Methods for measuring protein

Abstract

To provide a protein content measurement method which suppresses occurrence of bumping due to heating and does not require a long digestion process at low heating temperature when measuring protein content of a sample containing diatomaceous earth by the Kjeldahl method.SOLUTION: A protein content measurement method is provided, comprising Kjeldahl digestion step of adding 50-300 mg of boiling stones per 10 mL of concentrated sulfuric acid to a sample so as to cause sulfuric acid digestion of the sample at 300-480°C without causing bumping.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for measuring proteins. In particular, it relates to a method for measuring the amount of protein in a sample containing diatomaceous earth by the Kjeldahl method.

Background Art

[0002] According to the Dietary Fiber Society, lumina coid is defined as "a food component that is difficult to be digested and absorbed in the human small intestine and exhibits physiological effects useful for maintaining health through the digestive tract."

[0003] The components included in such a "lumina coid" concept are roughly classified into non-starch components and starch components. Non-starch components are classified into, in addition to the狭义 "dietary fiber" according to the definition of CCNFSDU, oligosaccharides, sugar alcohols, resistant proteins, and others. On the other hand, starch components are classified into resistant starch and resistant maltodextrin.

[0004] Among these "lumina coid" components, particular attention is paid to "resistant protein" which refers to a protein component or a protein complex component among non-starch components.

[0005] Known resistant proteins include soybean-derived proteins such as soybean high molecular weight protein fraction (HMF) and soybean resistant protein (SRP), buckwheat-derived protein, silk-derived protein, sake lees-derived protein, and the like.

[0006] Resistant protein is expected to be applied to various uses as food and beverages and pharmaceuticals having a health-promoting effect.

[0007] However, regarding its quantification method, only a few proposals have been made so far, and no established method has been proposed.

[0008] Non-patent document 1 proposes a quantitative method for determining the amount of resistant protein by enzymatically treating a sample and measuring the amount of protein in the remaining enzyme-treated residue using a combustion method. This method has the advantage of being able to quantify the amount of resistant protein regardless of its composition or physical properties. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Yuta Takahashi, Research on Resistant Proteins in Soybeans and Tofu, Nagano Prefectural Industrial Technology Center Research Report No. 15, pp. F28-F29 (2020) [Overview of the project] [Problems that the invention aims to solve]

[0010] In measuring the amount of protein, i.e., the amount of resistant protein, in a material containing resistant protein as a protein component, it is conceivable to perform a preliminary step, as proposed in Non-Patent Document 1, by enzymatically treating the material to obtain an enzymatic treatment residue, and then use the enzymatic treatment residue obtained in the preliminary step to measure the amount of protein.

[0011] Possible methods for measuring protein content in this case include the Kjeldahl method, which involves a step of decomposing the sample in concentrated sulfuric acid, and the combustion method. The protein content measurement method proposed in Non-Patent Document 1 employs the combustion method.

[0012] In the protein content measurement method proposed in Non-Patent Document 1, a step of filtering with diatomaceous earth is employed as a preliminary step to preparing the enzyme-treated residue to be used for protein content measurement by combustion. Therefore, the enzyme-treated residue used for protein content measurement contains diatomaceous earth.

[0013] Therefore, if one were to use the Kjeldahl method to measure protein content instead of the combustion method used in the protein content measurement method proposed in Non-Patent Document 1, the enzyme-treated residue containing diatomaceous earth, which was used as a filter material in the preceding process, would be subjected to sulfuric acid decomposition.

[0014] Concentrated sulfuric acid containing diatomaceous earth is prone to sudden boiling, and if you try to lower the heating temperature to prevent this, you will have to allow for a longer decomposition time.

[0015] Therefore, the aim of this invention is to propose a method for measuring the protein content of a sample containing diatomaceous earth using the Kjeldahl method, which suppresses the occurrence of sudden boiling due to heating and does not require a long decomposition process at a low heating temperature.

[0016] Furthermore, this invention aims to propose a protein content measurement method that suppresses the occurrence of bumping due to heating and eliminates the need for a long decomposition process at a low heating temperature, even when the enzyme-treated residue, which contains diatomaceous earth used in the filtration process prior to obtaining the enzyme-treated residue, is subjected to protein content measurement by the Kjeldahl method, in order to measure the amount of protein in a material containing resistant protein as a protein component, i.e., the amount of resistant protein, by enzymatically treating the material as a pretreatment and then measuring the amount of protein by the Kjeldahl method. [Means for solving the problem]

[0017] The inventors of this invention discovered that boiling chips, which are widely used to prevent bumping, can also be applied to this method, and thus completed the present invention.

[0018] In other words, the present invention provides, for example, the following embodiments. [1] A protein measurement method for measuring the amount of protein in a sample containing diatomaceous earth by the Kjeldahl method, A Kjeldahl decomposition step is provided in which 50 mg to 300 mg of boiling stones are added per 10 ml of concentrated sulfuric acid, and the sample is decomposed with sulfuric acid without causing bumping at a heating temperature of 300 °C to 480 °C. Protein measurement method.

[0019] [2] In the Kjeldahl decomposition step, 100 mg to 300 mg of boiling stones are added per 10 ml of concentrated sulfuric acid, and the sulfuric acid decomposition of the sample is carried out without causing bumping at a heating temperature of 480 °C. The protein measurement method of [1].

[0020] [3] A protein measurement method for measuring the amount of the protein in a material containing the protein as a component by the Kjeldahl method, An enzyme treatment step of treating the material with an enzyme to obtain an enzyme treatment residue, and A Kjeldahl decomposition step of Kjeldahl decomposing the enzyme treatment residue, and Since the step of filtering using diatomaceous earth is included in the enzyme treatment step, the enzyme treatment residue contains diatomaceous earth, In the Kjeldahl decomposition step, 50 mg to 300 mg of boiling stones are added per 10 ml of concentrated sulfuric acid, and the sulfuric acid decomposition of the enzyme treatment residue is carried out without causing bumping at a heating temperature of 300 °C to 480 °C. Protein measurement method.

[0021] [4] In the Kjeldahl decomposition step, 100 mg to 300 mg of boiling stones are added per 10 ml of concentrated sulfuric acid, and the sulfuric acid decomposition of the enzyme treatment residue is carried out without causing bumping at a heating temperature of 480 °C. The protein measurement method of [3].

[0022] [5] The protein measurement method of [3] or [4], wherein the material comprises any one or a combination of a plurality of kinds selected from soybeans, soy-derived foods, buckwheat, silk, and sake lees.

[0023] [6] The method for measuring a protein according to [1] or [2], wherein the protein is a resistant protein.

[0024] [7] The method for measuring a protein according to any one of [3] to [5], wherein the protein is a resistant protein. [Advantages of the Invention]

[0025] According to the present invention, when measuring the protein content of a sample containing diatomaceous earth by the Kjeldahl method, it is possible to provide a method for measuring the protein content that suppresses the occurrence of bumping due to heating and does not require a long decomposition process at a low heating temperature.

[0026] Further, according to the present invention, in the measurement of the protein content in a material containing a resistant protein as a protein component, that is, in the measurement of the protein content for measuring the amount of the resistant protein, when the material is enzymatically treated as a pretreatment and then the protein content is measured by the Kjeldahl method, even if the enzyme-treated residue containing diatomaceous earth used in the filtration in the previous step of obtaining the enzyme-treated residue by the enzyme treatment is subjected to the measurement of the protein content by the Kjeldahl method, it is possible to provide a method for measuring the protein content that suppresses the occurrence of bumping due to heating and does not require a long decomposition process at a low heating temperature. [Modes for Carrying Out the Invention]

[0027] Hereinafter, the present invention will be described based on its embodiments and examples. However, the present invention is not limited thereto, and any modification can be made and implemented within the technical scope grasped from the description of the claims.

[0028] [Overview] The present inventors have conducted various studies on a method for quantifying a resistant protein known to have a predetermined lipid metabolism improving effect.

[0029] In this study, as proposed in Non-Patent Document 1, we decided to use the enzyme-treated residue obtained by enzymatically decomposing a material containing resistant protein as a protein component for protein content measurement.

[0030] Furthermore, instead of the combustion method used in the protein quantity measurement method proposed in Non-Patent Document 1, we investigated a protein quantity measurement method that uses the Kjeldahl method, and that does not cause bumping due to heating during the sulfuric acid decomposition process, even though the enzyme-treated residue containing diatomaceous earth, which was used as a filter material in the process of obtaining the enzyme-treated residue, is decomposed by sulfuric acid using the Kjeldahl method, and does not require a long decomposition process at a low heating temperature.

[0031] As a result, we unexpectedly discovered that by applying conventionally known boiling chips, the quantification time could be shortened without affecting the quantitative value. This invention is based on this unexpected finding of the inventor.

[0032] [Resistant Protein] In this invention, "resistant protein" refers, as described above, to protein components or protein complex components among the non-starch components included in the concept of "luminacoids." In this invention, "luminacoid" is defined, in accordance with the definition of the Japanese Society for Dietary Fiber Research, as "a food component that is poorly digested and absorbed in the human small intestine and exerts physiological effects that contribute to maintaining health through the digestive tract."

[0033] Examples of resistant proteins include proteins derived from various raw materials such as soybeans, buckwheat, silk, and sake lees. All of these are targets for measurement in this invention.

[0034] (First embodiment) Based on the inventors' findings described above, one embodiment of the present invention is a protein measurement method for measuring the amount of protein in a sample containing diatomaceous earth by the Kjeldahl method.

[0035] This protein measurement method includes a Kjeldahl decomposition step in which 50 to 300 milligrams of boiling chips are added to 10 milliliters of concentrated sulfuric acid, and the sample is decomposed by sulfuric acid at a heating temperature of 300°C to 480°C without causing bumping.

[0036] In the above, there are no particular limitations on the boiling chips; any commercially available boiling chips used to prevent bumping when heating a liquid to a boil can be used.

[0037] Furthermore, the degradation accelerators conventionally used in Kjeldahl degradation (sulfate degradation) performed for protein quantity measurement can be added to the Kjeldahl degradation (sulfate degradation) process described above.

[0038] Concentrated sulfuric acid containing diatomaceous earth is prone to bumping, and lowering the heating temperature to prevent this requires a longer decomposition time. However, by adding 50 to 300 milligrams of boiling chips per 10 milliliters of concentrated sulfuric acid during sulfuric acid decomposition (Kjeldahl decomposition), sulfuric acid decomposition (Kjeldahl decomposition) can be performed at heating temperatures of 300°C to 480°C without causing bumping.

[0039] Since sulfuric acid decomposition is performed at heating temperatures of 300°C to 480°C, a protein quantity measurement method can be provided that does not require a long decomposition process at low heating temperatures.

[0040] Furthermore, by adding 50 to 300 milligrams of boiling chips per 10 milliliters of concentrated sulfuric acid, it is possible to suppress the occurrence of bumping due to heating and perform sulfuric acid decomposition (Kjeldahl decomposition).

[0041] According to the inventor's research, when measuring the amount of protein in a sample containing diatomaceous earth using the Kjeldahl method, employing the aforementioned specific steps in the sulfuric acid decomposition (Kjeldahl decomposition) process did not affect the measurement of the protein amount.

[0042] In the Kjeldahl decomposition step of the protein measurement method described above, 100 to 300 milligrams of boiling chips are added per 10 milliliters of concentrated sulfuric acid, and the sulfuric acid decomposition of the sample can be performed at a heating temperature of 480°C without causing bumping.

[0043] As mentioned above, when using the Kjeldahl method to measure the amount of protein in a sample containing diatomaceous earth, it has been confirmed that adding boiling chips can suppress the occurrence of bumping during the heating step of sulfuric acid decomposition, and that this does not affect the measurement of protein content. However, according to the inventor's research, the amount of boiling chips added was related to the height of the heating temperature at which bumping could be suppressed, and the length of time required to complete sulfuric acid decomposition without causing bumping.

[0044] Therefore, as described above, by adding 100 to 300 milligrams of boiling chips per 10 milliliters of concentrated sulfuric acid in the Kjeldahl decomposition process and heating at a temperature of 480°C, the sulfuric acid decomposition of the sample can be carried out without causing bumping.

[0045] By performing sulfuric acid decomposition at a heating temperature of 480°C, the time required for sulfuric acid decomposition (Kjeldahl degradation) can be shortened, providing a protein quantity measurement method that does not require a long decomposition process at a low heating temperature.

[0046] In the protein measurement method described above, the protein can be a resistant protein.

[0047] Therefore, according to the protein measurement method described above, the amount of protein in a material containing resistant protein as a protein component, i.e., the amount of resistant protein, can be effectively measured.

[0048] (Second Embodiment) Another embodiment of the present invention is a protein measurement method for measuring the amount of protein in a material containing protein as a component by the Kjeldahl method, comprising an enzymatic treatment step of enzymatically treating the material to obtain an enzymatic treatment residue, and a Kjeldahl decomposition step of Kjeldahl decomposing the enzymatic treatment residue.

[0049] In the protein measurement method of this embodiment, since the enzyme treatment step includes a step of filtration using diatomaceous earth, the enzyme treatment residue contains diatomaceous earth.

[0050] Furthermore, in the Kjeldahl decomposition process, 50 to 300 milligrams of boiling chips are added per 10 milliliters of concentrated sulfuric acid, and the sulfuric acid decomposition of the enzyme treatment residue is carried out at a heating temperature of 300°C to 480°C without causing bumping.

[0051] In this embodiment as well, there are no particular limitations on the boiling chips; any commercially available boiling chips used to prevent bumping when heating a liquid to a boil can be used.

[0052] Furthermore, the degradation accelerators conventionally used in Kjeldahl degradation (sulfate degradation) performed for protein quantity measurement can be added to the Kjeldahl degradation (sulfate degradation) process described above.

[0053] As described in the first embodiment above, concentrated sulfuric acid containing diatomaceous earth is prone to bumping, and if the heating temperature is lowered to prevent bumping, the decomposition time must be extended. In this embodiment, since the sample subjected to the Kjeldahl decomposition process is an enzyme treatment residue containing diatomaceous earth, if Kjeldahl decomposition (sulfuric acid decomposition) is performed using a conventionally known method, there is a risk of bumping, and if a conventionally known method is used, the heating temperature must be lowered and the decomposition time extended to prevent bumping.

[0054] In contrast, in this embodiment, in the Kjeldahl decomposition step, 50 to 300 milligrams of boiling chips are added per 10 milliliters of concentrated sulfuric acid, and the sulfuric acid decomposition (Kjeldahl decomposition) of the enzyme treatment residue can be performed at a heating temperature of 300°C to 480°C without causing bumping.

[0055] Since sulfuric acid decomposition is carried out at heating temperatures of 300°C to 480°C, it does not require a long decomposition process at low heating temperatures. By adding 50 to 300 milligrams of boiling chips per 10 milliliters of concentrated sulfuric acid, the occurrence of bumping due to heating can be suppressed, and sulfuric acid decomposition (Kjeldahl decomposition) can be performed.

[0056] According to the inventor's research, this embodiment also did not affect the measurement of protein content.

[0057] As explained in the first embodiment described above, when employing the Kjeldahl method to measure the amount of protein in a sample containing diatomaceous earth, adding boiling chips can suppress the occurrence of bumping during the heating step of sulfuric acid decomposition. This does not affect the measurement of protein content, and the amount of boiling chips added is related to the heating temperature at which bumping can be suppressed and the length of time required to complete sulfuric acid decomposition without causing bumping.

[0058] Therefore, in this embodiment as well, the amount of boiling chips added per 10 milliliters of concentrated sulfuric acid in the Kjeldahl decomposition step is 100 to 300 milligrams, and the sulfuric acid decomposition of the enzyme treatment residue can be carried out at a heating temperature of 480°C without causing bumping.

[0059] By performing sulfuric acid decomposition at a heating temperature of 480°C, the time required for sulfuric acid decomposition (Kjeldahl degradation) can be shortened, providing a protein quantity measurement method that does not require a long decomposition process at a low heating temperature.

[0060] In this embodiment, the material containing protein as an ingredient can be one or a combination of any of the following: soybeans, soybean-derived foods, buckwheat, silk, or sake lees.

[0061] Furthermore, the aforementioned protein can be a resistant protein.

[0062] Distant proteins, such as soybean high molecular weight protein fraction (HMF) and soybean resistant protein (SRP), as well as buckwheat protein, silk protein, and sake lees protein, are expected to have various applications as health-promoting foods, beverages, and pharmaceuticals. This invention provides a method for quantifying resistant proteins. [Examples]

[0063] Approximately 1 g of frozen tofu defatted powder was weighed out ("S" g), 50 mL of 0.08 M phosphate buffer (pH 6.0) and 0.1 mL of heat-resistant amylase solution (Fujifilm Wako Pure Chemical Industries) were added, and the mixture was reacted in a boiling water bath for 30 minutes, shaking every 5 minutes.

[0064] Next, after cooling to room temperature, 0.275 M sodium hydroxide was added until the pH reached 7.5.

[0065] 0.1 mL of protease solution (Fujifilm Wako Pure Chemical Industries) was added to this, and the mixture was reacted in a 60°C water bath for 30 minutes while shaking.

[0066] After cooling to room temperature, 0.325 M hydrochloric acid was added until the pH reached 4.5.

[0067] 0.3 mL of amyloglucosidase solution (Fujifilm Wako Pure Chemical Industries) was added to this, and the mixture was reacted in a 60°C water bath for 30 minutes while shaking.

[0068] Four times the volume of 95% (v / v) ethanol (60°C) was added to the liquid obtained from the above treatment, and the mixture was left at room temperature for 1 hour.

[0069] This suspension was filtered by suction using a crucible-type filter (G2G) lined with diatomaceous earth (Fujifilm Wako Pure Chemical Industries), and the residue collected on the glass filter was sequentially washed with 78% (v / v) ethanol, 95% (v / v) ethanol, and acetone.

[0070] The enzyme-treated residue containing diatomaceous earth obtained in this way was used to measure the protein content, i.e., the resistant protein content, by the Kjeldahl method ("T" g).

[0071] Resistant protein (RP) measurement (g / 100g) = T / S × 100 This was the request.

[0072] In the Kjeldahl method measurement, 10 mL of concentrated sulfuric acid and 1.5 g of Kjeldahl decomposition accelerator (Keltab) were added to the enzyme-treated residue containing diatomaceous earth prepared as described above, and sulfuric acid decomposition was carried out.

[0073] In this process, commercially available boiling chips (Wako Pure Chemical Industries) were added in the range of 0 mg to 300 mg, and the sulfuric acid decomposition was carried out by changing the heating temperature in the sulfuric acid decomposition process as shown in Table 1 below.

[0074] In preliminary studies, adding 301 mg or more of commercially available boiling chips (Wako Pure Chemical Industries) allowed for sulfuric acid decomposition without bumping at the heating temperatures shown in Table 1 below. However, this made the subsequent distillation step, used for measuring resistant protein content (protein amount) by the Kjeldahl method, difficult. Therefore, we investigated setting the upper limit for commercially available boiling chips (Wako Pure Chemical Industries) to be added per 10 mL of concentrated sulfuric acid to 300 mg.

[0075] [Table 1]

[0076] When measuring the protein content in a sample that does not contain diatomaceous earth using the Kjeldahl method, it is generally possible to complete the sulfuric acid decomposition in about 3 hours without causing bumping by heating the sample at 480°C.

[0077] As described above, the prepared sample (enzyme-treated residue) contains diatomaceous earth, which was used as a filter material in the process of obtaining the enzyme-treated residue.

[0078] Comparative Example 1 aimed to decompose sulfuric acid in a short time of about 3 hours by heating to 480°C, but bumping occurred.

[0079] To measure the protein content in samples containing diatomaceous earth, the Kjeldahl method is used. To prevent bumping during the sulfuric acid decomposition process, it is possible to lower the heating temperature in the sulfuric acid decomposition step.

[0080] Comparative Example 2 involves performing sulfuric acid decomposition without causing bumping by lowering the heating temperature to 200°C.

[0081] As mentioned above, when measuring the protein content in a sample without diatomaceous earth using the Kjeldahl method, it is generally possible to complete sulfuric acid decomposition in about 3 hours without causing bumping by heating at 480°C. However, in Comparative Example 2, sulfuric acid decomposition was performed at a heating temperature of 200°C, which required about 10 hours.

[0082] When Comparative Example 2 was heated to 200°C, it was possible to decompose the sample in 10 hours without causing bumping. The amount of resistant protein measured was 22.5 ± 1.3 g / 100 g.

[0083] As described above, when 10 mL of concentrated sulfuric acid and 1.5 g of Kjeldahl decomposition accelerator (Keltab) were added to the enzyme-treated residue containing diatomaceous earth, and then 25 mg of commercially available boiling chips (Wako Pure Chemical Industries) were added, sulfuric acid decomposition was carried out. Bumping occurred at both heating temperatures of 480°C and 300°C (Comparative Examples 3 and 4).

[0084] When 50 mg of commercially available boiling chips (Wako Pure Chemical Industries) were added, bumping occurred at a heating temperature of 480°C (Comparative Example 5), but no bumping occurred at a heating temperature of 300°C, and sulfuric acid decomposition was possible in 8 hours (Example 1).

[0085] Furthermore, as in Example 2, when 100 mg of boiling chips were added, the sulfuric acid decomposition was completed in about 3 hours without bumping even when heated to 480°C.

[0086] Even when 200 mg and 300 mg of boiling chips were added, the sulfuric acid decomposition was completed in about 3 hours without bumping when heated to 480°C (Examples 3 and 4).

[0087] The resistant protein content of Examples 1, 2, 3, and 4 was measured and found to be 22.1±0.9g / 100g, 22.4±1.0g / 100g, 22.8±1.1g / 100g, and 22.2±0.9g / 100g, respectively.

[0088] The measured values ​​of resistant protein in Examples 1, 2, 3, and 4, which were able to perform sulfuric acid decomposition without causing bumping, were equivalent to the measured values ​​of resistant protein in Comparative Example 2, where the heating temperature was lowered to 200°C and sulfuric acid decomposition was performed over approximately 10 hours. This confirmed that the methods of Examples 1 to 4 did not affect the measured values.

[0089] As a result, we confirmed that when using the Kjeldahl method to measure the amount of protein in a sample containing diatomaceous earth, adding boiling chips can suppress the occurrence of bumping during the heating step of sulfuric acid decomposition, that this does not affect the measurement of resistant protein amount, and that the amount of boiling chips added is related to the height of the heating temperature at which bumping can be suppressed and the length of time required to complete sulfuric acid decomposition without causing bumping.

Claims

[Claim 1] A protein measurement method for measuring the amount of protein in a sample containing diatomaceous earth by the Kjeldahl method, The system includes a Kjeldahl decomposition step in which 100 to 300 milligrams of boiling chips are added per 10 milliliters of concentrated sulfuric acid, and the sample is decomposed by sulfuric acid at a heating temperature of 480°C without causing bumping. Protein measurement methods.

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