Method for detecting content of starch in pumpkin pulp

Abstract

The invention belongs to the technical field of food detection, and particularly relates to a method for detecting the content of starch in pumpkin pulp. Comprising the following steps: S1, pretreating a pumpkin sample; S2, detecting starch; S2.1, cleaning with ethanol to remove glucose and maltodextrin in a sample to be detected; S2.2, dissolving resistant starch with DMSO; according to the method disclosed by the invention, the detection time is greatly shortened, the whole detection time is only 3-4 hours, and the actual detection operation time of a national standard method is more than 8 hours.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food detection, and particularly relates to a method for detecting starch content in pumpkin pulp. BACKGROUND

[0002] Starch is the main storage material of carbohydrates in higher plants, and exists in different tissue parts of plants, such as leaves, fruits, seeds and the like. Starch in plants is mainly composed of two different components, amylose and amylopectin. Starch content not only affects the yield of major crops such as rice, wheat, barley and potato, but also determines the taste quality of many vegetable crops such as pumpkin, sweet potato, taro and chestnut. Therefore, starch content is widely used as an important index for measuring the quality of various agricultural products.

[0003] Pumpkin is a common vegetable crop in Cucurbitaceae Cucurbita, which is drought and barren tolerant, has wide adaptability and high yield, and can be used as food and grain, and is an important economic crop. The fruit is rich in nutrients and has excellent taste, and is rich in starch, vitamins, lutein, carotene, phenols and flavonoids and other nutrients. Studies have shown that the starch content of pumpkin is positively correlated with the waxy nature of the fruit taste.

[0004] At present, the common methods for detecting the starch content in pumpkin pulp mainly include two types: the first is the national standard GB-5009.9-2023 method, which has high accuracy, but has the disadvantages of long test time and high cost; the second type is the wavelength absorption method and near-infrared spectroscopy method built by the laboratory, which requires high laboratory instruments, or through extraction and purification of pumpkin pulp starch for detection. These methods are complicated, and the reported studies have not been compared and detected with the national standard method, and the accuracy of the results is insufficient. Therefore, finding a simple and efficient method for detecting the starch content of pumpkin fruit has important theoretical significance and economic value for pumpkin breeding and the detection of pumpkin fruit quality on the market, and the development of related reagent kits. SUMMARY

[0005] The purpose of the present application is to provide a method for detecting the starch content in pumpkin pulp.

[0006] A method for detecting the starch content in pumpkin pulp, comprising the following steps: S1, pumpkin sample pretreatment: picking fresh pumpkins, removing the peel and cutting into thin slices, drying in an oven to constant weight, then grinding in a mortar to obtain a dried pumpkin pulp sample powder, which is the sample to be tested; S2, starch detection: S2.1, wash the sample to be tested with ethanol to remove glucose and maltodextrin, S2.2, dissolve the resistant starch in DMSO, In the test tube, immediately add 2 mL of DMSO, mix well, put into a boiling water bath for 5 min, take the test tube out of the boiling water bath, immediately add 3 mL of α-amylase solution diluted 30 times with MOPS Buffer, mix well on a vortex shaker for 20 s, and then put it into a boiling water bath for 6 min; S2.3, starch determination, Put the test tube into a 50℃ water bath for about 5 min; add 4 mL of sodium acetate Buffer with a concentration of 200 mM at pH 4.5, then add 100 μL of starch glucosidase solution, vortex mix, and incubate at 50℃ for 30 min; Transfer the solution in the test tube into a 25 mL volumetric flask, rinse the test tube with sodium acetate Buffer with a concentration of 200 mM at pH 4.5 and make up to volume, Take 1 mL of the mixed solution after making up to volume into a 1.5 mL EP tube, centrifuge at 12000 rcf for 2 min, take 100 μL of supernatant into the bottom of a 15 mL test tube, add 3 mL of GOPOD reagent in the test tube, incubate at 50℃ for 20 min, and obtain the sample solution, Take 100 μL of sodium acetate Buffer with a concentration of 200 mM at pH 4.5 into the bottom of a 15 mL test tube, add 3 mL of GOPOD reagent in the test tube, incubate at 50℃ for 20 min, and obtain the blank control solution, Take 100 μL of D-glucose standard solution into the bottom of a 15 mL test tube, add 3 mL of GOPOD reagent in the test tube, incubate at 50℃ for 20 min, and obtain the standard solution, Measure the absorbance values of the sample solution, blank control solution and standard solution at 510 nm; S3, starch content calculation, Use the absorbance value measured in step S2.3 to calculate the starch content of the pumpkin sample by substituting the following formula: Wherein: : the difference between the absorbance values of the sample solution and the blank control at 510 nm, i.e. , : the difference between the absorbance values of the standard solution and the blank control at 510 nm, i.e. , EV: dilution concentration, i.e. the volume number of the sample solution in step S2.3, which is 25 mL in this step, : convert the sample weight to 100mg, W is the actual weight of the pumpkin sample weighed in S2.1, in mg.

[0007] In the above method for detecting the starch content in pumpkin pulp, in S1, the thickness of the slices is 1-2mm, the drying temperature is 60-65℃, and the dried sample powder is sieved through a 40-mesh sieve.

[0008] In the above method for detecting the starch content in pumpkin pulp, in S2.1, the ethanol used to remove glucose and maltodextrin from the sample to be tested is 0.1g of the sample to be tested is weighed into a test tube, 5mL of 80v / v% ethanol solution is added to the test tube, mixed, incubated at 80-85℃ for 5min, and then 5mL of 80v / v% ethanol solution is added again, mixed again. The supernatant is discarded by centrifugation, 5mL of 80v / v% ethanol solution is added to the test tube, mixed, 5mL of 80v / v% ethanol solution is added again, mixed again, and then centrifuged again, and the supernatant is poured out.

[0009] In the above method for detecting the starch content in pumpkin pulp, S4 is further included, the experimental results are compared and calibrated with national standard data, 3 biological replicates are made for each sample, then the results of the acid hydrolysis method in the national standard GB-5009.9-2023 method are compared, and the detection results are verified and calibrated using the correlation curve.

[0010] In the above method for detecting the starch content in pumpkin pulp, the correlation curve is y=1.378x+0.5454, R 2 =0.9968, x is the starch content value of the pumpkin sample in S3, and y is the national standard determination value.

[0011] In the above method for detecting the starch content in pumpkin pulp, in S2, the MOPS Buffer, alpha-amylase solution, amyloglucosidase solution, sodium acetate Buffer, GOPOD reagent and D-glucose standard solution are configured according to the method in the instruction manual of the total starch detection kit of Megazyme company, model K-TSTA.

[0012] Compared with the prior art, the method has the advantages that: 1、The method greatly shortens the detection time: the entire detection time only needs 3-4h, and the actual operation time of the national standard method is more than 8h.

[0013] 2、The method of the present application greatly saves manpower, material resources and the degree of operation complexity, and the demand for laboratory facilities and reagents: the method of the present application only needs ordinary laboratory operation and simple several kinds of laboratory common basic instruments, the operation is simple, the reagent toxicity is smaller, but the reagent and operation step of the national standard method are complicated, the laboratory facilities and instrument requirements are also very high, it needs to be carried out in a fume hood, also needs many distillation equipment and the like which are only in biochemical laboratory or high-standard laboratory, the experimental operation requirements of personnel are also very high, it needs to master deep chemical experimental operation skills, which is not friendly to many agricultural and biological research scientific researchers.

[0014] 3、The method of the present application greatly reduces the detection cost: at present, the price of the market has the qualification of the detection agency using the national standard method to detect the starch content of pumpkin sample is 300 yuan per sample, and the detection using the method only needs 150 yuan.

[0015] 4、Most of the pumpkin starch contains resistant starch, so in the method, DMSO is added to dissolve the resistant starch, so as to ensure the accuracy of the detection method. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The detection method of the present application determines the correlation analysis diagram of the national standard data. DETAILED DESCRIPTION

[0017] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0018] Experimental instruments: one ten thousandth balance, a blast drying oven / oven, a constant temperature water bath, a vortex shaker, a centrifuge, a UV spectrophotometer, different range pipettes, a mortar, a 40-mesh stainless steel screen and the like.

[0019] Experimental reagents: total starch detection kit (Megazyme, K-TSTA), anhydrous ethanol, DMSO, glacial acetic acid, calcium chloride dihydrate, MOPS sodium salt, NaOH and the like.

[0020] Experimental materials: seven different starch content materials in a certain unit pumpkin germplasm resource library are selected as test materials (ST-01-07), are planted in a plastic greenhouse, and cultivation management refers to local habits, and fresh pumpkin fruits pollinated for 15-20 days are used for subsequent detection in the experiment.

[0021] The present application provides a method for detecting the starch content in pumpkin flesh, comprising the following steps: A method for detecting the starch content in pumpkin flesh, characterized in that it comprises the following steps: S1, pumpkin sample pretreatment: pick fresh pumpkin, remove the skin after cutting into 1-2 mm thin slices, put into the oven at 60-65℃ drying to constant weight, usually 36h, then put into a mortar and pestle, get pumpkin pulp dried sample powder, 40 mesh sieve, that is the sample to be tested; Only 60-65℃ constant temperature drying, one is to prevent the caramelization of sugar in the pumpkin pulp in the process of high temperature fixation, two is to make the amylase in the pulp itself inactivated at this temperature, reduce the degradation of starch, so that the experimental results are inaccurate. In addition, according to the different water content of different pumpkin samples, the drying time can be adjusted appropriately, that is, the drying time of 36h is not a fixed value, which can be appropriately extended or shortened according to the actual situation, such as 30h, 40h and so on.

[0022] S2, starch detection: This step is to adjust the scheme according to the actual detection suitable for pumpkin sample according to the specific steps in the instruction manual of total starch detection kit (Megazyme, K-TSTA). If necessary, understand and adjust the detection principle, preparation method of specific reagent, data calculation, matters needing attention and other contents, please refer to the instruction manual. This step of the method uses the method of K-TSTA total starch detection kit instruction manual of Megazyme company to configure MOPS Buffer, α-amylase solution, starch glucosidase solution, sodium acetate Buffer, GOPOD reagent and D-glucose standard solution.

[0023] Specific as follows: S2.1, use ethanol to clean the glucose and malt dextrin in the sample to be tested, Take 0.1g of the sample to be tested and add it to the test tube. Add 5mL of 80v / v% ethanol solution to the test tube, mix well, and incubate at 80-85℃ for 5min. Add another 5mL of 80v / v% ethanol solution and mix again. Centrifuge at 3250rcf to discard the supernatant. Add 5mL of 80v / v% ethanol solution to the test tube, vortex to mix, and add another 5mL of 80v / v% ethanol solution. Mix well and centrifuge again under the same conditions. Pour out the supernatant.

[0024] S2.2, DMSO dissolves resistant starch, Immediately add 2mL of DMSO to the test tube and mix well. Place it in a boiling water bath for 5min. Take the test tube out of the boiling water bath and immediately add 3mL of α-amylase solution diluted 30 times with MOPS Buffer. Mix vigorously on the vortex shaker for 20s, then place it in a boiling water bath for 6min. Mix vigorously every 2min for a total of 3 times to ensure complete uniformity of the solution. S2.3, starch determination, Put the test tube into 50℃ water bath for about 5min; add 4mL pH4.5 200mM sodium acetate Buffer, then add 100μL AMG solution, vortex mix, incubate at 50℃ for 30min; Transfer the solution in the test tube into a 25mL volumetric flask, rinse the test tube with pH4.5 200mM sodium acetate Buffer and make up to volume, it is noted that the volume of making up to volume in this step can be adjusted according to the starch content in pumpkin, for example, if the starch content is very low, the volume of making up to volume can be reduced to 10mL, if the starch content is very high, the volume of making up to volume can be increased to 50mL.

[0025] Take 1mL of the mixed solution after making up to volume into a 1.5mL EP tube, centrifuge at 12000rcf for 2min, take 100μL supernatant into the bottom of a 15mL test tube, add 3mL GOPOD reagent into the test tube, incubate at 50℃ for 20min, get sample solution, Take 100μL pH4.5 200mM sodium acetate Buffer into the bottom of a 15mL test tube, add 3mL GOPOD reagent into the test tube, incubate at 50℃ for 20min, get blank control solution, Take 100μL D-glucose standard solution into the bottom of a 15mL test tube, add 3mL GOPOD reagent into the test tube, incubate at 50℃ for 20min, get standard solution, Measure the absorbance values of sample solution, blank control solution and standard solution at 510nm; S3, calculate the starch content, Use the absorbance value measured in S2.3 step to calculate the starch content of pumpkin sample by the following formula: Wherein: : the difference between the absorbance values of sample solution and blank control at 510nm, i.e. , : conversion factor (see total starch detection kit instruction for details), : the difference between the absorbance values of standard solution and blank control at 510nm, i.e. , EV: dilution concentration, i.e. the volume of making up to volume in S2.3, which is 25mL in this step, but can be determined according to the dilution volume in actual determination, 0.1: loading volume 0.1mL, : unit conversion factor, i.e. convert μg to mg, : convert the loading amount to 100 mg, W is the actual mass of the pumpkin sample weighed in S2.1, in mg, DM%: dry matter content, i.e. the weight of the sample after drying / the weight of the sample before drying * 100%.

[0026] S4, comparison and calibration of experimental results with national standard data, Each sample was made in three biological replicates, then the results of the acid hydrolysis method in the national standard GB-5009.9-2023 method were compared, and the detection results were verified and calibrated by using the correlation curve.

[0027] Table 1 Total starch detection results of 7 pumpkin samples As Figure 1 shown in the correlation analysis diagram of the detection results of the detection method of the application and the national standard data, the x-axis is the detection value of the method, and the y-axis is the determination value of the national standard.

[0028] The correlation curve is y = 1.378x + 0.5454, R 2 = 0.9968, x is the starch content value of the pumpkin sample in S3, and y is the determination value of the national standard. It is proved that the fitting of the results of the method of the application and the national standard method is very good, which represents high accuracy, and in daily breeding and scientific research work, the data results obtained by the detection method can be directly used for material screening, and the national standard detection values converted by the correlation formula can be used for reference and comparison with the national standard detection data in other literatures, official data of varieties, etc. Therefore, the detection method of the patent can be used as a kind of fast and accurate method for determining the total starch content in pumpkin flesh in actual scientific research and breeding work.

[0029] In addition, resistant starch is a relatively stable form found in plants by scientists in recent years. Because it decomposes slowly, it also causes a slow release of glucose in the human digestive tract, so it is friendly to diabetic patients. Although there are few reports on pumpkin resistant starch at home and abroad at present. However, in our previous pre-experiment of detecting starch content, it was found that resistant starch existed in most pumpkin starch. Therefore, in this method, DMSO is added to dissolve resistant starch, which ensures the accuracy of the detection method.

[0030] Applicant optimized the preparation parameters of pumpkin fruit samples (including slice thickness, drying temperature and time) in the previous pre-experiment, and screened out the product with the best repeatability from various commercially available starch detection kits as the detection tool. On this basis, combined with the characteristics of pumpkin fruit itself, a set of experimental procedures suitable for the detection of starch content in pumpkin fruit pulp was established. In order to further verify the reliability of the method, the applicant compared it with the national standard method (GB 5009.9-2023), and based on the comparison results, a correlation curve was constructed which can be used for data calibration of the method.

[0031] In summary, the present application first develops a convenient, accurate and low-cost detection method for the starch content of pumpkin fruit pulp based on commercially available finished kits. This method is suitable for rapid judgment of the starch content of different pumpkin materials in actual scientific research and breeding work, providing an effective tool for large-scale germplasm resource screening.

[0032] The specific embodiments described herein are merely illustrative of the spirit of the present application. Those skilled in the art of the present application can make various modifications or supplements to the described specific embodiments or replace them with similar ways, without departing from the spirit of the present application.

Claims

1. A method for detecting starch content in pumpkin pulp, characterized by, The method comprises the following steps: S1, pumpkin sample pretreatment: pick fresh pumpkin, remove the skin and cut into thin slices, put into an oven to dry to constant weight, then put into a mortar to grind, get the dried pumpkin pulp powder, which is the sample to be tested; S2, starch detection: S2.1, use ethanol to clean and remove glucose and malt dextrin in the sample to be tested, S2.2, dissolve resistant starch in DMSO, Immediately add 2mL DMSO in the test tube, mix well, put into a boiling water bath for 5min, take the test tube out of the boiling water bath, immediately add 3mL α-amylase solution diluted 30 times with MOPS Buffer, mix well on a vortex oscillator for 20s, and then put into a boiling water bath for 6min; S2.3, starch determination, Put the test tube into a 50℃ water bath for about 5min; add 4mL sodium acetate Buffer with a concentration of 200mM at pH4.5, then add 100μL of starch glucosidase solution, mix well, and incubate at 50℃ for 30min; Transfer the solution in the test tube into a 25mL volumetric flask, rinse the test tube with sodium acetate Buffer with a concentration of 200mM at pH4.5 and make up to volume, Take 1mL of the mixed solution after making up to volume into a 1.5mL EP tube, centrifuge at 12000rcf for 2min, take 100μL of supernatant and transfer into the bottom of a 15mL test tube, add 3mL GOPOD reagent in the test tube, incubate at 50℃ for 20min, and get the sample solution, Take 100μL of sodium acetate Buffer with a concentration of 200mM at pH4.5 and transfer into the bottom of a 15mL test tube, add 3mL GOPOD reagent in the test tube, incubate at 50℃ for 20min, and get the blank control solution, Take 100μL of D-glucose standard solution and transfer into the bottom of a 15mL test tube, add 3mL GOPOD reagent in the test tube, incubate at 50℃ for 20min, and get the standard solution, Measure the absorbance values of the sample solution, blank control solution and standard solution at 510nm; S3, starch content calculation, Use the absorbance value measured in step S2.3 to calculate the starch content of the pumpkin sample by substituting into the following formula: Wherein: : the difference between the absorbance values of the sample solution and the blank at 510 nm, i.e. , : difference between absorbance values at 510 nm of the standard solution and the blank control, i.e. , EV: dilution concentration, i.e. the constant volume in step S2.3, which is 25mL in this step, : Convert the loading amount to 100 mg, W is the actual mass of the pumpkin sample weighed in S2.1, in mg, DM%: dry matter content, i.e. the sample weight after drying / the sample weight before drying*100%.

2. The method for detecting starch content in pumpkin pulp according to claim 1, characterized by, In S1, the thickness of the slices is 1-2mm, the drying temperature is 60-65℃, and the dried sample powder is sieved through a 40 mesh sieve.

3. The method for detecting starch content in pumpkin pulp according to claim 1, characterized by, In S2.1, the ethanol cleaning to remove glucose and malt dextrin in the sample to be tested is as follows: Take 0.1g of the sample to be tested and add into a test tube, add 5mL of 80v / v% ethanol solution into the test tube, mix well, incubate at 80-85℃ for 5min, add 5mL of 80v / v% ethanol solution again, mix well again; Centrifuge to discard the supernatant, add 5mL of 80v / v% ethanol solution into the test tube, mix well, add 5mL of 80v / v% ethanol solution again, mix well, centrifuge again, and pour out the supernatant.

4. The method for detecting starch content in pumpkin pulp according to claim 1, characterized by, Also includes S4, experimental results and national standard data comparison and calibration, each sample do 3 biological repeat, then use the acid hydrolysis method in the national standard GB-5009.9-2023 method result comparison, and use the correlation curve to verify and calibrate the detection result.

5. The method for detecting starch content in pumpkin pulp according to claim 4, characterized by, The correlation curve is y = 1.378x + 0.5454, R 2 = 0.9968, x is the starch content value of the pumpkin sample in S3, and y is the national standard determination value.

6. The method for detecting starch content in pumpkin pulp according to claim 1, characterized in that, In S2, the method in the total starch detection kit instruction manual of Megazyme company, model K-TSTA is used to configure MOPS Buffer, alpha-amylase solution, starch glucosidase solution, sodium acetate Buffer, GOPOD reagent and D-glucose standard solution.

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