A method for measuring solid matter in avian bile based on turbidity detection

Abstract

The present invention relates to the technical field of fowl bile processing, and in particular to a method for detecting the bile acid content in fowl bile based on turbidity, which comprises the following steps: adjusting a solution containing bound bile acid to alkaline to obtain a suspension; and then measuring the OD value of the suspension. 600 This solution can solve the technical problems of long time and high cost in detecting bile acid content in poultry bile. When the purchased poultry bile raw materials arrive at the inspection site, this method can be used to conduct a preliminary test on the bile acid content in the poultry bile, and complete the on-site inspection work. Unqualified products with too low bile acid content can be returned on the spot, which greatly saves time and cost. This solution can be applied to practical operations such as the quality inspection of poultry bile, the research and development and production of poultry bile powder and artificial bear bile powder.

Description

Technical Field

[0001] The invention relates to the technical field of poultry bile processing, and in particular to a method for detecting the bile acid content in poultry bile based on turbidity. Background Art

[0002] Taurochenodeoxycholic acid has strong anti-inflammatory, antitussive and expectorant effects, and is most effective for simple tracheitis. It can also be taken orally to treat cholelithiasis. Taurocholic acid can reduce the permeability of capillaries in inflamed tissues, inhibit inflammatory swelling, and inhibit the production of inflammatory mediators such as NO, PGE2, and histamine, thereby having an anti-inflammatory effect. Taurochenodeoxycholic acid and taurocholic acid are both conjugated bile acid components, widely present in the bile of many animals, and are the main active ingredients in poultry bile.

[0003] The current mainstream method for detecting bile acid in poultry bile is reversed-phase high-performance liquid chromatography-elastic saturation (HPLC-ELSD). Accurate quantification requires sample pretreatment and a calibration curve for the instrument, making the operation relatively complex. Testing is also lengthy, taking 6-8 hours per test. Only one sample can be tested at a time, often requiring more than 20 hours to complete when a large number of samples are sampled. This makes it difficult to provide results on the same day the raw bile arrives. Furthermore, the testing equipment is expensive, costing hundreds of thousands of yuan to purchase, along with high maintenance and operating costs and the high requirements for operator expertise. Due to this high cost, poultry bile suppliers often avoid purchasing HPLC-ELSD equipment to test bile acid content when purchasing from slaughterhouses. This results in a lack of quality control at the supplier end, increasing operating costs for downstream processing and production companies. (For example, bile may be found to contain substandard bile acid content only after delivery to a downstream processing plant, resulting in significant waste in transportation and storage costs.) In summary, there is an urgent need to develop a method that can quickly and inexpensively detect the content of bile acid components in poultry bile to overcome the shortcomings of the HPLC-ELSD method in the prior art. Summary of the Invention

[0004] The present invention aims to provide a method for detecting the bile acid content in avian bile based on turbidity, so as to solve the technical problems of long time consumption and high cost in detecting the bile acid content in avian bile.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for detecting bile acid content in avian bile based on turbidity, comprising the steps of: adjusting a solution containing bound bile acid to alkaline to obtain a suspension; and then measuring the OD of the suspension. 600 value.

[0007] The principle and advantage of this solution are: after a lot of research, the inventors found that the water solubility of free bile acid is poor, but the water solubility of bound bile acid is good. For example, chenodeoxycholic acid is not water soluble, while taurochenodeoxycholic acid is very water soluble. The inventors then analyzed the components of avian bile and found that the bound bile acid (mainly taurochenodeoxycholic acid and taurocholic acid) accounts for the vast majority of the bile acid in avian bile, and then used this property to detect the bile acid content in avian bile. The bound bile acid is converted into unbound bile acid (free bile acid) by the alkaline lysis method. Due to its poor water solubility, the free bile acid forms a large amount of precipitate. After the precipitate is dispersed in the solution, a suspension is formed, which can be detected by measuring the turbidity value (OD) in the solution. 600 The free bile acid content can be measured by turbidimetric analysis (HPLC), which can then be used to calculate the total bile acid content in the avian bile sample. The experimental results show that the turbidity-based method for detecting bile acid in avian bile is highly consistent with the results obtained by HPLC, making it reliable and feasible.

[0008] When the purchased poultry gallbladder raw materials arrive at the inspection site, this method can be used to conduct a preliminary test on the bile acid content in the poultry gallbladder, and the inspection work can be completed on the spot. Substandard products with too low bile acid content can be returned on the spot, greatly saving time and cost. If the detection method of the existing technology is adopted, the detection time will be relatively long, and it is impossible to quickly process the substandard products. The substandard products will take a long time to return and will take up warehouse space. In addition, if the storage method is not appropriate, the poultry gallbladder will become corrupt and deteriorate, which not only loses the corrupted raw materials, but also causes cross contamination to other raw materials and the storage environment, causing greater losses to the company.

[0009] Furthermore, the solution containing bound bile acid is obtained by the following method: avian bile is treated with an organic solvent, and the liquid phase is taken to obtain a solution containing bound bile acid.

[0010] Using the above technical solution, avian bile is treated with an organic solvent to precipitate components such as protein and lipids in the bile, leaving a large amount of bound bile acid in the liquid phase. The solution containing bound bile acid can be used in subsequent alkaline lysis and precipitation steps of free bile acid.

[0011] When avian bile is directly subjected to alkaline lysis and bile acid precipitation, the linear relationship between absorbance and bile acid content is poor. This is because some impurities, primarily proteins, precipitate during this process, affecting accurate quantification. Therefore, adding a pretreatment step before this operation can overcome this problem. Methods for removing proteins include isoelectric precipitation, salting out, and organic solvent precipitation. Bile contains a large number of protein types, making isoelectric precipitation unsuitable. Salting out will leave a large amount of salt ions in the sample, affecting the effectiveness of alkaline lysis. This protocol uses an organic solvent method to precipitate protein impurities, which also has a certain precipitation effect on other impurities, thereby eliminating the impact of impurities on the linear relationship between absorbance and bile acid content, making the detection process more accurate.

[0012] Furthermore, the organic solvent includes one or two of ethanol, isopropanol and acetone.

[0013] Using this technical solution, the three organic solvents can fully remove impurities such as protein and lipids from avian bile, reducing factors that affect the subsequent alkaline lysis and precipitation of free bile acid, thereby making bile acid measurement more accurate. All three solvents improved the linear relationship between absorbance and bile acid content, with isopropyl alcohol showing the best impurity removal effect, minimizing the impact on this linear relationship.

[0014] Furthermore, the avian bile is obtained by the following method: cutting the avian bile, filtering to obtain the liquid phase, and then centrifuging to obtain the supernatant to obtain the avian bile.

[0015] By adopting the above technical solution, impurities such as muscle tissue and connective tissue are removed by centrifugation, thereby fully reducing substances that affect bile acid detection in poultry bile.

[0016] Furthermore, the avian bile is diluted 20-100 times to obtain diluted avian bile, and then the diluted avian bile is treated with an organic solvent, and the liquid phase is taken to obtain a solution containing bound bile acid; the volume ratio of the diluted avian bile to the organic solvent is 1:2.

[0017] By using the above technical solution, diluting avian bile can avoid inaccurate detection caused by excessive concentration of the target component. By using the above volume ratio of diluted avian bile to organic solvent, impurities in avian bile that affect bile acid detection can be effectively removed.

[0018] Furthermore, the solution containing bound bile acid was heated at 60° C. for 10 minutes to evaporate the organic solvent; then, a sodium hydroxide solution was added to the solution containing bound bile acid, and the mixture was heated at 60° C. for 10 minutes in a sealed environment to obtain a suspension.

[0019] Using this technical solution, the organic solvent in the bound bile acid solution is first removed to prevent it from interfering with subsequent reactions. The bound bile acid then reacts with sodium hydroxide to form free bile acid. Due to the poor water solubility of the free bile acid, a large amount of precipitate forms, which disperses in the solution to form a suspension.

[0020] Furthermore, the method further comprises the steps of determining a standard curve: preparing a plurality of standard solutions containing standard avian bile powder, wherein the content of the standard avian bile powder in the standard solutions is different; treating the standard solutions with an organic solvent and an alkaline solution to obtain a standard suspension, and measuring the OD of the standard suspension. 600 The standard curve was established by fitting.

[0021] By adopting the above technical solution and establishing a standard curve, a quantitative relationship between the absorbance value and the content of avian bile powder can be obtained, which lays a foundation for measuring and calculating the specific content value of bile acid from unknown samples.

[0022] Furthermore, the mass fraction of bile acid in the standard poultry bile powder is ≥50%.

[0023] Using the above technical solution, a qualified product is one whose bile acid content in poultry bile powder is ≥50%. The bile acid in poultry bile powder is primarily present in the form of taurochenodeoxycholic acid, which accounts for approximately 85% of the total bile acid. Taurocholic acid also accounts for approximately 13% of the total bile acid. Generally speaking, the bile acid content of standard poultry bile powder is 50-70%.

[0024] Furthermore, the content of standard avian bile powder in the standard solution is 0.2-2 mg / ml.

[0025] By adopting the above technical solution, when the concentration of standard avian bile powder in the standard solution is 0.2-2 mg / ml, the correlation coefficient obtained by linear regression fitting of absorbance and standard concentration is more ideal, and the test result is more accurate.

[0026] Furthermore, the bile acid content in avian bile is obtained by the following method: obtaining avian bile from avian bile, treating the avian bile with an organic solvent, taking a liquid phase to obtain a solution containing bound bile acid; adjusting the solution containing bound bile acid to alkaline to obtain a suspension; and then measuring the OD value of the suspension. 600 Value; According to the OD of the suspension 600 The bile acid content in avian bile was calculated based on the values ​​and the standard curve.

[0027] Using the above technical solution, the OD 600 The bile acid content in avian bile can be calculated by substituting the value into the formula represented by the standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the standard curve of Example 1.

[0029] Figure 2 This is the linear fitting image of Experimental Example 1.

[0030] Figure 3 This is the linear fitting image of Experimental Example 2 (0.01-2 mg / ml).

[0031] Figure 4 This is the linear fitting image of Experimental Example 2 (0.08-1.5 mg / ml).

[0032] Figure 5 This is the linear fitting image of Experimental Example 3 (no pretreatment before alkaline lysis).

[0033] Figure 6 This is the linear fitting image of Experimental Example 3 (pre-treatment before alkaline lysis). DETAILED DESCRIPTION

[0034] Example 1:

[0035] 1. Standard curve determination:

[0036] Preparation of standard product: Take 100g of frozen avian bile, thaw it, cut the gallbladder with scissors, filter it through double-layer gauze, centrifuge the filtrate at 10,000rpm for 5 minutes, take the centrifugal supernatant and dry it into powder (about 4g). Determine the bile acid content (expressed as mass percentage W%) by reverse-phase high-performance liquid chromatography-elastic dynamics (RP-HPLC-ELSD) method (measured by the content of taurochenodeoxycholic acid and taurocholic acid). Obtain avian bile powder with a known qualified bile acid content and store it in a dry, sealed container at room temperature as a standard product.

[0037] Standard curve drawing: Take 7 15ml centrifuge tubes and mark them 1-7 respectively. Weigh 20mg of standard in centrifuge tube 1 and dissolve it in 2ml of purified water to make a standard solution with a concentration of 10mg / ml. Dilute it in centrifuge tubes 2-7 to obtain 1ml of standard solution at 0.2, 0.4, 0.8, 1.2, 1.6, and 2mg / ml respectively. Add 2ml of isopropanol to centrifuge tubes 2-7 respectively, mix well and centrifuge at 10000rpm for 5 minutes, take the supernatant, heat it in a water bath with the lid open at 60℃ for 10 minutes, add 1ml of 2M NaOH solution, tighten the tube cap, heat it in a water bath at 60℃ for 20 minutes, mix well and measure the OD 600 , draw the standard curve: y = Ax-B, where y is the standard concentration (unit: mg / ml); x is the absorbance value of the standard; A and B are both constants. Take the absorbance value of the standard as the independent variable and the standard concentration as the dependent variable, and perform a one-variable linear fit to obtain the values ​​of A and B.

[0038] 2. Sample testing:

[0039] Take 10g of frozen avian bile (sample mass is expressed in m), thaw and cut the gallbladder with scissors, filter through double gauze, centrifuge the filtrate at 10000rpm for 5 minutes, take the centrifugal supernatant (quantitative volume V), take 0.5ml and dilute it 50 times with purified water (the dilution factor can be selected according to the sample situation, and the dilution factor is expressed by N), take 1ml of the dilution and add 2ml of isopropanol, mix well and centrifuge at 10000rpm for 5 minutes, take the supernatant, heat it at 60℃ with a water bath with the lid open for 10 minutes, add 1ml of 2M NaOH solution, tighten the tube cap, heat it at 60℃ with a water bath for 20 minutes, mix well and measure the OD 600 (OD of the sample to be tested 600 The bile acid content of avian bile is calculated as follows: y' = (Ax'-B) × W% × N × V / m, where y' is the bile acid content per gram of avian bile frozen product (in mg / g); x' is the OD value of the sample to be tested. 600 value; V is the volume of the supernatant (unit: ml); N is the dilution factor; W% is the mass percentage of bile acid in the standard; m is the mass of the sample (frozen avian bile) (unit: g); A and B are constants calculated from the standard curve.

[0040] In this embodiment, the bile acid content in the standard sample selected is 60% (mass percentage, the bile acid specifically is taurochenodeoxycholic acid and taurocholic acid). The measurement data are shown in Table 1, and the standard curve is: y = 16.044x-0.0362 (y = concentration of avian bile powder standard before alcohol precipitation, x = measured corresponding OD 600 For standard curve images, see Figure 1 . Measure the OD of the bird bile sample 600 After the value is obtained, the OD value of the bird gallbladder sample is 600 The values ​​were substituted into the standard curve, and the bile acid content was calculated according to the following formula: y'=(16.044x'-0.0362)×60%×50×V / 10.

[0041] Table 1: Concentration and absorbance of standard samples of avian bile powder

[0042] Concentration of poultry bile powder (mg / ml) Absorbance (Abs) 0.2 0.015 0.4 0.027 0.8 0.052 1.2 0.077 1.6 0.102 2 0.127

[0043] Experimental Example 1: Study on the effect of impurity removal

[0044] Take 10g of frozen poultry bile, thaw it and cut the gallbladder with scissors. Filter it through double-layer gauze, centrifuge the filtrate at 10,000rpm for 5 minutes, take the centrifugal supernatant and dry it into powder (about 0.4g, the mass fraction of bile acid is 60% after measurement). Weigh 40mg of poultry bile powder in a centrifuge tube, add 2ml of purified water to dissolve it, and prepare a poultry bile powder solution with a concentration of 20mg / ml.

[0045] Ethanol precipitation to remove impurities: dilute to obtain 1 ml of 0.2, 0.4, 0.8, 1.2, and 1.6 mg / ml avian bile powder solutions, add 2 ml of ethanol to each, mix well, centrifuge at 10,000 rpm for 5 minutes, take the supernatant, heat at 60°C in a water bath with the lid open for 10 minutes, add 1 ml of 2M NaOH solution to each solution, cover the tube tightly, heat at 60°C in a water bath for 20 minutes, mix well, and measure the OD 600 , draw a linear curve and check the degree of linearity.

[0046] Isopropanol precipitation to remove impurities: dilute to obtain 1 ml of 0.2, 0.4, 0.8, 1.2, and 1.6 mg / ml avian bile powder solutions, add 2 ml of isopropanol to each, mix well, centrifuge at 10,000 rpm for 5 minutes, take the supernatant, heat at 60°C in a water bath with the lid open for 10 minutes, add 1 ml of 2M NaOH solution to each solution, cover the tube tightly, heat at 60°C in a water bath for 20 minutes, mix well, and measure the OD 600 , draw a linear curve and check the degree of linearity.

[0047] Acetone precipitation to remove impurities: dilute to obtain 1 ml of 0.2, 0.4, 0.8, 1.2, and 1.6 mg / ml avian bile powder solutions, add 2 ml of acetone to each, mix well, centrifuge at 10,000 rpm for 5 minutes, take the supernatant, heat at 60°C in a water bath with the lid open for 10 minutes, add 1 ml of 2M NaOH solution to each solution, cover the tube tightly, heat at 60°C in a water bath for 20 minutes, mix well, and measure the OD 600 , draw a linear curve and check the degree of linearity.

[0048] The measurement results are shown in Table 2, and the linear fitting images are shown in Figure 2 .Depend on Figure 2 It can be seen that after isopropanol precipitation, the correlation coefficient of linear fitting (R 2, greater than 0.999 is considered to be an ideal linear fit relationship) is best. After being treated with ethanol and acetone, the correlation coefficient is not ideal, and the content level of fowl bile powder cannot be accurately reflected by the absorbance value. In fowl bile powder, in addition to bile acid, it also contains a large amount of impurities such as protein and lipid. If these impurities cannot be removed before alkali treatment, or if the target component bile acid is also removed at the same time during the process of removing impurities, the accuracy of subsequent measurements will be seriously affected. For example, if impurities are not fully removed, non-bile acid substances will also precipitate during the subsequent alkali precipitation, which will cause the measured absorbance value to be high; if the target component bile acid is also removed at the same time during the process of removing impurities, the measured absorbance value will be low. Both of the above situations will cause the measured value of absorbance to not accurately reflect the amount of fowl bile powder, resulting in an undesirable correlation coefficient of linear fit. The most ideal state is to remove impurities, retain all bile acid substances, and after alkali precipitation, measure the turbidity of the suspension to accurately reflect the content of bile acid by turbidity. Research has found that using isopropyl alcohol as a decontamination solvent can fully remove impurities that affect the linear relationship while retaining the target components to the greatest extent possible.

[0049] Table 2: Absorbance measurement results under different impurity removal methods

[0050] Concentration of poultry bile powder (mg / ml) Absorbance (ethanol) Absorbance (isopropanol) Absorbance (acetone) 0.1 0.015 0.012 0.011 0.2 0.037 0.025 0.020 0.4 0.071 0.051 0.045 0.6 0.107 0.077 0.063 0.8 0.158 0.103 0.097

[0051] Experimental Example 2: Study on the linear range of detection

[0052] Take 10g of frozen poultry bile, thaw it and cut the gallbladder with scissors, filter it with double gauze, centrifuge the filtrate at 10000rpm for 5 minutes, take the centrifugal supernatant, add 2 times the volume of isopropanol, mix it and centrifuge it at 10000rpm for 5 minutes, take the supernatant and dry it into powder, use reversed-phase high performance liquid chromatography-ELSD to detect the bile acid content (e.g., 60%), and obtain poultry bile powder with qualified bile acid content (e.g., 0.4g). Weigh 40mg of poultry bile powder in a centrifuge tube, add 2ml of purified water to dissolve it, and prepare a poultry bile powder solution with a concentration of 20mg / ml. Dilute it to obtain 1ml of poultry bile powder solution at 0.02, 0.08, 0.16, 0.2, 0.8, 1.6, 2, 3, and 4mg / ml, respectively, add 1ml of 2M NaOH solution, tighten the tube cap, heat it in a water bath at 60℃ for 20 minutes, mix it and measure OD 600 , draw a linear curve and find the best linear range. The dilution concentration of avian bile powder is: 0.01, 0.04, 0.08, 0.1, 0.4, 0.8, 1, 1.5, 2 mg / ml. Table 3 is the absorbance test results, Figure 3 and Figure 4 is the linear fitting result.

[0053] Table 3: Different concentrations of poultry bile powder and absorbance measurements

[0054] Concentration of poultry bile powder (mg / ml) Absorbance (Abs) 0.01 0.006 0.04 0.008 0.08 0.011 0.1 0.014 0.4 0.052 0.8 0.102 1 0.127 1.5 0.189 2 0.227

[0055] Experimental Example 3: Pretreatment Process Research

[0056] Take 10g of frozen poultry gallbladder, thaw it and cut the gallbladder with scissors, filter it with double gauze, centrifuge the filtrate at 10000rpm for 5 minutes, take 1ml of the centrifugal supernatant, dilute it 20 times with purified water, take 0.1, 0.2, 0.3, 0.4, and 0.5ml of the dilution respectively, add purified water to 1ml, add 1ml of 2M NaOH solution respectively, cover the tube tightly, heat it in a water bath at 60℃ for 20 minutes, mix well and measure OD 600 , draw a linear curve to check the linearity. Bile dilution concentration (ml / ml): 0.005, 0.01, 0.015, 0.02, 0.025. The absorbance measurement results are shown in Table 4, and the linear fitting graph is shown in Figure 5 shown.

[0057] Table 4: Concentration and absorbance of different avian bile

[0058] Avian bile concentration (ml / ml) Absorbance (Abs) 0.005 0.152 0.01 0.181 0.015 0.224 0.02 0.287 0.025 0.310

[0059] Take 10g of frozen poultry bile, thaw it and cut the gallbladder with scissors, filter it with double gauze, centrifuge the filtrate at 10000rpm for 5 minutes, take the centrifugal supernatant, dilute it 20 times with purified water, take 0.1, 0.2, 0.3, 0.4, and 0.5ml of the dilution solution and add purified water to 1ml, add 2ml of isopropanol respectively, mix well and centrifuge at 10000rpm for 5 minutes, take the supernatant, heat it at 60℃ with a water bath with the lid open for 10 minutes, add 1ml of 2M NaOH solution respectively, cover the tube tightly, heat it at 60℃ with a water bath for 20 minutes, mix well and measure OD 600 , draw a linear curve to check the degree of linearity. The absorbance measurement results are shown in Table 5, and the linear fitting image is shown in Figure 6 shown.

[0060] Table 5: Concentration and absorbance of different poultry bile

[0061] Avian bile concentration (ml / ml) Absorbance (Abs) 0.005 0.033 0.01 0.064 0.015 0.095 0.02 0.126 0.025 0.158

[0062] contrast Figure 5 and Figure 6 The experimental results showed that after alcohol precipitation and alkaline lysis, the concentration of avian bile and OD 600In other words, in order for the turbidity value to better reflect the bile acid content, the poultry bile needs to be treated with alcohol precipitation to remove other impurities that affect the precipitation of free bile acid. These impurities cannot be removed by simple centrifugation or filtration. Only by adding a special organic solvent treatment step before the alkaline precipitation step can the impact of impurities on detection accuracy be fully avoided.

[0063] Experimental Example 4: Unknown Sample Verification Experiment

[0064] The detection process of this experimental example is shown in Example 1. Take 10g of frozen poultry bile from manufacturer 1, thaw it and cut the gallbladder with scissors, filter it through double-layer gauze, centrifuge the filtrate at 10,000rpm for 5 minutes, and take the centrifugal supernatant (quantitative volume V = 8.1ml). According to the summary of multiple experiments, it was found that the bile yield of different batches from different manufacturers was not much different. Generally, about 8ml of poultry bile (8±0.5ml) can be obtained from 10g of frozen poultry bile. Take 1ml of supernatant and add 2ml of isopropanol. After mixing, centrifuge at 10,000rpm for 5 minutes, take the supernatant, dry it into powder (weighing 0.049g), and use HPLC to detect the bile acid content (measured 63%). Take another 1ml of avian bile supernatant and dilute it 50 times with purified water. Take 1ml of the dilution and add 2ml of isopropanol. Mix well and centrifuge at 10000rpm for 5 minutes. Take the supernatant and heat it in a water bath with the lid open at 60℃ for 10 minutes. Add 1ml of 2M NaOH solution and tighten the tube cap. Heat it in a water bath at 60℃ for 20 minutes. Mix well and measure the OD. 600 Substituting the linear equation (measured as 0.066) into the equation (see Example 1), the bile acid content of the fowl bile was calculated as: y' = (16.044x' - 0.0362) × 60% × 50 × 8.1 / 10 = 24.852 (mg / g). The bile acid content of the frozen fowl bile per gram measured by HPLC was: 0.049 × 1000 × 8.1 × 63% / 10 = 25.005 (mg / g).

[0065] Take 10g of the frozen product of poultry bile from manufacturer 2, thaw it and cut the gallbladder with scissors, filter it through double-layer gauze, centrifuge the filtrate at 10,000rpm for 5 minutes, take the centrifugal supernatant (quantitative volume V=8.1ml), take 1ml and add 2ml isopropanol, mix well and centrifuge at 10,000rpm for 5 minutes, take the supernatant, dry it into powder (weighing 0.043g), and use HPLC to detect the bile acid content (measured 50%). Take another 1ml of poultry bile supernatant and dilute it 50 times with purified water, take 1ml of the dilution and add 2ml isopropanol, mix well and centrifuge at 10,000rpm for 5 minutes, take the supernatant, heat it at 60℃ for 10 minutes with a water bath with the lid open, add 1ml of 2M NaOH solution, tighten the tube cap, heat it at 60℃ for 20 minutes with a water bath, mix well and measure the OD 600(measured 0.047), substituted into the linear equation, the bile acid content of poultry bile y = (16.044 × 0.047-0.0362) × 60% × 50 × 8.1 / 10 = 17.444 (mg / g); HPLC method measured the bile acid content of each gram of poultry bile frozen product as: 0.043 × 1000 × 8.1 × 50% / 10 = 17.415 (mg / g).

[0066] The experimental results showed that the method based on turbidity to detect bile acid content in poultry bile was highly consistent with the results obtained by HPLC, and was reliable and feasible.

[0067] In order to further verify the accuracy of this protocol for detecting bile acid content in avian bile, the following comparative experiments were conducted:

[0068] (1) Take the frozen product of poultry gallbladder from manufacturer 1 and use the following method to detect the content of poultry gallbladder powder: take 10g of frozen product of poultry gallbladder, thaw it and cut the gallbladder with scissors, filter it with double-layer gauze, centrifuge the filtrate at 10000rpm for 5 minutes, take the supernatant of centrifugation, take 0.5ml of it and dilute it 50 times with purified water, take 1ml of the diluted supernatant and add 1ml of 2M NaOH solution, cover the tube tightly, heat it in a water bath at 60℃ for 20 minutes, mix it and measure the OD 600 That is, in this protocol, no alcohol precipitation was performed before alkaline lysis. The experiment was repeated three times, and the average bile acid content (mg / g) was calculated.

[0069] (2) Take the frozen product of poultry bile from manufacturer 1 and use the following method to detect the content of poultry bile powder: take 10g of frozen product of poultry bile, thaw it and cut the gallbladder with scissors, filter it with double-layer gauze, centrifuge the filtrate at 10000rpm for 5 minutes, and take the supernatant. Dilute 1ml of supernatant 50 times with purified water, take 1ml of dilution and add 2ml of ethanol, mix well and centrifuge at 10000rpm for 5 minutes, take the supernatant, heat it in a water bath with the lid open at 60℃ for 10 minutes, add 1ml of 2M NaOH solution, tighten the tube cap, heat it in a water bath at 60℃ for 20 minutes, mix well and measure the OD 600 That is, ethanol was used instead of isopropanol in the alcohol precipitation treatment of this protocol. The experiment was repeated three times, and the average bile acid content (mg / g) was calculated.

[0070] (3) Take the frozen product of poultry gallbladder from manufacturer 1 and use the following method to detect the content of poultry gallbladder powder: take 10g of frozen product of poultry gallbladder, thaw it and cut the gallbladder with scissors, filter it with double-layer gauze, centrifuge the filtrate at 10000rpm for 5 minutes, and take the supernatant. Dilute 1ml of supernatant 50 times with purified water, take 1ml of dilution and add 2ml of acetone, mix well and centrifuge at 10000rpm for 5 minutes, take the supernatant, heat it at 60℃ in a water bath with the lid open for 10 minutes, add 1ml of 2M NaOH solution, tighten the tube cap, heat it at 60℃ in a water bath for 20 minutes, mix well and measure the OD 600That is, this protocol uses acetone to remove impurities instead of isopropanol. The experiment was repeated three times, and the average bile acid content (mg / g) was calculated.

[0071] In order to compare whether the results obtained by the above detection methods can accurately reflect the actual content of bile acid, we calculated the measurement deviation as follows: bile acid content measured by turbidimetry - bile acid content measured by HPLC / bile acid content measured by HPLC × 100%. The measurement deviation of (1) was 236.52%; the measurement deviation of (2) was 43.72%; and the measurement deviation of (3) was 14.70%. It can be seen that the measurement results obtained by the measurement methods (1), (2), and (3) are quite different from the measurement results of HPLC, indicating that the use of isopropyl alcohol to precipitate impurities is very important for the implementation of this solution.

[0072] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A method for detecting bile acid content in avian bile based on turbidity, characterized in that: The method comprises the following steps: adjusting the solution containing bound bile acid to alkaline to obtain a suspension; and then measuring the OD of the suspension. 600 value.

2. The method for detecting bile acid content in avian bile based on turbidity according to claim 1, wherein: The solution containing bound bile acid is obtained by the following method: avian bile is treated with an organic solvent, and a liquid phase is taken to obtain a solution containing bound bile acid.

3. The method for detecting bile acid content in avian bile based on turbidity according to claim 2, wherein: The organic solvent includes one or two of ethanol, isopropanol and acetone.

4. The method for detecting bile acid content in avian bile based on turbidity according to claim 3, wherein: The avian bile is obtained by the following method: cutting the avian bile, filtering to obtain the liquid phase, and then centrifuging to obtain the supernatant to obtain the avian bile.

5. The method for detecting bile acid content in avian bile based on turbidity according to claim 4, characterized in that: The avian bile is diluted 20-100 times to obtain diluted avian bile, and then the diluted avian bile is treated with an organic solvent, and a liquid phase is taken to obtain a solution containing bound bile acid; the volume ratio of the diluted avian bile to the organic solvent is 1:

2.

6. The method for detecting bile acid content in avian bile based on turbidity according to claim 1, wherein: The solution containing bound bile acid was heated at 60° C. for 10 minutes to evaporate the organic solvent; then, a sodium hydroxide solution was added to the solution containing bound bile acid, and the mixture was heated at 60° C. for 10 minutes in a sealed environment to obtain a suspension.

7. The method for detecting bile acid content in avian bile based on turbidity according to claim 1, wherein: The method further comprises the steps of: preparing a plurality of standard solutions containing standard avian bile powder, wherein the content of the standard avian bile powder in the standard solutions is different; treating the standard solutions with an organic solvent and an alkaline solution to obtain a standard suspension; and measuring the OD of the standard suspension. 600 The standard curve was established by fitting.

8. The method for detecting bile acid content in avian bile based on turbidity according to claim 7, characterized in that: The mass fraction of bile acid in the standard poultry bile powder is ≥50%.

9. The method for detecting bile acid content in avian bile based on turbidity according to claim 8, characterized in that: The content of the standard avian bile powder in the standard solution is 0.2-2 mg / ml.

10. The method for detecting bile acid content in avian bile based on turbidity according to claim 9, characterized in that: The bile acid content in avian bile is obtained by the following method: obtaining avian bile from avian bile, treating the avian bile with an organic solvent, taking the liquid phase to obtain a solution containing bound bile acid; adjusting the solution containing bound bile acid to alkaline to obtain a suspension; and then measuring the OD value of the suspension. 600 Value; According to the OD of the suspension 600 The bile acid content in avian bile was calculated based on the values ​​and the standard curve.

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