Method for simultaneously determining total acid and protein solubility of fermented soybean meal

Abstract

The invention discloses a method for simultaneously measuring total acid and protein solubility of fermented soybean meal. The method comprises the following steps: S1, measuring the initial pH of a 0.2% potassium hydroxide solution; s2, weighing two fermented soybean meal samples with the same mass, respectively putting the two fermented soybean meal samples into a beaker A and a beaker B, and adding an equivalent 0.2% potassium hydroxide solution; substances in the beaker A are stirred and centrifuged, and supernate is taken to obtain to-be-detected liquid A; s3, adding a potassium hydroxide standard titration solution into the to-be-detected solution A for titration, observing the pH change of the to-be-detected solution A at any time by using an acidimeter, recording the volume of the consumed potassium hydroxide standard titration solution when the titration end point is reached, and meanwhile, performing a blank test; s4, adding a potassium hydroxide standard titration solution into the beaker B, stirring, centrifuging, taking supernate, digesting, and measuring crude protein contents W1 and W2; and S5, calculating a result. According to the method for simultaneously determining the total acid and the protein solubility of the fermented soybean meal, the accuracy of detecting the protein solubility of the fermented soybean meal is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality analysis of feed raw materials, in particular to a method for simultaneously determining total acid and protein solubility of fermented soybean meal. BACKGROUND

[0002] The fermented soybean meal is a high-end feed raw material obtained by inoculating specific microorganisms such as lactic acid bacteria, bacillus and yeast on soybean meal. As a new type of biological feed, it does not add antibiotics and is rich in probiotics, lactic acid and other nutrients, and is a good substitute for fish meal and other animal proteins. Fermented soybean meal can degrade cellulose and protein in soybean meal through various enzymes produced by microbial fermentation, remove various anti-nutritional factors, and improve the nutritional value of soybean meal. At the same time, well-fermented soybean meal can emit a certain acid aroma, which has a feeding effect and can further increase the feed intake of livestock and poultry, reduce resource waste and create huge economic value, and has a broad application prospect in the feed industry.

[0003] The current published national standard "GB / T 43745-2024 Feed Raw Materials Fermented Soybean Meal" does not specify the determination method of total acid and protein solubility of fermented soybean meal, and the "NY / T 2218-2022 Feed Raw Materials Fermented Soybean Meal" only specifies that the potassium hydroxide protein solubility is executed according to the provisions of Appendix A of "GB / T 19541-2017 Feed Raw Materials Soybean Meal", and does not specify the determination of total acid content. Moreover, "GB / T 19541-2017 Feed Raw Materials Soybean Meal" does not consider the high total acid content of fermented soybean meal, which directly affects the detection results of potassium hydroxide protein solubility.

[0004] Therefore, the present application provides a method for simultaneously determining total acid and protein solubility of fermented soybean meal, which describes the determination method and calculation method of total acid content of fermented soybean meal, improves the accuracy of potassium hydroxide protein solubility detection of fermented soybean meal, and provides practical and effective reference data for the purchase and use of fermented soybean meal. SUMMARY

[0005] The present application provides a method for simultaneously determining total acid and protein solubility of fermented soybean meal, which describes the determination method and calculation method of total acid content of fermented soybean meal, improves the accuracy of potassium hydroxide protein solubility detection of fermented soybean meal, and provides practical and effective reference data for the purchase and use of fermented soybean meal.

[0006] To achieve the above-mentioned purpose, the present application provides a method for simultaneously determining total acid and protein solubility of fermented soybean meal, comprising the following steps: S1, determination of pH of 0.2% potassium hydroxide solution: 2.35 g of potassium hydroxide with a purity of 85% was weighed, dissolved in deionized water and diluted to 1 L to obtain 0.2% potassium hydroxide solution; the pH value was determined by a pH meter, and the initial pH of 0.2% potassium hydroxide solution was recorded.

[0007] S2, extraction of sample: two portions of fermented soybean meal with a mass of 1.5000-2.1000 g were accurately weighed to obtain the sample, and the sample weights of the two portions were kept consistent; the mass of one portion of the sample was recorded as m, and then the two portions of the sample were respectively placed in a 250 mL beaker A and a 250 mL beaker B; 75-100 mL of 0.2% potassium hydroxide solution was added to the beaker A containing the sample, and the volume of the added 0.2% potassium hydroxide solution was recorded as V0; after stirring on a magnetic stirrer, it was transferred to a 100 mL centrifuge tube A with a cover, and then centrifuged in a centrifuge; 25-50 mL of supernatant A was taken by a pipette and placed in a 100 mL beaker C to obtain the test solution A, and the volume of the taken supernatant A was recorded as V1.

[0008] S3, determination of total acid: (1) the beaker C containing the test solution A was placed on a magnetic stirrer, and the acidity meter electrode was immersed in the test solution A; the magnetic stirrer was turned on and the pH change of the test solution A was observed; (2) 0.1000-0.1100 mol / L of potassium hydroxide standard titration solution was quickly added to the test solution A for titration, and the pH change of the test solution A was observed at any time; when approaching the titration endpoint, the titration speed was slowed down, and half a drop was added at a time until the pH of the test solution A reached the titration endpoint; the volume of the consumed potassium hydroxide standard titration solution at this time was recorded as V1. KOH ; (3) a blank test was performed, in which no fermented soybean meal sample was added, and the rest of the steps were the same as S2 and S3; the volume of the consumed potassium hydroxide standard titration solution was recorded as V2.

[0009] S4, determination of protein solubility: (1) after adding the 0.2% potassium hydroxide solution with a volume of V0 to the beaker B containing the fermented soybean meal sample, the potassium hydroxide standard titration solution in S3 was added, and the volume of the added was [ × (V KOH -V2) ], and after stirring on a magnetic stirrer, the test solution B was obtained; (2) the test solution B was transferred to an 80 mL centrifuge tube B with a cover, and then centrifuged in a centrifuge; 10-15 mL of supernatant B was taken by a pipette and placed in a digestion tube, and the volume of the taken supernatant B was recorded as V3; immediately, mixed catalyst and concentrated sulfuric acid were added to the digestion tube according to the provisions of GB / T 6432-2018, the content of crude protein W1 in the 0.2% potassium hydroxide solution was determined, and the total content of crude protein W2 in the same fermented soybean meal was determined according to the provisions of GB / T 6432-2018.

[0010] S5, result calculation: (1) the content of total acid is calculated according to the following formula: X= × ; In the formula: X-the content of total acid in the sample, units of grams per kilogram (g / kg) or grams per liter (g / L); C-the concentration of potassium hydroxide standard titration solution, units of moles per liter (mol / L); V KOH -the volume of potassium hydroxide standard titration solution consumed when titrating the sample solution A, units of milliliters (mL); V2-the volume of potassium hydroxide standard titration solution consumed when titrating the blank, units of milliliters (mL); V1-the volume of supernatant after centrifugation, units of milliliters (mL); V0-the total volume of 0.2% potassium hydroxide solution, units of milliliters (mL); m-the mass of the sample, units of grams (g); k-the conversion coefficient of acid, units of kilograms per mole (kg / mol) according to different types of acid; 1000-conversion coefficient; (2) the solubility of potassium hydroxide protein is calculated according to the following formula: W= ×100; In the formula: W1-the crude protein content of the sample dissolved in 0.2% potassium hydroxide solution, %; W2-the total crude protein content of the sample, %.

[0011] As a preferred technical solution of the present application, the stirring speed of the magnetic stirrer in S2 and S4 is 300 r / min, and the stirring time is 20 min.

[0012] As a preferred technical solution of the present application, the speed of the centrifuge in S2 and S4 is 2700 r / min, and the centrifugation time is 10 min.

[0013] As a preferred technical solution of the present application, the stirring speed of the magnetic stirrer in S3 is 200 r / min.

[0014] As a preferred technical solution of the present application, the potassium hydroxide standard titration solution in S3 and S4 should be titrated with a potassium hydroxide standard titration solution with a concentration of 0.01 mol / L or 0.05 mol / L when the total acid is ≤4 g / kg.

[0015] As a preferred technical solution of the present application, the pH of the titration end point in S3 is the initial pH of the 0.2% potassium hydroxide solution in S1.

[0016] As a preferred technical solution of the present application, the preparation method of the mixed catalyst in S4 is: weighing 0.4g of copper sulfate pentahydrate, 6.0g of potassium sulfate or sodium sulfate, grinding and mixing uniformly to obtain a mixed catalyst; the concentration of the concentrated sulfuric acid is 98%.

[0017] As a preferred technical solution of the present application, the specific numerical value of the conversion coefficient k of the acid in S5 is: malic acid, 0.067 kg / mol; acetic acid, 0.060 kg / mol; tartaric acid, 0.075 kg / mol; citric acid, 0.064 kg / mol; citric acid containing one molecule of crystal water, 0.070 kg / mol; lactic acid, 0.090 kg / mol; hydrochloric acid, 0.036 kg / mol; sulfuric acid, 0.049 kg / mol; phosphoric acid, 0.049 kg / mol.

[0018] Compared with the prior art, the present application has the following beneficial effects: 1、The present application can detect double indicators at one time by weighing two equal quality samples, and can simultaneously measure the total acid content and potassium hydroxide protein solubility in fermented soybean meal, and improve the accuracy of protein solubility detection. The existing standard "GB / T 19541-2017 Feedstuff Soybean Meal" is aimed at detecting ordinary soybean meal, and does not produce total acid in the fermentation process, and only specifies the method for separately measuring protein solubility; if "GB / T 19541-2017 Feedstuff Soybean Meal" is used to detect fermented soybean meal, it does not include the method for measuring the total acid content of fermented soybean meal, and does not consider the influence of the total acid of fermented soybean meal on the detection result of potassium hydroxide protein solubility - the organic acid in fermented soybean meal will react with 0.2% potassium hydroxide solution, resulting in a decrease in the alkalinity of the protein dissolution environment, and the soluble protein is not fully dissolved, so that the measured potassium hydroxide protein solubility is lower than the true value. The determination method of the present application systematically sets forth the determination method and calculation method of the total acid content of fermented soybean meal according to the principle of acid-base neutralization reaction, determines the total acid content, obtains the required alkali volume for neutralizing the total acid, eliminates the interference of trace acid in 0.2% potassium hydroxide solution through a blank test, and provides an optimal pH environment for protein dissolution when measuring protein solubility, thereby avoiding the influence of total acid on the detection result of potassium hydroxide protein solubility and improving the accuracy of the detection result.

[0019] 2. The method for simultaneously determining total acid and protein solubility in fermented soybean meal according to the present invention improves detection efficiency and ensures a safe and environmentally friendly experimental process. By designing to weigh two equal-mass samples, the pretreatment of one sample can be used for the determination of two indicators, reducing repetitive weighing, extraction, and centrifugation operations. In batch testing, multiple samples can undergo the same pretreatment, further shortening the operation cycle and improving the detection efficiency of total acid and protein solubility in fermented soybean meal. Furthermore, the reagents used in the entire determination process are mainly diluted alkaline solutions. Except for the protein solubility determination step, which requires the addition of concentrated sulfuric acid and a catalyst according to GB / T 6432-2018, the use of strong acids, highly corrosive, or toxic organic solvents is avoided, improving operational safety and environmental friendliness. Attached Figure Description

[0020] Figure 1 This is a flowchart of the method for simultaneously determining the total acidity and protein solubility of fermented soybean meal according to the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of the phrase "embodiment" in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0023] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0024] like Figure 1 As shown, a method for simultaneously determining the total acidity and protein solubility of fermented soybean meal includes the following steps: S1. Weigh potassium hydroxide, dissolve it in water, and determine the initial pH of the resulting 0.2% potassium hydroxide solution.

[0025] S2, two portions of the same mass of fermented soybean meal sample are weighed into beakers A and B, and an equal amount of 0.2% potassium hydroxide solution is added; the material in beaker A is stirred and centrifuged to obtain supernatant to obtain a test solution A.

[0026] S3, potassium hydroxide standard titration solution is added to the test solution A for titration, and the pH change of the test solution A is observed at any time using an acidity meter until the pH of the test solution A reaches the initial pH of the 0.2% potassium hydroxide solution in S1, that is, the titration end point, and the volume of the potassium hydroxide standard titration solution consumed at this time is recorded as V1. KOH A blank test is performed without adding the fermented soybean meal sample, and the volume of the potassium hydroxide standard titration solution consumed is recorded as V2.

[0027] S4, potassium hydroxide standard titration solution is added to beaker B, and after stirring, a test solution B is obtained; the test solution B is transferred to a centrifuge tube and centrifuged to obtain supernatant for digestion, and the crude protein content W1 in the 0.2% potassium hydroxide solution and the total crude protein content W2 in the same fermented soybean meal are determined.

[0028] S5, the total acid content and the protein solubility of the fermented soybean meal are calculated according to the measured data.

[0029] The raw materials used in the present application are all commercially available raw materials.

[0030] Example 1: A method for simultaneously determining the total acid and protein solubility of fermented soybean meal, comprising the following steps: S1, determination of the pH of 0.2% potassium hydroxide solution: 2.35 g of 85% purity potassium hydroxide is dissolved in deionized water and diluted to 1 L to obtain a 0.2% potassium hydroxide solution; the pH value is measured using a pH meter, and the initial pH of the 0.2% potassium hydroxide solution is recorded.

[0031] S2, sample extraction: two portions of fermented soybean meal with a mass of 1.5040 g are accurately weighed to obtain samples, and the mass of one of the samples is recorded as m; then the two portions of samples are placed in 250 mL beakers A and 250 mL beakers B, respectively; 75 mL of 0.2% potassium hydroxide solution is added to beaker A containing the sample, and the volume of the 0.2% potassium hydroxide solution added is recorded as V0; after stirring at a speed of 300 r / min on a magnetic stirrer for 20 min, it is transferred to a 100 mL centrifuge tube A with a cover, and then centrifuged at a speed of 2700 r / min for 10 min; 50 mL of supernatant A is removed using a pipette and placed in a 100 mL beaker C to obtain a test solution A, and the volume of the supernatant A removed is recorded as V1.

[0032] S3, determination of total acid: (1) the beaker C containing the test solution A was placed on the magnetic stirrer, the acidity electrode was immersed in the test solution A, the magnetic stirrer was started at a speed of 200 r / min, and the pH change of the test solution A was observed; (2) 0.1077 mol / L standard titration solution of potassium hydroxide was quickly added to the test solution A for titration, and the pH change of the test solution A was observed at any time; when approaching the titration end point, the titration speed was slowed down, half drop was added at a time, until the pH of the test solution A reached the initial pH of the 0.2% potassium hydroxide solution in S1, that is, the titration end point was reached, and the volume value of the consumed standard titration solution of potassium hydroxide at this time was recorded as V KOH ; (3) a blank test was carried out, in which no fermented soybean meal sample was added, and the rest of the steps were the same as S2 and S3, and the volume value of the consumed standard titration solution of potassium hydroxide was recorded as V2.

[0033] S4, determination of protein solubility: (1) after adding the volume V0 of 0.2% potassium hydroxide solution to the beaker B containing the fermented soybean meal sample, the standard titration solution of potassium hydroxide in S3 was added, and the volume was [ × (V KOH -V2) ], after stirring on the magnetic stirrer at a speed of 300 r / min for 20 min, the test solution B was obtained; (2) the test solution B was transferred to an 80 mL centrifugal tube B with a cover, and then centrifuged at 2700 r / min for 10 min, 10 mL of supernatant B was taken out with a pipette and placed in a digestion tube, the volume of the supernatant B was recorded as V3, and immediately 6.4 g of mixed catalyst and 12 mL of concentrated sulfuric acid were added to the digestion tube according to the provisions of GB / T 6432-2018, the content of crude protein in the 0.2% potassium hydroxide solution W1 was determined, and the total content of crude protein W2 in the same fermented soybean meal was determined according to the provisions of GB / T 6432-2018.

[0034] S5, result calculation: (1) the content of total acid is calculated according to the following formula: X= × ; , wherein: X is the content of total acid in the sample, in units of grams per kilogram (g / kg) or grams per liter (g / L); c is the concentration of the standard titration solution of potassium hydroxide, in units of moles per liter (mol / L); V KOH is the volume of the standard titration solution of potassium hydroxide consumed when titrating the test solution A, in units of milliliters (mL); V2 is the volume of the standard titration solution of potassium hydroxide consumed when titrating the blank, in units of milliliters (mL); V1 is the volume of the supernatant after centrifugation, in units of milliliters (mL); V0 — Total volume of 0.2% potassium hydroxide solution added, in milliliters (mL); m — Mass of the sample, in grams (g); k – the conversion factor for acids, calculated as lactic acid, with a value of 0.090 kg / mol; 1000 – Conversion factor; (2) The solubility of potassium hydroxide in proteins is expressed as a mass fraction (W), with the value expressed as a percentage (%), and is calculated using the following formula: W= ×100; Where: W1—the crude protein content of the sample dissolved in 0.2% potassium hydroxide solution, %; W2—Total crude protein content in the sample, %.

[0035] Example 2: A method for simultaneously determining total acidity and protein solubility in fermented soybean meal includes the following steps: S1. Determination of pH of 0.2% potassium hydroxide solution: Weigh 2.35g of potassium hydroxide with a purity of 85%, dissolve it in deionized water and make up to 1L to obtain a 0.2% potassium hydroxide solution; measure its pH value with a pH meter and record it as the initial pH of the 0.2% potassium hydroxide solution.

[0036] S2. Sample Extraction: Accurately weigh two portions of fermented soybean meal, each weighing 1.5022 g, to obtain samples. Keep the weights of the two samples consistent and record the mass of one sample as m. Then, place the two samples into 250 mL beaker A and 250 mL beaker B, respectively. Add 75 mL of 0.2% potassium hydroxide solution to beaker A containing the samples. Record the volume of the added 0.2% potassium hydroxide solution as V0. Stir at 300 r / min for 20 min on a magnetic stirrer. Transfer the solution to a 100 mL capped centrifuge tube A and centrifuge at 2700 r / min for 10 min. Transfer 50 mL of the supernatant A to a 100 mL beaker C using a pipette to obtain the test solution A. Record the volume of the transferred supernatant A as V1.

[0037] S3. Determination of total acidity: (1) Place beaker C containing test solution A on a magnetic stirrer, immerse the pH meter electrode in test solution A, turn on the magnetic stirrer at a speed of 200 r / min, and observe the pH change of test solution A; (2) Quickly add 0.1077 mol / L potassium hydroxide standard titration solution to test solution A for titration, and observe the pH change of test solution A at any time; when approaching the titration endpoint, slow down the titration speed, add half a drop at a time, until the pH of test solution A reaches the initial pH of 0.2% potassium hydroxide solution in S1, which is the titration endpoint. Record the volume of potassium hydroxide standard titration solution consumed at this time as V. KOH(3) Blank test, no fermented soybean meal sample is added in the blank test, and the rest of the steps are the same as S2 and S3, and the value of the consumed potassium hydroxide standard titration solution is recorded as V2.

[0038] S4, determination of protein solubility: (1) after adding the volume V0 of 0.2% potassium hydroxide solution to the beaker B containing the fermented soybean meal sample, the volume of the added potassium hydroxide standard titration solution in S3 is [V × (V KOH -V2)], after stirring at a speed of 300 r / min on a magnetic stirrer for 20 min, the measured liquid B is obtained; (2) the measured liquid B is transferred to the 80 mL centrifuge tube B with a cover, and then centrifuged at 2700 r / min for 10 min, 10 mL of supernatant B is removed from the digestion tube with a pipette, the volume of the removed supernatant B is recorded as V3, and immediately 6.4 g of mixed catalyst and 12 mL of concentrated sulfuric acid are added to the digestion tube according to the provisions of GB / T 6432-2018, the content W1 of crude protein in the 0.2% potassium hydroxide solution is determined, and the total content W2 of crude protein in the same fermented soybean meal is determined according to the provisions of GB / T 6432-2018.

[0039] S5, result calculation: (1) the content of total acid is calculated according to the following formula: X= × ; In the formula: X is the content of total acid in the sample, with units of grams per kilogram (g / kg) or grams per liter (g / L); c is the concentration of the potassium hydroxide standard titration solution, with units of moles per liter (mol / L); V KOH is the volume of the consumed potassium hydroxide standard titration solution when titrating the measured liquid A, with units of milliliters (mL); V2 is the volume of the consumed potassium hydroxide standard titration solution when titrating the blank, with units of milliliters (mL); V1 is the volume of the removed supernatant after centrifugation, with units of milliliters (mL); V0 is the total volume of the added 0.2% potassium hydroxide solution, with units of milliliters (mL); m is the mass of the sample, with units of grams (g); k is the conversion coefficient of acid, with a value of 0.090 kg / mol, calculated as lactic acid; 1000 is the conversion coefficient; (2) the solubility of potassium hydroxide protein is calculated according to the following formula: W= ×100; W1 - crude protein content of the sample dissolved in 0.2% potassium hydroxide solution, %; W2 - total crude protein content in the sample, %.

[0040] Example Three A method for simultaneously determining total acid and protein solubility of fermented soybean meal, comprising the following steps: S1, determination of pH of 0.2% potassium hydroxide solution: 2.35 g of potassium hydroxide with a purity of 85% was weighed, dissolved in deionized water and made up to 1 L to obtain 0.2% potassium hydroxide solution; the pH value was determined by a pH meter, and the initial pH of the 0.2% potassium hydroxide solution was recorded.

[0041] S2, extraction of the sample: two portions of fermented soybean meal with a mass of 1.5019 g were accurately weighed to obtain the sample, and the weighing amount of the two portions of the sample was kept consistent; the mass of one portion of the sample was recorded as m, and then the two portions of the sample were respectively placed into 250 mL beaker A and 250 mL beaker B; 75 mL of 0.2% potassium hydroxide solution was added to the beaker A containing the sample, and the volume of the added 0.2% potassium hydroxide solution was recorded as V0; after stirring at a speed of 300 r / min for 20 min on a magnetic stirrer, it was transferred to 100 mL centrifuge tube A with a cover, and then centrifuged at a speed of 2700 r / min for 10 min; 50 mL of supernatant A was removed by a pipette and placed in 100 mL beaker C to obtain the test solution A, and the volume of the removed supernatant A was recorded as V1.

[0042] S3, determination of total acid: (1) the beaker C containing the test solution A was placed on a magnetic stirrer, the acidity meter electrode was immersed in the test solution A, the magnetic stirrer was started at a speed of 200 r / min, and the pH change of the test solution A was observed; (2) 0.1077 mol / L potassium hydroxide standard titration solution was quickly added to the test solution A for titration, and the pH change of the test solution A was observed at any time; when approaching the titration endpoint, the titration speed was slowed down, half a drop was added at a time, and the pH of the test solution A reached the initial pH of the 0.2% potassium hydroxide solution in S1, which was the titration endpoint, and the value of the consumed potassium hydroxide standard titration solution volume at this time was recorded as V KOH ; (3) a blank test was performed, in which no fermented soybean meal sample was added, and the remaining steps were the same as S2 and S3, and the value of the consumed potassium hydroxide standard titration solution volume was recorded as V2.

[0043] S4, determination of protein solubility: (1) after adding 0.2% potassium hydroxide solution with a volume of V0 to the beaker B containing the fermented soybean meal sample, the potassium hydroxide standard titration solution in S3 was added, and the volume of the added was[ × (V KOHV2), after stirring at a speed of 300 r / min on a magnetic stirrer for 20 min, to obtain a to-be-tested liquid B; (2) transferring the to-be-tested liquid B to an 80 mL centrifuge tube B with a cover, centrifuging at 2700 r / min for 10 min, and then using a pipette to transfer 10 mL of supernatant B in a digestion tube, the volume of the transferred supernatant B being recorded as V3, and immediately adding 6.4 g of mixed catalyst and 12 mL of concentrated sulfuric acid into the digestion tube according to the provisions of GB / T 6432-2018, determining the content W1 of crude protein in the 0.2% potassium hydroxide solution, and determining the total content W2 of crude protein in the same fermented soybean meal according to the provisions of GB / T 6432-2018.

[0044] S5, result calculation: (1) the content of total acid is calculated in X, and the formula is as follows: X= × ; In the formula, X is the content of total acid in the sample, in units of grams per kilogram (g / kg) or grams per liter (g / L); c is the concentration of the potassium hydroxide standard titration solution, in units of moles per liter (mol / L); V KOH is the volume of the potassium hydroxide standard titration solution consumed when titrating the to-be-tested liquid A, in units of milliliters (mL); V2 is the volume of the potassium hydroxide standard titration solution consumed when titrating the blank, in units of milliliters (mL); V1 is the volume of the supernatant after centrifugation, in units of milliliters (mL); V0 is the total volume of the 0.2% potassium hydroxide solution added, in units of milliliters (mL); m is the mass of the sample, in units of grams (g); k is the conversion coefficient of acid in terms of lactic acid, with a value of 0.090 kg / mol; 1000 is the conversion coefficient; (2) the solubility of potassium hydroxide protein is calculated in mass fraction W, and the value is expressed in %, and the formula is as follows: W= ×100; In the formula, W1 is the content of crude protein dissolved in the 0.2% potassium hydroxide solution, %; W2 is the total content of crude protein in the sample, %.

[0045] Example Four: A method for simultaneously determining total acid and protein solubility of fermented soybean meal, comprising the following steps: S1, determination of pH of 0.2% potassium hydroxide solution: 2.35 g of potassium hydroxide with a purity of 85% was weighed, dissolved in deionized water and diluted to 1 L to obtain 0.2% potassium hydroxide solution; the pH value was determined by a pH meter, and the initial pH of the 0.2% potassium hydroxide solution was recorded.

[0046] S2, extraction of sample: two portions of fermented soybean meal with a mass of 2.0043 g were accurately weighed to obtain the sample, and the weighing amount of the two portions of sample was kept consistent, and the mass of one portion of sample was recorded as m, and then the two portions of sample were respectively placed in a 250 mL beaker A and a 250 mL beaker B; 100 mL of 0.2% potassium hydroxide solution was added to the beaker A containing the sample, and the volume of the added 0.2% potassium hydroxide solution was recorded as V0, and after stirring at a speed of 300 r / min on a magnetic stirrer for 20 min, it was transferred to a 100 mL centrifuge tube A with a cover, and then centrifuged at a speed of 2700 r / min for 10 min, and 50 mL of supernatant A was removed by a pipette into a 100 mL beaker C to obtain a test solution A, and the volume of the removed supernatant A was recorded as V1.

[0047] S3, determination of total acid: (1) the beaker C containing the test solution A was placed on a magnetic stirrer, the acidity meter electrode was immersed in the test solution A, the magnetic stirrer was started at a speed of 200 r / min, and the pH change of the test solution A was observed; (2) 0.1077 mol / L of potassium hydroxide standard titration solution was quickly added to the test solution A for titration, and the pH change of the test solution A was observed at any time; when approaching the titration end point, the titration speed was slowed down, half a drop was added at a time, until the pH of the test solution A reached the initial pH of the 0.2% potassium hydroxide solution in S1, that is, the titration end point was reached, and the value of the volume of the consumed potassium hydroxide standard titration solution at this time was recorded as V KOH ; (3) a blank test was performed, in which no fermented soybean meal sample was added, and the rest of the steps were the same as S2 and S3, and the value of the volume of the consumed potassium hydroxide standard titration solution was recorded as V2.

[0048] S4, determination of protein solubility: (1) after adding 0.2% potassium hydroxide solution with a volume of V0 to the beaker B containing the fermented soybean meal sample, the potassium hydroxide standard titration solution in S3 was added, and the volume of the added was[ × (V KOHV2), after stirring at a speed of 300 r / min on a magnetic stirrer for 20 min, to obtain a test liquid B; (2) transferring the test liquid B to an 80 mL centrifuge tube B with a cover, centrifuging at 2700 r / min for 10 min, and then using a pipette to transfer 10 mL of supernatant B in a digestion tube, the volume of the transferred supernatant B being recorded as V3, and immediately adding 6.4 g of mixed catalyst and 12 mL of concentrated sulfuric acid into the digestion tube according to the provisions of GB / T 6432-2018, determining the content W1 of crude protein in the 0.2% potassium hydroxide solution, and determining the total content W2 of crude protein in the same fermented soybean meal according to the provisions of GB / T 6432-2018.

[0049] S5, result calculation: (1) the content of total acid is calculated in X, and the formula is as follows: X= × ; In the formula, X is the content of total acid in the sample, in units of grams per kilogram (g / kg) or grams per liter (g / L); c is the concentration of the potassium hydroxide standard titration solution, in units of moles per liter (mol / L); V KOH is the volume of the potassium hydroxide standard titration solution consumed when titrating the test liquid A, in units of milliliters (mL); V2 is the volume of the potassium hydroxide standard titration solution consumed when titrating the blank, in units of milliliters (mL); V1 is the volume of the supernatant after centrifugation, in units of milliliters (mL); V0 is the total volume of the 0.2% potassium hydroxide solution added, in units of milliliters (mL); m is the mass of the sample, in units of grams (g); k is the conversion coefficient of acid in terms of lactic acid, with a value of 0.090 kg / mol; 1000 is the conversion coefficient; (2) the solubility of potassium hydroxide protein is calculated in mass fraction W, and the value is expressed in %, and the formula is as follows: W= ×100; In the formula, W1 is the content of crude protein dissolved in the 0.2% potassium hydroxide solution, %; W2 is the total content of crude protein in the sample, %.

[0050] Example Five: A method for simultaneously determining total acid and protein solubility of fermented soybean meal, comprising the following steps: S1, determination of pH of 0.2% potassium hydroxide solution: 2.35 g of potassium hydroxide with a purity of 85% was weighed, dissolved in deionized water and diluted to 1 L to obtain 0.2% potassium hydroxide solution; the pH value was determined by a pH meter, and the initial pH of the 0.2% potassium hydroxide solution was recorded.

[0051] S2, extraction of sample: two portions of fermented soybean meal with a mass of 2.0571 g were accurately weighed to obtain samples, and the weighing amounts of the two samples were kept consistent; the mass of one sample was recorded as m, and then the two samples were respectively placed in 250 mL beaker A and 250 mL beaker B; 100 mL of 0.2% potassium hydroxide solution was added to the beaker A containing the sample, and the volume of the added 0.2% potassium hydroxide solution was recorded as V0; after stirring at a speed of 300 r / min for 20 min on a magnetic stirrer, it was transferred to 100 mL centrifuge tube A with a cover, and then centrifuged at a speed of 2700 r / min for 10 min; 50 mL of supernatant A was removed by a pipette and placed in 100 mL beaker C to obtain the test solution A, and the volume of the removed supernatant A was recorded as V1.

[0052] S3, determination of total acid: (1) the beaker C containing the test solution A was placed on a magnetic stirrer, the acidity meter electrode was immersed in the test solution A, the magnetic stirrer was started at a speed of 200 r / min, and the pH change of the test solution A was observed; (2) 0.1077 mol / L of potassium hydroxide standard titration solution was quickly added to the test solution A for titration, and the pH change of the test solution A was observed at any time; when approaching the titration end point, the titration speed was slowed down, half a drop was added at a time, until the pH of the test solution A reached the initial pH of the 0.2% potassium hydroxide solution in S1, that is, the titration end point was reached, and the volume value of the consumed potassium hydroxide standard titration solution at this time was recorded as V KOH ; (3) a blank test was performed, in which no fermented soybean meal sample was added, and the rest of the steps were the same as S2 and S3, and the volume value of the consumed potassium hydroxide standard titration solution was recorded as V2.

[0053] S4, determination of protein solubility: (1) after adding 0.2% potassium hydroxide solution with a volume of V0 to the beaker B containing the fermented soybean meal sample, the potassium hydroxide standard titration solution in S3 was added, and the volume of the added was[ × (V KOHV2), after stirring at a speed of 300 r / min on a magnetic stirrer for 20 min, to obtain a to-be-tested liquid B; (2) transferring the to-be-tested liquid B to an 80 mL centrifuge tube B with a cover, centrifuging at 2700 r / min for 10 min, and using a pipette to transfer 10 mL of supernatant B in a digestion tube, the volume of the transferred supernatant B being recorded as V3, immediately adding 6.4 g of mixed catalyst and 12 mL of concentrated sulfuric acid into the digestion tube according to the provisions of GB / T 6432-2018, determining the content W1 of crude protein in the 0.2% potassium hydroxide solution, and determining the total content W2 of crude protein in the same fermented soybean meal according to the provisions of GB / T 6432-2018.

[0054] S5, result calculation: (1) the content of total acid is X, which is calculated according to the following formula: X= × ; In the formula: X is the content of total acid in the sample, with units of grams per kilogram (g / kg) or grams per liter (g / L); c is the concentration of the potassium hydroxide standard titration solution, with units of moles per liter (mol / L); V KOH is the volume of the potassium hydroxide standard titration solution consumed when titrating the to-be-tested liquid A, with units of milliliters (mL); V2 is the volume of the potassium hydroxide standard titration solution consumed when titrating the blank, with units of milliliters (mL); V1 is the volume of the supernatant after centrifugation, with units of milliliters (mL); V0 is the total volume of the 0.2% potassium hydroxide solution added, with units of milliliters (mL); m is the mass of the sample, with units of grams (g); k is the conversion coefficient of acid, with a value of 0.090 kg / mol, calculated as lactic acid; 1000 is a conversion coefficient; (2) the solubility of potassium hydroxide protein is W, with a mass fraction, and the value is expressed as %, which is calculated according to the following formula: W= ×100; In the formula: W1 is the content of crude protein dissolved in the 0.2% potassium hydroxide solution, %; W2 is the total content of crude protein in the sample, %.

[0055] Comparative Example 1 The solubility of potassium hydroxide protein was determined by the method described in Appendix A of GB / T 19541-2017 Feedstuff Raw Materials Soybean Meal, wherein the mass of the fermented soybean meal sample was 1.5038 g.

[0056] Comparative Example Two: The potassium hydroxide protein solubility of the fermented soybean meal was determined by the method described in Appendix A of GB / T 19541-2017 Feedstuff Soybean Meal, wherein the mass of the fermented soybean meal sample was 1.5021 g.

[0057] The total acid content of the fermented soybean meal was determined by the method described in the application in Example One, Two, Three, Four and Five, and the results are shown in Table 1.

[0058] Table 1: Total acid content of fermented soybean meal

[0059] The potassium hydroxide protein solubility of the fermented soybean meal was determined by the method described in the application in Example One, Two, Three, Four and Five, and the results are shown in Table 2.

[0060] Table 2: Potassium hydroxide protein solubility of fermented soybean meal As shown in Table 2, the crude protein content W1 of the fermented soybean meal sample determined by the determination method of Example One, Two, Three, Four and Five was slightly higher than W1 determined by the determination method of Comparative Example One and Two, and the total crude protein content W2 of the fermented soybean meal sample determined by the determination method of Example One, Two, Three, Four and Five was basically the same. Thus, it can be calculated that the potassium hydroxide protein solubility W of the fermented soybean meal of Example One, Two, Three, Four and Five was slightly higher than the potassium hydroxide protein solubility W of the fermented soybean meal of Comparative Example One and Two.

[0061] In summary, the method for simultaneously determining the total acid and protein solubility of fermented soybean meal according to the application takes into account the influence of the total acid of fermented soybean meal on the detection results of potassium hydroxide protein solubility, and describes the determination method and calculation method of the total acid content of fermented soybean meal, thereby improving the accuracy of the detection of potassium hydroxide protein solubility of fermented soybean meal.

[0062] The above is only a specific embodiment of the application, but the technical features of the application are not limited thereto. Any simple change, equivalent replacement or modification made on the basis of the application to solve the same technical problem and achieve the same technical effect is covered by the protection scope of the application.

Claims

1. A method for simultaneously determining the total acidity and protein solubility of fermented soybean meal, characterized in that, Includes the following steps: S1. Determination of pH of 0.2% potassium hydroxide solution: Weigh 2.35g of potassium hydroxide with a purity of 85%, dissolve it in deionized water and make up to 1L to obtain a 0.2% potassium hydroxide solution; use a pH meter to measure its pH value and record it as the initial pH of the 0.2% potassium hydroxide solution; S2. Sample Extraction: Accurately weigh two portions of fermented soybean meal, each weighing 1.5000–2.1000 g, to obtain samples. Keep the weights of the two samples consistent and record the mass of one sample as m. Then, place the two samples into 250 mL beaker A and 250 mL beaker B, respectively. Add 75–100 mL of 0.2% potassium hydroxide solution to beaker A containing the samples. Record the volume of the added 0.2% potassium hydroxide solution as V0. After stirring on a magnetic stirrer, transfer the solution to a 100 mL capped centrifuge tube A. Centrifuge the tube and then transfer 25–50 mL of the supernatant A into a 100 mL beaker C using a pipette to obtain the test solution A. Record the volume of the transferred supernatant A as V1. S3. Determination of total acidity: (1) Place beaker C containing test solution A on a magnetic stirrer, immerse the pH meter electrode in test solution A, turn on the magnetic stirrer and observe the pH change of test solution A; (2) Quickly add potassium hydroxide standard titration solution with a concentration of 0.1000-0.1100 mol / L to test solution A for titration, and observe the pH change of test solution A at any time; when approaching the titration endpoint, slow down the titration speed and add half a drop at a time until the pH of test solution A reaches the titration endpoint, and record the volume of potassium hydroxide standard titration solution consumed at this time as V. KOH (3) Perform a blank test. No fermented soybean meal sample is added in the blank test. The remaining steps are the same as S2 and S3. Record the volume of potassium hydroxide standard titration solution consumed as V2. S4. Determination of protein solubility: (1) Add a volume of 0.2% potassium hydroxide solution of volume V0 to beaker B containing the fermented soybean meal sample, and then add the potassium hydroxide standard titration solution from S3. The volume added is [ ×(V) KOH (2) After stirring on a magnetic stirrer, the test solution B is obtained; (3) The test solution B is transferred to an 80mL centrifuge tube B with a cap, and centrifuged in a centrifuge. Then, 10-15mL of supernatant B is transferred to a digestion tube using a pipette. The volume of supernatant B transferred is recorded as V3. Immediately, according to GB / T6432-2018, mixed catalyst and concentrated sulfuric acid are added to the digestion tube. The crude protein content W1 in 0.2% potassium hydroxide solution is determined, and the total crude protein content W2 in the same fermented soybean meal is determined according to GB / T 6432-2018. S5. Calculation of results: (1) The total acid content is expressed as X and calculated using the following formula: X= × ； In the formula: X — the total acid content in the sample, in grams per kilogram (g / kg) or grams per liter (g / L). c — Concentration of potassium hydroxide standard titration solution, in moles per liter (mol / L). V KOH —The volume of potassium hydroxide standard titration solution consumed when titrating test solution A, in milliliters (mL); V2 — The volume of potassium hydroxide standard titration solution consumed during blank titration, in milliliters (mL); V1 — Volume of supernatant transferred after centrifugation, in milliliters (mL). V0 — Total volume of 0.2% potassium hydroxide solution added, in milliliters (mL); m — the mass of the sample, in grams (g); k – the conversion factor for acids, expressed in kilograms per mole (kg / mol) for different types of acids. 1000 – Conversion factor; (2) The solubility of potassium hydroxide in proteins is expressed as a mass fraction (W), with the value expressed as a percentage (%), and is calculated using the following formula: W= ×100； Where: W1—the crude protein content of the sample dissolved in 0.2% potassium hydroxide solution, %; W2—Total crude protein content in the sample, %.

2. The method for simultaneously determining total acidity and protein solubility in fermented soybean meal according to claim 1, characterized in that, The magnetic stirrers described in S2 and S4 both have a stirring speed of 300 r / min and a stirring time of 20 min.

3. The method for simultaneously determining total acidity and protein solubility in fermented soybean meal according to claim 1, characterized in that, The centrifuge speeds described in S2 and S4 are both 2700 r / min, and the centrifugation time is 10 min.

4. The method for simultaneously determining total acidity and protein solubility in fermented soybean meal according to claim 1, characterized in that, The magnetic stirrer described in S3 has a stirring speed of 200 r / min.

5. The method for simultaneously determining total acidity and protein solubility in fermented soybean meal according to claim 1, characterized in that, For the potassium hydroxide standard titration solutions described in S3 and S4, when the total acid content is ≤4 g / kg, titration should be performed using a potassium hydroxide standard titration solution with a concentration of 0.01 mol / L or 0.05 mol / L.

6. The method for simultaneously determining total acidity and protein solubility in fermented soybean meal according to claim 1, characterized in that, The pH of the titration endpoint described in S3 is the initial pH of the 0.2% potassium hydroxide solution in S1.

7. The method for simultaneously determining the total acidity and protein solubility of fermented soybean meal according to claim 1, characterized in that, The preparation method of the mixed catalyst in S4 is as follows: weigh 0.4g of copper sulfate pentahydrate and 6.0g of potassium sulfate or sodium sulfate, grind and mix them to obtain the mixed catalyst; the concentration of the concentrated sulfuric acid is 98%.

8. The method for simultaneously determining the total acidity and protein solubility of fermented soybean meal according to claim 1, characterized in that, The specific values ​​of the conversion factor k for acids in S5 are as follows: malic acid, 0.067 kg / mol; acetic acid, 0.060 kg / mol; tartaric acid, 0.075 kg / mol; citric acid, 0.064 kg / mol; citric acid containing one molecule of water of crystallization, 0.070 kg / mol; lactic acid, 0.090 kg / mol; hydrochloric acid, 0.036 kg / mol; sulfuric acid, 0.049 kg / mol; phosphoric acid, 0.049 kg / mol.

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