JP5936957B2 - Method for measuring milk fat content - Google Patents

Description

  The present invention relates to a method for measuring milk fat content in foods, and more specifically, 1,2 contained in foods by a method (LC-MS / MS) combining liquid chromatography and tandem mass spectrometry. -Dipalmitoyl-3-butyroyl-sn-glycerol (sn-PPBu) and 1-butyroyl-2,3-dipalmitoyl-sn-glycerol (sn-BuPP) were quantified, and measurement of such sn-PPBu and sn-BuPP The present invention relates to a method for calculating the content of milk fat contained in food from the value and a kit for use in measurement of milk fat content in food.

  Milk fat such as butter is one of important edible oils and fats, and since it has a characteristic flavor, milk fat is used in foods such as cookies, biscuits and bread to improve the flavor. Some processed fat products including margarine have added butter.

  Since the milk fat content in such foods affects the commercial value, it is necessary and important for manufacturers and sellers to know the content.

  Milk fat is rich in short chain fatty acids such as butyric acid and caproic acid, and this is a characteristic of milk fat not found in other fats and oils. As a conventional method for measuring milk fat content, as disclosed in Iverson, J. and Sheppard, AJJ of the AOAC, 1977, Vol. 60, pages 284-288, fats and oils are derivatized by transesterification, and the amount of butyrate ester is A method for measuring gas by gas chromatography (GC) is generalized. However, in the method of derivatizing into butyric acid ester, short-chain fatty acid esters such as butyric acid ester are highly volatile and easily soluble in water, so they are lost during derivatization treatment, and accurate milk fat There exists a problem that content may not be calculated | required.

  In recent years, a method has been reported in which fats and oils are not directly derivatized but directly introduced into GC. Buchgraber, M., Androni, S., and Anklam, EJ Agric. Food Chem. 2007, Volume 55, pages 3275-3283 include triacylglycerols containing butyric acid, such as 1-palmitoyl-2-stearoyl-3. -A method for measuring milk fat content using butyroyl-glycerol (PSB) as an index is disclosed. However, in the method of measuring the milk fat content by directly introducing PSB into GC, special equipment for preventing thermal decomposition of triacylglycerol and introducing it into GC is required.

  In addition, Kemppinen, A., and Kalo, PJ Chromatogr. A 2006, 1134, 260-283, discloses a method for measuring triacylglycerol in butter in combination with gas chromatography and mass spectrometry (GC- MS) discloses a method for determining the content of triacylglycerol. However, in order to determine the total amount of milk fat in butter by such a method using GC-MS, it is necessary to measure the content of a plurality of triacylglycerols, and there is a problem that accuracy is lacking.

  In addition, due to recent advances in oil processing technology, oil processing by transesterification is also possible with advanced transesterification technology. However, in the above method using GC, the positional isomer of triacylglycerol cannot be separated, and therefore it is impossible to detect the presence or absence of a fat processing step by transesterification.

  As a method for separating the positional isomers of triacylglycerol and measuring the content of each positional isomer, a method combining GC measurement and LC-MS / MS measurement is disclosed in International Publication No. 2008/010543. And it is disclosed by Unexamined-Japanese-Patent No. 2009-153425. In these methods, after measuring the content of each triacylglycerol in the oil and fat composition by GC, each positional isomer of oleyldipalmitoylglycerol, namely 3-oleyl-1,2-dipalmitoylglycerol ( Disclosed is a method for determining the abundance ratio of PPO) and 2-oleyl-1,3-dipalmitoylglycerol (POP) by measurement by LC-MS / MS, and determining the contents of PPO and POP in the fat and oil composition. ing.

  On the other hand, a method for measuring milk fat content using only LC-MS / MS is also disclosed in Kalo, P., Kemppinen, A., Ollilainen, V., and Kuksis, A. Lipids 2004, 39, 915-928. Is disclosed. This method uses high performance liquid chromatography (HPLC) using a normal phase column and LC-MS / MS using electron ionization, and includes a triacyl containing a short-chain fatty acid prepared by transesterification. A coefficient called a molar correction factor (MCF) is calculated from the glycerol mixture, and this can be used to quantify the content of milk fat in food samples. According to this method, milk fat in a food sample is divided into four fractions after extraction using a silica gel flash column, which are subjected to LC-MS / MS, and each detected peak is compared with MCF. Thus, the content of milk fat in the sample is determined by determining the amount of triacylglycerol corresponding to each ion. On the other hand, this method requires complicated operations such as measurement of various triacylglycerol isomer ratios of the synthesized triacylglycerol mixture by GC, and is not versatile.

International Publication 2008/010543 Pamphlet JP 2009-153425 A

Iverson, J. and Sheppard, A. J. J. of the AOAC 1977, 60, 284-288 Buchgraber, M., Androni, S., and Anklam, E. J. Agric. Food Chem. 2007, 55, 3275-3283 Kemppinen, A, and Kalo, P. J. Chromatogr. A 2006 1134 260-283 Kalo, P., Kemppinen, A., Ollilainen, V., and Kuksis, A. Lipids 2004 39: 915-928

  As shown above, conventional methods for measuring milk fat content in foods require derivatization, require special equipment, combine multiple measurement methods, or complex measurements. Is the method that is needed. Further, in the conventional method for measuring the milk fat content in food, it is necessary to extract the lipid component from the food by the Folch method or the like when measuring the milk fat content. For this reason, development of the measuring method of milk fat content with high generality and simpler was desired. That is, an object of the present invention is to provide a method for measuring milk fat content in foods that is more general and simpler.

  In order to solve the above-mentioned problems, the present inventors have intensively studied, obtained knowledge that the contents of sn-PPBu and sn-BuPP in milk fat are constant values, and used sn-PPBu and sn-BuPP as an index. The present invention was completed by finding that the contents of sn-PPBu and sn-BuPP in foods were quantified by LC-MS / MS, and the milk fat content in foods could be calculated from these quantitative values. .

That is, the present invention is (1) a method for measuring milk fat content in food, comprising the following steps (a) and (b) in sequence: (a) quantifying sn-PPBu and sn-BuPP contained in a food sample The step of:
(B) calculating the milk fat content according to the following formula 1 from the measured values of sn-PPBu and sn-BuPP;
Milk fat content (g / 100 g) = {sn-PPBu measured value (g) + sn-BuPP measured value (g)} / K × 100 [Formula 1]
(Where K is the content of sn-PPBu and sn-BuPP contained in 100 g of milk fat)
Regarding
(2) In the step (a), sn-PPBu and sn-BuPP are quantified by LC-MS / MS, and the method for measuring the milk fat content in the food according to (1) above,
(3) In the step (a), an internal standard is used, and the method for measuring the milk fat content in the food according to (1) or (2),
(4) In the step (a), the food sample is directly diluted with an organic solvent without isolating fats and oils from the food sample, and sn-PPBu and sn-BuPP are quantified (1) About the measuring method of the milk fat content in the foodstuff in any one of-(3),
(5) The method for measuring milk fat content in food according to any one of (1) to (4), further comprising (c) a step of detecting sn-PBuP in the food sample. Regarding
(6) Before the step (b), the above-mentioned (5), wherein sn-PPBu and sn-BuPP and sn-PBuP are separated by reverse phase liquid chromatography using a C28 column or a C30 column. ) Regarding the method for measuring the milk fat content in the food
(7) A kit for measuring milk fat content in a food comprising a standard solution containing a mixture of any ratio of sn-PPBu and sn-BuPP as a control,
(8) The above description (7), wherein the mixture of any ratio of sn-PPBu and sn-BuPP is 1,2-dipalmitoyl-3-butyroyl-rac-glycerol (rac-PPBu) Regarding a kit for measuring milk fat content in foods of
(9) The kit for measuring milk fat content in the food according to (7) or (8) above, further comprising a standard solution containing sn-PBuP,
(10) A standard solution containing a mixture of any ratio of sn-PPBu and sn-BuPP, a standard solution containing rac-PPBu, or a standard solution containing sn-PBuP is a standard solution containing an internal standard. It is related with the kit for measuring the milk fat content in the foodstuff in any one of said (7)-(9) characterized by the above-mentioned.

  In a conventional method for measuring milk fat contained in food, for example, a method for measuring milk fat content using butyl butyrate as an index described in Non-Patent Document 1, n volume of sodium hydroxide is added to a constant volume food sample. -A complicated pretreatment is required in which a sample solution is prepared by adding butanol solution, treating with boron trifluoride-butanol reagent, and re-volumetricizing butyl butyrate extracted using centrifugation. Further, in the method of directly applying triacylglycerol shown in Non-Patent Document 2 to GC, a cold on-column injection method must be used for GC injection, and special equipment is required. In addition to this, in the method for measuring triacylglycerol content in milk fat using LC-MS / MS described in Non-Patent Document 4, milk fat in butter is separated by silica gel thin layer chromatography (TLC). ) Or silica gel flash column chromatography to extract only the triacylglycerol component and analyze this triacylglycerol component using LC-MS / MS.

  Furthermore, in order to measure the milk fat content in foods using the conventional method, a sample sample extracted by suspending, eluting, or centrifuging the lipid components in food by the Folch method or the like is used. A complicated pretreatment such as use is necessary.

  On the other hand, according to the method for measuring milk fat content of the present invention, the food containing the target milk fat, etc., is crushed as needed, and the food is obtained only by a very simple pretreatment of volumetric adjustment. The measurement of milk fat content in can be performed. Moreover, milk fat content in a foodstuff can be calculated only by measuring the quantity of sn-PPBu and sn-BuPP. Furthermore, by using the positional isomer of sn-PPBu and sn-BuPP, that is, sn-PBuP, it is possible to qualitatively detect the presence or absence of a fat processing step by transesterification.

It is a figure which shows the outline of the preparation method of a sample solution. Method A—Extraction method by conventional method and Method B—One pot extraction method. 2 is a chromatogram of a standard solution containing rac-PPBu (rt 17.4 min) and sn-PBuP (rt 15.7 min) shown in Example 1. FIG. 2 is a calibration curve of rac-PPBu obtained by Method A of Example 1. 2 is a calibration curve of rac-PPBu obtained by Method B of Example 1.

[Measurement method of milk fat content in food]
As a method for measuring the milk fat content in the food of the present invention, the step (a) for quantifying sn-PPBu and sn-BuPP contained in the food sample, and the following formula from the measured values of sn-PPBu and sn-BuPP: 1 is not particularly limited as long as it is a method that sequentially includes the step (b) of calculating the milk fat content according to 1. Here, food refers to food products including foods, food materials, and food raw materials, including beverages. , Liquid, solid, gel, granule, powder, tablet and the like. Moreover, milk fat is lipid derived from milk of ruminants such as cows, and particularly lipid derived from cow milk can be preferably exemplified. Milk fat can be used as a raw material for butter and butter oil.

  Milk fat content (g / 100 g) = {sn-PPBu measured value (g) + sn-BuPP measured value (g)} / K × 100 [Formula 1]

  In Formula 1, the K value is the total content of sn-PPBu and sn-BuPP contained in 100 g of milk fat. Separately, the total content of sn-PPBu and sn-BuPP contained in 100 g of milk fat raw material By quantifying the amount by step (a) of the method for measuring milk fat content of the present invention, the K value can be determined.

  For example, in Example 3 in this specification, the K value is K = 3.91 when the method A is used as the sample solution preparation method for three types of butter oil, and the sample solution preparation method is as follows. When method B is used, K = 3.99. In addition, the practitioner can determine the content of the milk fat in the food by calculating the K value of the same raw material, particularly when the milk fat raw material contained in the food is known. Can be requested. In addition, when the milk fat raw material is unknown, the K value obtained by the method described in the examples in the present specification can also be used.

  In step (a), the amount of sn-PPBu and sn-BuPP in the food is determined by (i) a standard solution containing a certain amount of a mixture of sn-PPBu and sn-BuPP at an arbitrary ratio, preferably sn-PPBu: sn- A standard curve containing a certain amount of rac-PPBu with BuPP = 1: 1 is applied to LC-MS / MS, and a calibration curve is prepared. (Ii) A constant volume food sample is applied to LC-MS / MS. The determination of sn-PPBu and sn-BuPP contained in the sample can be performed by comparing with the calibration curve.

  As a method for preparing the calibration curve of (i) above, any commonly used method may be used. For example, a calibration curve can be created by plotting the concentration of rac-PPBu on the X axis and the peak intensity of rac-PPBu observed by LC-MS / MS on the Y axis. The detection limit and the quantification limit can be calculated from the ratio of peak intensity to noise (sn ratio). In addition, either an external standard method or an internal standard method may be used for preparing the calibration curve, but it is preferable to use the internal standard method.

  As a method for quantifying sn-PPBu and sn-BuPP contained in the food sample of (ii) above, the content of sn-PPBu and sn-BuPP contained in the food sample under the same conditions as the preparation of the calibration curve is determined by LC-MS. The sn-PPBu and sn-BuPP content in the food sample can be determined by measuring by / MS and plotting the obtained sn-PPBu and sn-BuPP content values on a calibration curve.

(Liquid chromatography)
As a method for quantifying sn-PPBu and sn-BuPP in a food sample in step (a), LC-MS / MS, a method combining liquid chromatography and mass spectrometry (LC-MS), gas chromatography and mass Although not particularly limited, such as a method combining analytical methods (GC-MS), a gas chromatography method (GC), a liquid chromatography method (LC), a supercritical fluid chromatography method (SFC), LC-MS / MS is preferred. . As a column used in liquid chromatography (LC) in such LC-MS / MS, a column capable of separating sn-PPBu and sn-BuPP from other components, preferably sn-PPBu, sn-BuPP and sn-PBuP, is used. As long as the column can be separated, either a normal phase column or a reverse phase column can be used, but a reverse phase column is preferably used. As the reversed-phase column, a column in which the carrier is silica gel modified with a linear hydrocarbon group is preferable, and the hydrocarbon group has 8 to 32 carbon atoms, preferably 18 to 32 carbon atoms, more preferably 28 to 30 carbon atoms. It is. As the column grade, any column of any grade can be used without any problem as long as it can separate sn-PPBu and sn-BuPP from other components, preferably sn-PPBu and sn-BuPP and sn-PBuP. It is preferable to use a column for liquid chromatography (HPLC).

  In addition, since sn-PPBu and sn-BuPP are in a relationship of enantiomers (enantiomers), they exhibit the same behavior in LC and are detected as the same peak. Similarly, they exhibit the same behavior in the following MS / MS.

  The elution solvent used in the LC in step (a) is not particularly limited, but when a normal phase column is used, for example, hexane, heptane, ethyl acetate, acetone, tetrahydrofuran, tetrahydropyran, diethyl ether, tert-butyl methyl ether , Toluene, benzene, xylene, carbon tetrachloride, chloroform, dichloromethane, dimethylformamide, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetonitrile, propionitrile, etc. It can be used as a mixed solvent.

  When using a reverse phase column, as an elution solvent, for example, water, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetonitrile, propionitrile alone, Alternatively, it can be used as a mixed solvent thereof. If necessary, an organic acid such as formic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, or a base such as triethylamine, tributylamine, N, N-diisopropylethylamine, or N-methylmorpholine is added to these solvents. be able to. Among them, a mixed solvent system of acetone / acetonitrile is preferable as an elution solvent, and the mixing ratio is acetone / acetonitrile-20: 1 to 2: 1, more preferably 10: 1 to 3: 1, more preferably 8 by volume ratio. : 1-4: 1, most preferably 4: 1.

(Tandem mass spectrometry)
The ionization method in tandem mass spectrometry (MS / MS) used in step (a) is not particularly limited as long as it is an ionization method capable of detecting the ion peaks of sn-PPBu and sn-BuPP. For example, electron ionization (EI) Method, chemical ionization (CI) method, electrolytic desorption (FD) method, electrospray ionization (ESI) method, atmospheric pressure chemical ionization (APCI) method and the like, and preferable ionization methods are CI method and FD method. The ESI method and the APCI method, more preferably the ESI method and the APCI method, and most preferably the APCI method.

As an ion peak of sn-PPBu and sn-BuPP, m / z is detected as M ⁺ = 639 which is a molecular ion peak of sn-PPBu and sn-BuPP. In addition to this, depending on the ionization method, an ammonia-added ion peak such as [M + NH ₄ ] ⁺ = 656, an ion peak from which a butyric acid group is eliminated from a molecular ion peak (m / z = 551), a molecule An ion peak (m / z = 383) from which the palmitoyl group is eliminated from the ion peak, [M + H] ⁺ = 640 which is a protonated ion peak, [M + Na] ⁺ = 662 which is a sodium ion peak, and LC which is a molecular ion peak In some cases, an ion peak or the like obtained by adding the molecular weight of the solvent used in the above is detected. Examples of a method for monitoring these product ions include multiple reaction monitoring (MRM) or selective reaction monitoring (SRM), preferably MRM.

  Advantages of using LC-MS / MS in the step (a) include the following points. When measuring sn-PPBu and sn-BuPP in food samples, since various triacylglycerols are contained in foods, sn-PPBu and sn-BuPP are separated as a single peak only by using LC. It is difficult to do. However, when LC-MS / MS is used, sn-PPBu and sn-BuPP in food are separated from other triacylglycerols in LC and then subjected to MS / MS. Even if other compounds are contained in the peaks of sn-PPBu and sn-BuPP, the contents of sn-PPBu and sn-BuPP can be measured. The principle is as follows.

  MS / MS is an apparatus in which two mass spectrometers (MS) are arranged in series and have a collision activation chamber between them. In the LC-MS / MS used in the present invention, each peak separated by LC is ionized with a specific mass number derived from sn-PPBu and sn-BuPP after first ionizing the sample with the first MS. Can be detected by the second MS. For this reason, even if other compounds are included in the peaks of sn-PPBu and sn-BuPP separated by LC, the ion peak derived from this compound is not selected when the second MS is introduced. From this, even if other components are mixed in n-PPBu and sn-BuPP after LC, the same data as that obtained by measuring only n-PPBu and sn-BuPP can be obtained by using MS / MS. it can.

(Internal standard method)
In the step (a), an internal standard method can be used as necessary to facilitate the quantification of sn-PPBu and sn-BuPP. As a method for measuring the milk fat content using the internal standard method, a mixture of any ratio of sn-PPBu and sn-BuPP, preferably a standard solution containing rac-PPBu, which is an equivalent mixture thereof, and a sample solution are used. Preferably, the same internal standard is added.

The internal standard is not particularly limited as long as it can quantitate sn-PPBu and sn-BuPP when using LC-MS / MS, and preferably has an odd total acyl carbon number that does not exist in natural fats and oils. Triacylglycerol. As the triacylglycerol as the internal standard, commercially available triacylglycerol may be used, or one prepared according to the application may be used. In preparation, because of its simplicity, all three acyl groups are preferably triacylglycerols, more preferably triacylglycerols having C ₉ , C ₁₁ , C ₁₃ , C ₁₅ acyl groups, C ₁₁ , more preferably triacylglycerol having an acyl group of C _13, triacylglycerol having an acyl group of C ₁₁ (tri undecapeptide Noin) are most preferred. On the other hand, triacylglycerols consisting of even-chain fatty acids with an even total acyl carbon number are naturally abundant, so it is considered better to avoid using them as internal standards, but prohibiting their use. Absent.

(Sample preparation)
In the step (a), the extraction method of the lipid component from the food sample is not particularly limited as long as the lipid component is sufficiently extracted. For example, the conventional extraction method (Method A) and the method developed in the present invention are used. One-pot extraction method (Method B) can be used. FIG. 1 shows a schematic sample solution preparation method (method A and method B) from a food sample in the method for measuring milk fat content of the present invention.

  As the above method A, after accurately measuring the mass of the sample, the sample is extracted with an organic solvent, washed with an aqueous solvent, concentrated, and then the solution of the concentrate whose volume has been accurately measured is obtained. A sample solution for measurement of milk fat content of the present invention can be obtained.

  As a method for extracting a lipid component from a sample, any extraction method can be used as long as the lipid component is not reduced. For example, a Folch method, a Bligh & Dyer method, or the like can be used. Extraction solvents are not limited to these, but include methanol, ethanol, 1-propanol, 2-propanol, diethyl ether, tert-butyl methyl ether, tetrahydrofuran, dichloromethane, chloroform, pentane, hexane, petroleum ether, acetone, ethyl acetate. , Benzene, toluene and the like can be used alone or in combination. Among them, when using the Folch method or the Bligh & Dyer method, a chloroform / methanol-2: 1 (v / v) mixed solvent is used as the extraction solvent. On the other hand, it is considered better to avoid the use of non-volatile or high-boiling solvents such as dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, but the use of these is not prohibited.

  The solution after extraction is concentrated after washing the oil phase with an aqueous solvent. By this washing operation, impurities such as salt and sugar can be removed. As a method for separating the oil phase and the aqueous phase, centrifugal separation may be used as long as it is difficult to separate the oil phase and the aqueous phase using a separatory funnel. The oil phase after washing with water is preferably dehydrated with a desiccant and then concentrated, and anhydrous sodium sulfate, anhydrous magnesium sulfate, or the like may be used as the dehydrating agent. Examples of the concentration method of the extraction solution include a method of concentrating using an evaporator and volatilizing the solvent by blowing an inert gas such as argon or nitrogen to the extraction solution.

  The residue after concentration can be used as a sample solution by dissolving in the measurement solvent. When preparing a sample solution, it is necessary to accurately prepare the volume of the solution by using a volumetric flask or the like. In this case, examples of the solvent that can be used include methanol, ethanol, 1-propanol, 2-propanol, acetone, acetonitrile, and the like, preferably ethanol, 1-propanol, 2-propanol, acetone, more preferably 2- Propanol, acetone, most preferably 2-propanol.

  As the above-mentioned method B, after accurately measuring the mass of the sample, the sample solution used for the LC-MS / MS analysis can be easily obtained by extracting with a solvent used for the direct LC-MS / MS analysis. In this case, examples of the solvent that can be used include methanol, ethanol, 1-propanol, 2-propanol, acetone, acetonitrile, and the like, preferably ethanol, 1-propanol, 2-propanol, acetone, more preferably 2- Propanol, acetone, most preferably 2-propanol.

(Characteristics of milk fat)
Milk fat is rich in short chain fatty acids such as butyric acid and caproic acid, and this is a characteristic of milk fat not found in other fats and oils. Milk fat contains sn-PPBu and sn-BuPP, but does not contain sn-PBuP, which is a positional isomer thereof. On the other hand, since the oil and fat composition that has undergone the oil and fat processing step by transesterification contains both sn-PPBu and sn-BuPP and sn-PBuP, the step of detecting sn-PBuP in the method for measuring milk fat content of the present invention By providing (c), it is possible to qualitatively determine the presence or absence of an oil processing step by transesterification.

  In step (c), the qualitative determination of the presence or absence of the fat-and-oil processing step by transesterification is performed in the above-mentioned step (a). In addition, sn-PBuP is added to the column, and the column used for LC is reversed phase liquid chromatography using a C28 column or C30 column. At this time, sn-PPBu and sn-BuPP and sn-PBuP showed good separation when the eluent was an acetone / acetonitrile mixed solvent, and the mixing ratio was acetone / acetonitrile-20: It is 1-2: 1, more preferably 10: 1-3: 1, still more preferably 8: 1-4: 1, and most preferably 4: 1.

  In step (c), sn-PPBu and sn-BuPP can be quantified in the same manner as in step (a), and an internal standard method can also be used for quantification. When using the internal standard method, it is preferred to add the same internal standard to a standard solution and sample solution containing a mixture of any proportion of sn-PPBu and sn-BuPP, preferably rac-PPBu and sn-PBuP.

  Furthermore, since triacylglycerol derived from vegetable oils and fats such as margarine does not contain sn-PPBu or sn-BuPP, butter and margarine etc. are mixed by using the present invention using sn-PPBu and sn-BuPP as an index. Also when measuring the milk fat contained in foods used (for example, cookies, biscuits, sachets, breads, etc.), the milk fat content in the food can be easily measured.

[Kit for measuring milk fat content in foods]
The kit for measuring the milk fat content in the food of the present invention is not particularly limited as long as it is a kit having a standard solution containing a mixture of any ratio of sn-PPBu and sn-BuPP, preferably rac-PPBu. Although not, it may further include sn-PBuP. When sn-PBuP is contained in the standard solution, a mixture of any ratio of sn-PPBu and sn-BuPP, preferably a mixing ratio of rac-PPBu and sn-PBuP is a molar ratio, preferably 9: 1. ˜1: 9, more preferably 4: 6 to 6: 4, still more preferably an equal mixture.

  The sn-PPBu, sn-BuPP, and sn-PBuP can be naturally derived or synthesized, and rac-PPBu can be synthesized. Furthermore, the standard solution can be a standard solution containing the internal standard described above. The kit of the present invention can be provided with a package insert explaining the method for measuring the milk fat content in the food of the present invention.

(Synthesis of rac-PPBu)
Examples of the method for synthesizing rac-PPBu and sn-PPBu include, but are not limited to, the following methods.
As a method of chemically synthesizing rac-PPBu, a method of esterifying 1,2-dipalmitoyl-rac-glycerin and butyric acid, or a basic condition of 1,2-dipalmitoyl-rac-glycerin and butyric chloride is used. There is a method of condensing below. In addition, it can be synthesized by esterifying rac-1-monobutyrin and palmitic acid, or by condensing rac-1-monobutyrin and palmitoyl chloride under basic conditions. As a general synthesis method, a target rac-PPBu can be synthesized by dissolving a substrate in an organic solvent and allowing a dehydrating agent, a condensing agent, or a base to act in an inert gas atmosphere. Here, as the inert gas, argon, dried nitrogen, or the like can be used. Examples of the organic solvent that can be used include, but are not limited to, dichloromethane, chloroform, toluene, N, N-dimethylformamide, and the like, preferably dichloromethane. As the dehydrating agent, acid-based dehydrating agents such as concentrated sulfuric acid and paratoluenesulfonic acid can be used. As the condensing agent, a carbodiimide-based condensing agent or the like can be used. If necessary, pyridine, 4-N, N-dimethylpyridine, 4-pyrrolidinopyridine or the like may be used. As the base, organic bases such as triethylamine, tributylamine, ethyldiisopropylamine, N-methylmorpholine, and pyridine can be used.

(Synthesis of sn-PPBu)
In the above synthesis method, an enantiomer of sn-PPBu is synthesized by using an enantiomer of 1,2-dipalmitoyl-sn-glycerine instead of 1,2-dipalmitoyl-rac-glycerin or rac-1-monobutyrin. You can also.

(Synthesis of sn-PBuP)
Examples of a method for synthesizing sn-PBuP include a method of esterifying 2-monobutyrin and palmitic acid, and a condensation of 2-monobutyrin and palmitoyl chloride under basic conditions. As the reaction conditions, the same conditions as in the method for synthesizing rac-PPBu can be used.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
[Example]
In the examples, the following analytical instruments were used.
LC-MS / MS
LC system: Alliance e2695 (Waters Corporation)
Column: Sunrise C28, 4.6 mm ID x 250 mm, 5 μm, (ChromaNik Technologies Inc.)
UV-visible light detector: 2489 UV Visible Detector (manufactured by Waters Corporation)
APCI / MS system: Quattro micro API (Waters Corporation)
Analysis software: MassLynx Ver. 4.1 (manufactured by Waters Corporation)
GC
GC system: HP6890 (manufactured by Agilent Technologies)
Column: CP-WAX 52 CB, 30m x 0.25mm ID (Varian)
Detector: Flame ionization detector (Agilent Technologies)

Preparation of calibration curve Seven concentrations of rac-PPBu and sn-PBuP were dissolved in 2-propanol and the final concentrations were 250, 200, 150, 100, 50, 10, and 1 μg / mL, respectively. A standard solution was prepared. To each standard solution, triundecanoin was added to a final concentration of 10 μg / mL, and used as an internal standard. 10 μL of the prepared standard solution was subjected to LC-MS / MS. Separation of rac-PPBu and sn-PBuP by LC was as shown in FIG. 1, and each peak showed good separation.
MS / MS ionization was performed by atmospheric pressure chemical ionization (APCI), and multiple reaction monitoring (MRM) was used as a data collection mode. The detection ions are these ammonia-added molecular ions, [M + NH ₄ ] ⁺ = 656. Based on this result, the concentration of the standard solution is plotted on the x-axis, and the ratio of the ionic strength of rac-PPBu or sn-PBuP and triundecanoin is plotted on the y-axis, thereby calibrating rac-PPBu and sn-PBuP. A line was created (FIGS. 3 and 4). The detection limit and the quantification limit were calculated from the ratio of peak intensity to noise (sn ratio), and were sn = 3 and 10, respectively.

The LC-MS / MS measurement at the time of preparing the calibration curve was performed under the following conditions.
LC
Solvent: acetone / acetonitrile-80: 20 (v / v)
Flow rate: 1 mL / min Column temperature: 15 ° C
MS
Ion source: polarity, APCI positive; corona current, 3.0 μA
Ion source temperature: 120 ° C
Desolvation temperature: 450 ° C
cone gas flow: 50L / hour
desolvation gas flow: 200L / hour Data collection mode: Multiple reaction monitoring (MRM)

Synthesis of rac-PPBu In a 100 mL four-necked flask purged with argon, 1,2-dipalmitoyl-rac-glycerin (1 g), butyric acid (0.17 g), 4-dimethylaminopyridine (0.24 g), and Dichloromethane (10 mL) was added. N-ethyldiisopropylamine (0.25g) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.37g) were added there, and it stirred at -20 degreeC overnight. The oily compound obtained by directly concentrating the reaction solution was subjected to flash column chromatography to obtain colorless oily rac-PPBu (0.8 g, yield 72%).
CI-MS: m / z 551 (45%), 384 (100%)
APCI-MS: m / z 656 [M + NH ₄ ] ⁺

Synthesis of PBuP 1,3-dipalmitin (1 g), butyric acid (0.17 g), 4-dimethylaminopyridine (0.24 g), and dichloromethane were added to a 100 mL four-necked flask replaced with an inert gas (argon). (10 mL) was added. N-ethyldiisopropylamine (0.25g) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.37g) were added there, and it stirred at -20 degreeC overnight. The oily compound obtained by directly concentrating the reaction solution was subjected to flash column chromatography to obtain a colorless oily sn-PBuP (0.5 g, yield 45%).
CI-MS: m / z 551 (25%), 384 (100%)
APCI-MS: m / z 656 [M + NH ₄ ] ⁺

Measurement of sn-PPBu content in butter

Method A
As measurement samples, lipids were extracted from three commercially available butters by the Folch method. First, butter was diluted 9 times (volume ratio) with distilled water and added to a double amount of chloroform / methanol solution (volume ratio 2: 1) in a test tube equipped with a screw stopper. This mixed solution was vigorously mixed using a vortex mixer, and centrifuged at 1,500 G for 10 minutes. After collecting the lower layer, it was concentrated using a nitrogen stream at room temperature, and the lipid was weighed to obtain butter oil. A sample solution was prepared by dissolving this butter oil in 2-propanol to a final concentration of 5 mg / mL. At the same time, triundecanoin was added to the sample solution to a final concentration of 10 μg / mL, and used as an internal standard. The sample solution was subjected to LC-MS / MS, and the contents of sn-PPBu and sn-BuPP in butter oil are shown in Table 1 based on the obtained ion peak and the calibration curve of Example 1 (FIG. 3). Could be decided. At this time, rac-PBuP was not detected.

Method B
As measurement samples, three types of commercially available butters identical to the butters used in Method A were dissolved in 2-propanol, and sample solutions were prepared so as to have final concentrations of 5 mg / mL, respectively. At the same time, triundecanoin was added to the sample solution to a final concentration of 10 μg / mL, and used as an internal standard. The sample solution was subjected to LC-MS / MS, and the contents of sn-PPBu and sn-BuPP in butter oil are shown in Table 1 based on the obtained ion peak and the calibration curve of Example 1 (FIG. 4). Could be decided. At this time, rac-PBuP was not detected.

Measurement of milk fat content of commercial margarine (using 15% butter)

Method A
When the contents of sn-PPBu and sn-BuPP in margarine with 15% commercial butter were measured under the same conditions as in Method A of Example 3, sn-PPBu was 0.58 ± 0. It was found that 01 g was contained. When this was applied to Formula 1, the milk fat content of commercially available butter 15% margarine was 14.76 ± 0.26 g, and this margarine contained almost the same amount of butter as indicated. I understood.

Method B
When the content of sn-PPBu in commercially available butter 15% margarine was measured under the same conditions as in Method B of Example 3, it was found that 0.59 ± 0.01 g of sn-PPBu was contained in 100 g of margarine extracted oil. I understood. When this was applied to Formula 1, the milk fat content of commercially available butter 15% margarine used was 14.81 ± 0.19 g, and this margarine contained almost the same amount of butter as indicated. I understood.

[Reference Experiment 1]
Measurement of butyric acid content in butter by GC method (butyric acid measurement method) 100 mg of three commercially available butters used in Example 1 were dissolved in 2 mL of n-hexane, and 1 mL of butyl isovalerate n-hexane standard A solution (0.5 mg / mL) and 2M potassium hydroxide in n-butanol were added. After the previous solution was vigorously stirred with a vortex mixer, distilled water was added and centrifuged at 3,000 G for 10 minutes. The n-hexane layer was collected and dried over anhydrous sodium sulfate to prepare a sample solution. This sample solution was subjected to GC, and the butyric acid content in butter oil could be determined as shown in Table 2.

Measurement of milk fat content in mixed sample of butter oil (BO) and margarine oil (MO)

Method A
Lipid was extracted from commercially available butter and margarine by the Folch method in the same manner as in Method A of Example 3 to prepare butter oil (BO) and margarine oil (MO). Four types of samples in which BO and MO were mixed at a weight ratio (BO: MO) of 5:95, 20:80, 50:50, and 80:20 were dissolved in 2-propanol, respectively, to a final concentration of 5 mg / A sample solution was prepared to be mL. At the same time, triundecanoin was added to the sample solution to a final concentration of 10 μg / mL, and used as an internal standard. This sample solution was subjected to LC-MS / MS, and the content of sn-PPBu in each sample solution could be determined from the obtained ion peak and the calibration curve of Example 1 (FIG. 3). As a result, the milk fat content in each sample could be determined as shown in Table 3.

GC method (butyric acid measurement method)
100 mg of each of the above four types of samples was dissolved in 2 mL of n-hexane, and 1 mL of a standard solution of butyl isovalerate in n-hexane (0.5 mg / mL) and 2M potassium hydroxide in n-butanol were added. After the previous solution was vigorously stirred with a vortex mixer, distilled water was added and centrifuged at 3,000 G for 10 minutes. The n-hexane layer was collected and dried over anhydrous sodium sulfate to prepare a sample solution. The sample solution was subjected to GC, and the content of dairy butyl in each sample solution was quantified, whereby the content of milk fat in each sample could be determined as shown in Table 3.

  The milk fat content in the mixed sample of butter oil and margarine oil, obtained by using the method A and the GC method, was almost the same value, and they almost coincided with the milk fat amount (calculated value) in the mixed sample. From this, it can be seen that this method is extremely effective as a method for measuring the milk fat content in food.

Milk fat content of commercially available butter cookies (using 3% butter)

Method A
For a butter cookie that is labeled as using 3% butter in a commercially available package, 1 g of the butter cookie is crushed, diluted 9 times (volume ratio) with distilled water, and doubled in a test tube with a screw stopper. It was added to a chloroform / methanol solution (volume ratio 2: 1). This mixed solution was vigorously mixed using a vortex mixer, and centrifuged at 1,500 G for 10 minutes. After collecting the lower layer, it was concentrated using a nitrogen stream at room temperature, and the lipid was weighed to obtain butter oil. A sample solution was prepared by dissolving this butter oil in 2-propanol to a final concentration of 5 mg / mL. At the same time, triundecanoin was added to the sample solution to a final concentration of 10 μg / mL, and used as an internal standard. As a result, 0.38 ± 0.02 g of sn-PPBu is contained in 100 g of butter cookie extract oil, and K _A = 3.91 obtained in Example 3 and 100 g of butter cookie milk from the formula I. The fat content was determined to be 9.64 ± 0.42 g.

Method B
1 g of the butter cookie was crushed and diluted with 2-propanol to obtain a 40 mg / mL sample solution. At the same time, triundecanoin was added to the sample solution to a final concentration of 10 μg / mL, and used as an internal standard. This sample solution was filtered through a 0.45 μm membrane filter and then subjected to LC-MS / MS, and it was confirmed that the content of sn-PPBu in 100 g of butter cookies was 0.39 ± 0.00 g. Moreover, milk fat content of butter cookies 100g from _K B = 3.99 and the formula I obtained in Example 3 was determined to be 9.73 g ± 0.10.

  In both method A and method B, the milk fat content in the butter cookie is almost the same as the content described in the package, and this method is suitable as a method for measuring milk fat in food ingredients. Recognize.

The method for measuring the content of milk fat according to the present invention comprises quantifying sn-PPBu and sn-BuPP contained in food by combining liquid chromatography and tandem mass spectrometry (LC-MS / MS). The content of milk fat contained therein can be easily measured. For this reason, conventional methods for measuring the content of milk fat in foods require complicated pretreatment, derivatization, or special equipment, those that combine multi-step measurement methods, and complicated measurements. On the other hand, the method for measuring the content of milk fat according to the present invention is a very simple pretreatment in which a food containing milk fat is pulverized and fixed as necessary. As a result, the milk fat content in the food can be measured, so that the working efficiency can be greatly improved.
Thus, the present invention is useful in the food industry.

Claims (10)

The measuring method of the milk fat content in foodstuffs provided with the following processes (a) and (b) sequentially.
(A) Quantification of 1,2-dipalmitoyl-3-butyroyl-sn-glycerol (sn-PPBu) and 1-butyroyl-2,3-dipalmitoyl-sn-glycerol (sn-BuPP) contained in food samples The step of:
(B) calculating the milk fat content from the measured values of sn-PPBu and sn-BuPP by the following formula;
Milk fat content (g / 100 g) = {measured value of sn-PPBu (g) + measured value of sn-BuPP (g)} / K × 100
(Where K is the content of sn-PPBu and sn-BuPP contained in 100 g of milk fat) The method for measuring milk fat content in foods according to claim 1, wherein in the step (a), sn-PPBu and sn-BuPP are quantified by LC-MS / MS. The method for measuring milk fat content in foods according to claim 1 or 2, wherein an internal standard is used in step (a). In the step (a), the food sample is directly diluted with an organic solvent without isolating fats and oils from the food sample, and sn-PPBu and sn-BuPP are quantified. A method for measuring milk fat content in foods. The method according to claim 1, further comprising: (c) detecting 1,3-dipalmitoyl-2-butyroyl-sn-glycerol (sn-PBuP) in a food sample. Of milk fat content in Japanese food. 6. The food according to claim 5, wherein, prior to step (b), sn-PPBu and sn-BuPP and sn-PBuP are separated by reverse phase liquid chromatography using a C28 column or C30 column. Method for measuring the milk fat content. A mixture of 1,2-dipalmitoyl-3-butyroyl-sn-glycerol (sn-PPBu) and 1-butyroyl-2,3-dipalmitoyl-sn-glycerol (sn-BuPP) as a control in any ratio A kit for measuring a milk fat content in a food, characterized in that it comprises a standard solution containing. 8. The food product according to claim 7, wherein the mixture of any ratio of sn-PPBu and sn-BuPP is 1,2-dipalmitoyl-3-butyroyl-rac-glycerol (rac-PPBu). Kit for measuring milk fat content. The kit for measuring a milk fat content in a food according to claim 7 or 8, wherein the standard solution further comprises 1,3-dipalmitoyl-2-butyroyl-sn-glycerol (sn-PBuP). . A standard solution containing a mixture of any ratio of sn-PPBu and sn-BuPP, a standard solution containing a rac-PPBu standard solution, or a standard solution containing sn-PBuP is a standard solution containing an internal standard. The kit for measuring the milk fat content in the food according to any one of claims 7 to 9.

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