determination of fat in an emulsion which contains fat par¬ ticles, in which method the infrared absorption of the emulsion sample is measured and the fat is determined on the basis of a specific absorption peak in the absorption spectrum by elimi¬ nating from it any error effect possibly caused by the other constituents of the emulsion.

The principal targets of quantitative fat determinations are milk and products made from milk. The natural fat concentration in milk is usually within the range 3.0-5.5 %, but the fat concentrations in different milk products may vary from close to 0 to more than 40 %. Measurement of the fat concentration is required as a basis for the milk price to be paid to the pro¬ ducer and additionally in the quality control of products pre¬ pared from milk.

The determination of the fat concentration in milk is possible by traditional chemical means, of which so-called Gerber and Rose-Gottlieb methods can be mentioned as examples. However, infrared absorption is currently used in routine determina¬ tions, although chemical methods continue to be necessary for the calibration of the IR analyzers. Most commonly the absorp¬ tion peak, at a wave number of approx. 1750 (1/cm) (at a wave¬ length of approx. 5.7 μ_n) , of the carbon-oxygen double bonds of the triglyceride part contained in the fat molecule is used in IR determinations. In addition to this there is used a method described in EP Patent publication 0012492, which is based on the absorption peak, at a wave number of approx. 2870 (1/cm) (at a wavelength of approx. 3.5 μ_τt), of the carbon-hydrogen bonds in the fat molecule. The drawback in the use of the carbon-oxygen double bond in IR determinations is that the absorption peak is not dependent on the size of the fat molecules, and therefore, for example, any variation in the milk fat composition due to changes of season or to different feeding of the livestock will cause errors in the determinations. Furthermore, the said bond cannot be used in connection with fermented milk products, since the lactic acid present disturbs the determination. When the carbon- hydrogen bond is used as the basis of determination, the first- mentioned drawback is avoided, since the number of the said bonds in a fat molecule is directly proportional to the size of the molecule. On the other hand, when the C-H bond is used, it is necessary to take into account the contribution to the ab¬ sorption peak by other substances present in milk, mainly pro¬ teins and lactose, which contain C-H bonds. This can be done by measuring, in addition to the absorption peak of the C-H bond also the IR absorption peaks characteristic of the said other substances and by calculating on the basis of them a correction of the C-H bond absorption peak, thereby arriving at the fat concentration aimed at in the determination.

The use of the C-H bond absorption peak involves, however, drawbacks which worsen the method as compared with, for ex¬ ample, the previous method based on the C=0 double bond. In the infrared spectrum of milk, the C-H absorption peak is located at a highly inconvenient place on the slope of a strong absorp¬ tion peak of water, where it is difficult to distinguish. Fur¬ thermore, as the composition of milk, i.e. the amounts of fat, proteins, lactose, minerals, and other constituents, varies, the proportion of water in the sample subject to determination is not always precisely known, and this may cause in the result an uncontrolled error which is further multiplied in the cor¬ rections calculated for proteins and lactose. Another error source which hampers especially the use of the C-H bond is the scatter occurring in the sample alongside IR absorption. The scatter caused by fat particles strongly affects the signals to be registered and thereby disturbs the determination of fat when the wavelength of the absorption peak corresponds to the diameter of the particles or is smaller than it. When the di¬ ameter of the fat particles in raw milk is in the order of 0.5- 5 urn, it follows from this that scatter strongly disturbs C-H bond absorption occurring at a wavelength of 3.5 μ , unless the fat particles can be reduced to a diameter clearly below 3.5 μm in a homogenization before fat determination. Therefore it is stated in the above-mentioned EP patent publication 0012492 that the mean diameter of the fat particles may be at maximum 2 μm, and usually the IR analyzers used for milk fat determina¬ tions are indeed equipped with homogenizers by means of which the fat particles can be chopped to the size level mentioned. However, when the analyzers are in continuous use, the opera¬ tion of the homogenizers gradually deteriorates so that, with time, more and more poorly homogenized milk samples, containing also fat particles larger than 2 μm in diameter and giving erroneous results, arrive in the measuring cuvette. The problem could be in part diminished by, for example, homogenizing the sample several times, but this would lower the analyzer capaci¬ ty to such an extent that the system would not be practicable. Determination based on the C=0 absorption peak is not as badly hampered by scatter even in deficiently homogenized samples, since the peak is located at a clearly higher wavelength, 5.7 μm, at which the absorbance curve depicting the concentration of fat as a function of absorption is clearly straighter.

The object of the present invention is to provide a new method for quantitative determination of fat by an infrared absorption technique in which the above-mentioned disadvantages based on the use of the C-H bond are avoided. The method according to the invention is characterized in that the determination is made from an absorption peak of fat-molecule carbon-carbon bonds at a wave number of approx. 1440 (1/cm).

The C-C bond absorption peak, at a wave number of approx. 1440, corresponding to a wavelength of approx. 7.0 μm, used according to the invention is located in the IR absorption spectrum at a place substantially more advantageous for analysis than the absorption peak of the C-H bond. When milk or milk products are being measured, the absorption of water does not disturb the measurement, and therefore the reading of the absorbance of the peak is easier. At the same time the peak yields a more reli¬ able determination result, which is not dependent on the amount of water in the sample. The wavelength of the absorption peak, 7.0 μm, is also clearly greater than the maximum diameter, approx. 5.0 μm, of the fat particles, which means that, in the measuring of raw milk, the method according to the invention is relatively insensitive to errors due to the scatter of light. The method thus yields a result in the correct direction even if the homogenization of the milk sample is deficient or is not done at all, albeit that in order to obtain precise results the homogenization of the sample to an average particle size pref¬ erably less than 3.0 μm continues to be recommended. Further¬ more, it is a significant advantage in the invention that, owing to lesser scatter, the proportion of the change in ab¬ sorption to the change in concentration is, especially at higher fat concentrations, greater than in the C-H absorption peak; this signifies a correspondingly improved determination precision.

It should be noted further that, as compared with the use of the C=0 absorption peak, the same advantage is achieved with the invention as is achieved with C-H absorption, namely that the variation in the size of fat molecules will not cause an error in the result obtained. When milk or milk products are measured, the effect of other constituents of milk which con¬ tain C-C bonds, i.e. primarily proteins and lactose, on the C-C absorption peak must be compensated for in the same manner as in determination based on C-H absorption. In these respects the method according to the invention is thus substantially of the same value as the said prior-art method. When the absorption peak of the C-C bond is being measured, regardless of the type of the emulsion, it is preferable to measure the absorption spectrum over a somewhat wider range, for example a wave number range of approx. 1380-1440, from which there is obtained the necessary reference wavelength, th absorption of which is subtracted from the absorption peak obtained.

When the determination is carried out on milk or on a milk product, for example, full milk containing fat 3.0-5.5 %, the milk is first heated to 40 °C and mixed carefully in order to obtain as representative a sample as possible for the analysis. Homogenization is then carried out, whereafter the sample is analyzed by measuring its IR absorption at the desired wave¬ lengths. In this case, according to the invention, it is pos¬ sible to measure the characteristic absorption peak, at a wave number of approx. 1550, of the protein present in the milk and the characteristic absorption peak, at a wave number of approx. 1050, of the lactose present in the milk, whereupon on the basis of them the contributions of proteins and lactose to the absorption peak of the C-C bond at a wave number of approx. 1440 can be compensated for. Depending on the IR spectrometer used, it may in this case be advantageous to measure from the sample a continuous IR absorption spectrum within a wave numbe range of approx. 1050-1550.

Even though the above discussion has mainly been about the use of the invention for the determination of the concentration of fat in milk or in a milk product, the method according to the invention can also be used for measuring emulsions containing other fats, such as vegetable fats. The determination of the fat concentrations of such emulsions may be in practice sim¬ pler, since the compensations due to other constituents, neces sary in the case of milk, are not necessary. In some cases it may be advantageous to base the determination of the fat concentration of an emulsion simultaneously on sev¬ eral different absorption peaks which are due to fat molecule bonds absorbent at different wave numbers. The idea is in prin¬ ciple known from US patent publication 4 447 725, in which both the absorption peak of the C=0 bond and the absorption peak of the C-H bond are used.

According to the present invention the procedure may be that, in addition to measuring the absorption peak of the C-C bond at a wave number of approx. 1440, also the.absorption peak of the C=0 double bond of the fat molecule ester groups at a wave number of approx. 1750 and/or the absorption peak of the single C-0 bond of the ester groups at a wave number of approx. 1160- 1190 and/or the absorption peak of the C-H bond of the fat molecule at a wave number of approx. 2870 are/is measured, in which case the determination of the fat is carried out on the basis of two or more absorption peaks in the spectrum. Most preferably, besides the C-C absorption peak, also the absorp¬ tion peak of the C=0 and/or C-0 bond is taken into account in such a determination, the absorption peak of the C-C bond hav¬ ing, however, the greatest weight in the determination.

In the accompanying drawings,

Figure 1 depicts an infrared absorption spectrum of full milk (fat concentration 3.4 %) measured by means of a Mattsson FTIR spectrometer within a wavelength range of approx. 3.0-10 μm (wave number range of approx. 1000-3000),

Figure 2 depicts an infrared absorption spectrum measured from the same full milk by means of the same spectrometer within a wave number range of approx. 1000-3000, the effect of the water in the background having been eliminated.

Figure 3 depicts an IR absorption spectrum measured in accor¬ dance with Figure 2 from a milk sample having a fat concentra¬ tion of lower than 0.05 %, and Figure 4 depicts graphically the dependence between the absor- bance and the fat concentration at the absorption peaks of the C-C bond, the C=0 bond, and the C-H bond.

In the absorption spectrum according to Figure 1, the absorp¬ tion peak 1 of the C-H bond is seen at a wavelength of approx. 3.5 μm (wave number of approx. 2870), the absorption peak 2 of the C=0 double bond at a wavelength of approx. 5.7 μm (wave number of approx. 1750), the absorption peak 3 of the C-C bond at a wavelength of approx. 7.0 μm (wave number of approx. 1440), and the absorption peak 4 of the single C-0 bond at a wavelength of approx. 8.4-8.6 μm (wave number of approx. 1160- 1190). It is observed that the absorption peak 1 of the C-H bond is located on the slope of a strong absorption peak of water, contrary to the C-C bond absorption peak 3 used accord¬ ing to the invention, at which peak absorption due to water does not occur.

Figure 2, the absorption spectrum of which was measured from the same sample of full milk as was the spectrum according to Figure 1, shows the absorption peaks 1-4 produced by the dif¬ ferent bonds of the fat molecule without the disturbing back¬ ground effect of water. In addition, the curve contains an absorption peak 5 due to the peptide bond of proteins at a wave number of approx. 1550 and an absorption peak 6 due to lactose at a wave number of approx. 1050. In Figure 3, the absorption spectrum of which was measured from a milk sample containing fat less than 0.05 %, the absorption peaks 1-4 due to fat bonds are clearly lowered as compared with the peaks in Figure 2, whereas the peaks 5 and 6 of lactose are substantially un¬ changed.

In Figure 4, curve 7 depicts the dependence of absorbance on the fat concentration in the sample at the absorption peak of the C-C bond, curve 8 depicts the dependence of absorbance on the fat concentration at the absorption peak of the C=0 bond, and curve 9 the dependence of absorbance on the fat concentra¬ tion at the absorption peak of the C-H bond. It can be seen that, when the absorption peak of the C-C bond is used in ac¬ cordance with the invention, the absorbance increases together with the fat concentration more strongly than at the peaks of the C=0 and C-H bonds, whereby a higher precision in the deter¬ mination is attained, especially with higher fat concentra¬ tions.

According to the basic idea of the invention, the fat con¬ centration in the milk is determined from the C-C bond absorp¬ tion peak at a wave number of 1440, seen in Figures 2 and 3, by subtracting from this the absorption at a reference wavelength of approx. 1380, and by calculating the fat concentration using a formula having the form

F=ax³ +bx² +cx-d

where F is the fat concentration and x = absorbance, and by further making, in the result obtained, corrections due to the protein peak 5 and the lactose peak 6. When a DaiLab IR-2000 spectrometer is used, the said calculation formula is

F=11.3x³ +9.Ox² +6.7x

and the protein and lactose correction coefficients are ob¬ tained from the following table:

Table

Fat Protein Lactose

Fat 1 0.020 0.015

Protein -0.007 1 -0.007

Lactose -0.005 ^•0.039 1

The effects of protein and lactose are taken into account by using linear corrections, in which the absorbances of peaks 5 and 6 are multiplied by coefficients according to the Table and are added to the fat concentration calculated, using the above- mentioned formula, from the C-C absorption peak. According to the Table, the protein peak absorbance is multiplied by 0.007 and the lactose peak absorbance by 0.005, and the obtained values are subtracted from the value calculated using the for¬ mula, whereby the final fat concentration is arrived at.

If absorption peaks due to other bonds of the fat molecule are used in the fat determination in addition to the absorption peak of the C-C bond, the fat concentration is calculated using a formula having the form:

F(-CC), (C-0), (C=0). (C-H) = k X F(C-C) + 1 x F(C-O) + m x F(C=0) + n x F(C-H)

where F(C-C), F(C-O), F(C=0) and F(C-H) are the fat concentra¬ tions calculated using the said bonds, the calculation being carried out in each case by using the formula

F=ax³ +bx² +cx-d

where F represents the quantitative amount of fat and x the ab¬ sorbance of the peak concerned.

In the formula, k, 1, m and n are experimental constants the sum of which must be approx. 1 and by varying which the con¬ tributions of the different bonds can be weighted in the deter¬ mination. Practical experiments have shown that it is prefer¬ able to weight the value F(C-C) calculated from the absorption peak of the C-C bond and to combine with it one or both of the quantities F(C=0) and F(C-O).

It is clear for an expert in the art that the different embodi¬ ments of the invention are not limited to that presented above as an example; they may vary within the scope of the accompany- ing patent claims. It should be noted in particular that the coefficients of the calculation formulae presented are appa¬ ratus-specific values which change when a shift is made to the use of some other spectrometer.

Claims

1. A method for quantitative determination of fat from an emulsion which contains fat particles, in which method the infrared absorption of an emulsion sample is measured and the fat is determined, on the basis of a specific absorption peak contained in the absorption spectrum, by eliminating from it any error effect possibly caused by the other constituents of the emulsion, characterized in that the determination is car¬ ried out from the absorption peak of the fat molecule carbon- carbon bonds (C-C) at a wave number of approx. 1440 (1/cm).

2. A method according to -Claim 1, characterized in that the infrared absorption spectrum of the emulsion is measured at least within a wavelength range of approx. 1380-1440.

3. A method according to Claim 1 or 2, characterized in that the emulsion is milk or a milk product.

4. A method according to Claim 3, characterized in that the emulsion is full milk having a fat concentration of approx. 3.0-5.5 %.

5. A method according to Claim 3 or 4, characterized in that the absorption peaks, at wave numbers of approx. 1550 and approx. 1050, of the proteins and lactose present in the milk are measured, and on the basis of them the contribution of the said substances to the absorption peak at a wavelength of approx. 1440 is compensated for.

6. A method according to any of Claims 3-5, characterized in that a continuous infrared absorption spectrum is measured within a wave number range of approx. 1050-1550.

7. A method according to any of Claims 3-6, characterized in that the milk is homogenized before the measuring of the infrared absorption spectrum, so that the size of the fat par- tides in the measuring step will in the main be less than 3 μm.

8. A method according to Claim 1 or 2, characterized in that the fat is determined from an emulsion containing vege¬ table fat.

9. A method according to any of the above claims, charac¬ terized in that, in addition to the absorption peak of carbon- carbon bonds ( C-C ) at a wave number of approx. 1440, the ab¬ sorption peak of the carbon-oxygen double bond (C=0) of the fat molecule ester groups at a wave number of approx. 1750, and/or the absorption peak of the single carbon-oxygen bond (C-0) of the ester groups at a wave number of approx. 1160-1190, and/or the absorption peak of the carbon-hydrogen bonds (C-H) of the fat molecule at a wave number of approx. 2870 are/is measured, the determination of fat being carried out on the basis of two or more absorption peaks in the spectrum.

10. A method according to Claim 9, characterized in that, besides the absorption peak of the carbon-carbon bonds, the absorption peak of the C=0 bond and/or the C-0 bond are/is taken into account in the determination; however, the deter¬ mination is in the main based on the IR absorption of the carbon-carbon bonds.