Raspberry wine-roasted processing method based on deterministic screening experiment design and raspberry wine-roasted processing product detection method based on deterministic screening experiment design

Abstract

The invention discloses a raspberry wine roasting processing method based on deterministic screening experiment design and a product detection method thereof. The raspberry wine roasting processing method comprises the following steps: adding cleaned and selected raspberries into yellow rice wine, uniformly stirring, moistening for 1-3 hours, putting into a frying container, processing at 100-150 DEG C for 10-30 minutes, cooling, and screening out dust, wherein the use amount of the yellow rice wine is 10-40% of the mass of the raspberries. According to the present invention, the optimal wine roasting processing process condition is obtained through the analysis by combining with the Design-Expert 12 software, the ellagic acid content and the linden glycoside content after the wine roasting are improved, and the content change of the kaempferol-3-O-rutinoside and the astragalus smicus glycoside before and after the wine roasting is not significant. Scientific basis is provided for quality standard formulation and clinical application of the wine raspberry.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal material processing technology and component detection technology thereof, and in particular to a raspberry wine processing method based on a deterministic screening experimental design and a product detection method thereof. Background Art

[0002] Raspberry, the dried fruit of Rubus chingii Hu, a plant of the Rosaceae family, is a edible and medicinal herb. Rich in nutrients, it can be used in beverages and yogurts containing raspberries. Furthermore, raspberries possess significant medicinal value, being included in the Chinese Pharmacopoeia. They are commonly used in Traditional Chinese Medicine as astringents, benefiting from kidney-tonifying, sperm-strengthening, urinary retention, liver-nourishing, and vision-enhancing properties. They are commonly used to treat conditions such as nocturnal emission, enuresis, frequent urination, impotence, premature ejaculation, and blurred vision. Modern pharmacological research has demonstrated that raspberries possess antioxidant, kidney-tonifying, liver-protecting, and lipid-lowering properties. Studies have also shown that phenolic acids and flavonoids are the active ingredients in raspberries. Ellagic acid and kaempferol-3-O-rutinoside are listed as indicator components in the pharmacopoeia's quality standards for raspberries. Astragalin has a wide range of pharmacological activities, including anti-inflammatory, antioxidant, neuroprotective, cardioprotective, and hepatoprotective activities. Literature also reports that astragalin protects against spermatogenesis impairment in diabetic mice. Therefore, astragalin is a pharmacologically active component of raspberry and is recommended for inclusion in quality evaluation indicators. Currently, only liquid chromatography-mass spectrometry is used to determine astragalin content in raspberry, which is not conducive to improving raspberry quality standards. Therefore, it is necessary to establish an HPLC method for the determination of these four components.

[0003] Processing has a great influence on the efficacy of Chinese herbal medicines. Wine-fried raspberry is one of the traditional processing methods, but the processing method of raspberry is not specified in the pharmacopoeia at present. The processing method of raspberry has been specified in the processing specifications of Chinese herbal medicine slices in many provinces, but there is no unified wine-fried raspberry process standard, which affects the stability of the quality of wine-fried raspberry. There is no report on the in-depth study of the wine-fried raspberry process. Therefore, the present invention uses the overall desirability (OD) of the content of four active ingredients in raspberry, ellagic acid, kaempferol-3-O-rutinoside, astragaloside and linaloside as the evaluation index, and adopts the deterministic screening design (DSD) to optimize the process parameters of raspberry wine-fried, including the amount of rice wine, the time of steaming, the processing time and the processing temperature, on the basis of the single factor experiment. After processing, the contents of two active ingredients, ellagic acid and basidiin, in wine-raspberry fruit increased. The contents of kaempferol-3-O-rutinoside and astragaloside did not change significantly before and after wine-roasting. Furthermore, the method for determining the contents of the four active ingredients in raspberry fruit established in the present invention is simple, reproducible, and highly applicable. The preferred wine-roasting process for raspberry fruit is stable and reliable, providing a scientific basis for the development of quality standards and clinical application of wine-roasted raspberry fruit. Summary of the Invention

[0004] In order to address the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing raspberry wine based on a deterministic screening design (DSD). The present invention also provides a method for simultaneously detecting the contents of three flavonoid components in raspberries, namely kaempferol-3-O-rutinoside, astragaloside and basidioside.

[0005] The technical solution of the present invention for solving the above technical problems is as follows: a method for preparing raspberry wine based on a deterministic screening experimental design, comprising the following steps: adding cleaned raspberries to rice wine and mixing evenly, wherein the amount of rice wine used is 10% to 40% of the mass of the raspberries, stewing for 1 hour to 3 hours, placing the raspberries in a stir-frying container, and preparing the raspberries at a temperature of 100° C. to 150° C. for 10 minutes to 30 minutes. The raspberries are cooled, and sifted to remove ash to obtain the wine.

[0006] Furthermore, the mass of raspberries is 50 g, and the amount of rice wine used is 10% to 30% of the mass of the raspberries.

[0007] Furthermore, the single factor investigation results in the process of raspberry wine roasting were combined with the deterministic screening experimental design to conduct experimental analysis, and the optimal parameter of rice wine dosage was obtained to be 19.4%.

[0008] Furthermore, the moistening time is 1 hour to 2 hours.

[0009] Furthermore, the single-factor investigation results of the raspberry wine roasting process were combined with the deterministic screening experimental design to conduct an experimental analysis, and the optimal parameter of the steaming time was obtained to be 1.8h.

[0010] Furthermore, the processing temperature is 125°C to 150°C.

[0011] Furthermore, the single-factor investigation results of the raspberry wine roasting process were combined with the deterministic screening experimental design for experimental analysis, and the optimal processing temperature parameter was obtained as 149℃.

[0012] Furthermore, the preparation time is 10 minutes to 20 minutes.

[0013] Furthermore, the single-factor investigation results of the raspberry wine roasting process were combined with the deterministic screening experimental design to conduct an experimental analysis, and the optimal parameter of the processing temperature was obtained as 10.5min.

[0014] On the other hand, the present invention also provides a method for detecting the content of a product prepared by the raspberry wine roasting method, the method comprising: determining the content of free ellagic acid; determining the content of kaempferol-3-O-rutinoside, astragaloside and basidiin;

[0015] (1) The method for determining the free ellagic acid content comprises the following steps:

[0016] 1) Preparation of reference substance stock solution

[0017] Weigh the ellagic acid reference substance and prepare it with an aqueous solution containing 70% methanol to a concentration of 20.0 μg mL -1 250mL of reference substance stock solution;

[0018] 2) Preparation of linear solution

[0019] Ellagic acid reference stock solution was measured and prepared with an aqueous solution containing 70% methanol and 0.1% formic acid to a concentration of 0.450 μg mL -1 , 0.900 μg·mL -1 , 1.80 μg·mL -1 , 2.70 μg·mL -1 , 4.50 μg·mL -1 , 9.00 μg·mL -1 , 13.5 μg·mL -1 and 18.0 μg·mL -1 5 mL of each reference solution;

[0020] 3) Preparation of test solution

[0021] Weigh approximately 100 mg of raspberry powder passed through a No. 4 sieve, accurately weigh it, and place it in a round-bottom flask. Add 50 mL of an extraction solvent containing 70% methanol by volume in water, weigh it, heat it under reflux for 1 hour, let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, take an appropriate amount of the solution, centrifuge it at 12,000 rpm for 10 minutes, accurately measure 3 mL of the supernatant, and add 3 μL of formic acid to obtain a test solution containing 0.1% formic acid by volume;

[0022] 4) HPLC-UV conditions

[0023] The chromatographic column was a Waters CORTECS C18 (4.6×150 mm, 2.7 μm); the mobile phase A was a 0.1% by volume formic acid-water solution, and the mobile phase B was a 0.1% by volume formic acid-acetonitrile solution; the flow rate was 0.5 mL / min; the mobile phase elution gradient was: 5% B at 0 min; 10% B at 10.0 min; 15% B at 25.0 min; 17% B at 30.0 min; 100% B at 31.0 min; and 100% B at 40.0 min. The column temperature was 35°C, and the injection volume was 3 μL. The detection wavelength of the UV detector was 254 nm.

[0024] (2) The method for determining the content of kaempferol-3-O-rutinoside, astragaloside and basidioside comprises the following steps:

[0025] 1) Preparation of mixed reference substance stock solution

[0026] Weigh the reference substances of kaempferol-3-O-rutinoside, astragaloside and basidioside respectively, and prepare them into the concentration of 406 μg·mL with 70% methanol in water solution. -1 , 402 μg·mL -1 and 400 μg·mL -1 25 mL of each reference substance stock solution was taken; 25 mL of each of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference substance stock solutions were measured and prepared with an aqueous solution containing 70% methanol by volume to a concentration of 142 μg mL -1 , 100 μg·mL -1 and 152 μg·mL -1 10 mL of mixed reference stock solution;

[0027] 2) Preparation of linear solution

[0028] Take an appropriate amount of the mixed reference stock solution and dilute it with an aqueous solution containing 70% methanol to a concentration of 1.42 μg mL-1 of kaempferol-3-O-rutinoside. -1 , 7.11 μg·mL -1 , 14.2 μg·mL-1 , 21.3 μg·mL -1 , 42.6 μg·mL -1 , 85.3 μg·mL -1 and 142 μg·mL -1 , astragalin concentrations were 1.00 μg·mL -1 , 5.02 μg·mL -1 , 10.0 μg·mL -1 , 15.1 μg·mL -1 , 30.1 μg·mL -1 , 60.2 μg·mL -1 and 100 μg·mL -1 and basilicin concentrations were 1.52 μg·mL -1 , 7.61 μg·mL -1 , 15.2 μg·mL -1 , 22.8 μg·mL -1 , 45.6 μg·mL -1 , 91.3 μg·mL -1 and 152 μg·mL -1 5 mL of each mixed reference solution;

[0029] 3) Preparation of test solution

[0030] Weigh 200 mg of raspberry powder passed through a No. 4 sieve, accurately weigh it, and place it in a stoppered conical flask. Add 25 mL of an extraction solvent containing 70% methanol by volume in water, weigh it, sonicate for 2 hours, let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, centrifuge it at 5000 rpm for 30 minutes, measure 20 mL of the supernatant, concentrate it under reduced pressure, and reconstitute it with the extraction solvent in a 2 mL volumetric flask.

[0031] 4) HPLC-UV conditions

[0032] The chromatographic column was Waters CORTECS C18 (4.6×150 mm, 2.7 μm); the mobile phase A was a 0.1% by volume formic acid-water solution, and the mobile phase B was a 0.1% by volume formic acid-acetonitrile solution; the flow rate was 0.5 mL / min; the mobile phase elution gradient was: 10% B at 0 min; 15% B at 13.0 min; 17% B at 25.0 min; 25% B at 28.0 min; 35% B at 35.0 min; 35% B at 37.0 min; 37% B at 38.0 min; 100% B at 45.0 min; the column temperature was 35°C, the injection volume was 3 μL, and the detection wavelength of the UV detector was 266 nm.

[0033] The present invention has the following beneficial effects:

[0034] 1. Based on a single-factor investigation, the present invention studies the processing method of raspberry wine-braised by deterministic screening experimental design (DSD). The optimal processing parameters are 19.4% rice wine dosage (50g raspberry), 1.8h steaming time, 10.5min processing time, and 149°C processing temperature. After wine-braised, the contents of ellagic acid and basidiin are increased, and the changes in the contents of kaempferol-3-O-rutinoside and astragalin before and after wine-braised are not significant. This provides some scientific basis for the formulation of quality standards and clinical application of wine-braised raspberry.

[0035] 2. The present invention also discloses a method for simultaneously detecting the content of three flavonoid components in raspberry, namely kaempferol-3-O-rutinoside, astragaloside and basidioside. The method is simple to operate and highly practical, and can provide a reference for improving the quality standards of raspberry. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The following are HPLC-UV analysis chromatograms of raspberry; A is the HPLC chromatogram of ellagic acid, and B is the HPLC chromatogram of three flavonoid components: 1. ellagic acid; 2. kaempferol-3-O-rutinoside; 3. astragaloside; 4. basidioside.

[0037] Figure 2 This is a graph showing the contents of four components in the single-factor investigation of raspberry wine.

[0038] Figure 3 A is the interactive response surface and contour map of each factor; A is the interactive response surface map of each factor, and B is the contour map. DETAILED DESCRIPTION

[0039] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0040] Example 1: Determination of the Contents of Ellagic Acid, Kaempferol-3-O-rutinoside, Astragaloside and Basiloside in Raspberries

[0041] 1. Specificity test

[0042] (1) Preparation of reference solution

[0043] Determination of ellagic acid content:

[0044] Accurately weigh an appropriate amount of ellagic acid reference substance and prepare it with an aqueous solution containing 70% methanol by volume to a concentration of 20.0 μg mL -1 250mL of reference substance stock solution.

[0045] Accurately measure an appropriate amount of ellagic acid reference stock solution and prepare it with diluent (an aqueous solution containing 70% methanol and 0.1% formic acid by volume) to a concentration of 0.450 μg mL -1 , 0.900 μg·mL -1 , 1.80 μg·mL -1 , 2.70 μg·mL -1 , 4.50 μg·mL -1 , 9.00 μg·mL -1 , 13.5 μg·mL -1 and 18.0 μg·mL -1 5mL of each reference solution.

[0046] Determination of 3 flavonoid contents:

[0047] Accurately weigh appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference substances, and prepare them with a diluent (aqueous solution containing 70% methanol by volume) to a concentration of 406 μg mL -1 , 402 μg·mL -1 and 400 μg·mL -1 Accurately measure appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference solution and use a diluent (aqueous solution containing 70% methanol by volume) to prepare kaempferol-3-O-rutinoside, astragaloside, and basidioside concentrations of 142 μg·mL, respectively. -1 , 100 μg·mL -1 and 152 μg·mL -1 Prepare 10 mL of mixed reference stock solution.

[0048] Accurately measure an appropriate amount of the mixed reference stock solution and dilute it with a diluent (an aqueous solution containing 70% methanol by volume) to a concentration of 1.42 μg mL-1 of kaempferol-3-O-rutinoside. -1 , 7.11 μg·mL -1 , 14.2 μg·mL -1 , 21.3 μg·mL -1 , 42.6 μg·mL -1 , 85.3 μg·mL -1 and 142 μg·mL -1 , astragalin concentrations were 1.00 μg·mL -1 , 5.02 μg·mL -1 , 10.0 μg·mL -1 , 15.1 μg·mL -1 , 30.1 μg·mL -1, 60.2 μg·mL -1 and 100 μg·mL -1 and basilicin concentrations were 1.52 μg·mL -1 , 7.61 μg·mL -1 , 15.2 μg·mL -1 , 22.8 μg·mL -1 , 45.6 μg·mL -1 , 91.3 μg·mL -1 and 152 μg·mL -1 5mL of each mixed reference solution.

[0049] (2) Preparation of test solution:

[0050] Preparation of test solution 1 (ellagic acid content determination)

[0051] Weigh about 100 mg of raspberry sample powder passed through a No. 4 sieve, accurately weigh it, place it in a round-bottom flask, accurately add 50 mL of extraction solvent (an aqueous solution containing 70% methanol by volume), weigh the weight, heat and reflux for 1 hour, cool, weigh again, make up the lost weight with the extraction solvent, shake well, take an appropriate amount of solution and centrifuge at 12000 rpm for 10 minutes, accurately measure 3 mL of supernatant, add 3 μL of formic acid to obtain a test solution containing 0.1% formic acid by volume.

[0052] Preparation of test solution 2 (determination of the content of three flavonoid components)

[0053] Take about 200 mg of raspberry sample powder passed through No. 4 sieve, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of extraction solvent (an aqueous solution containing 70% methanol by volume), weigh it, ultrasonicate for 2 hours, cool it, weigh it again, make up the lost weight with the extraction solvent, shake it well, centrifuge it at 5000 rpm for 30 minutes, accurately measure 20 mL of the supernatant, concentrate it under reduced pressure, and redissolve it in a 2 mL volumetric flask with the extraction solvent.

[0054] (3) HPLC-UV conditions:

[0055] Determination of ellagic acid content:

[0056] The HPLC column used was a Waters CORTECS C18 column (4.6×150 mm, 2.7 μm), and the sample solution was separated using a mobile phase gradient elution method. The mobile phase gradient elution method was as follows: at 0 min, mobile phase A was 95%, mobile phase B was 5%; at 10.0 min, mobile phase A was 90%, mobile phase B was 10%; at 25.0 min, mobile phase A was 85%, mobile phase B was 15%; at 30.0 min, mobile phase A was 83%, mobile phase B was 17%; at 31.0 min, mobile phase A was 0%, mobile phase B was 100%; at 40.0 min, mobile phase A was 0%, mobile phase B was 100%; mobile phase A and mobile phase B were expressed in volume percentages; mobile phase A was a formic acid-water solution with a formic acid concentration of 0.1%; mobile phase B was a formic acid-acetonitrile solution with a formic acid concentration of 0.1%. The mobile phase flow rate was 0.5 mL / min; the column temperature was 35°C; the injection volume was 3 μL; and the UV detection wavelength was 254 nm.

[0057] Determination of 3 flavonoid contents:

[0058] The HPLC column used was a Waters CORTECS C18 column (4.6×150 mm, 2.7 μm), and the sample solution was separated using a mobile phase gradient elution method. The specific method of mobile phase gradient elution is: at 0 min, mobile phase A is 90%, and mobile phase B is 10%; at 13.0 min, mobile phase A is 85%, and mobile phase B is 15%; at 25.0 min, mobile phase A is 83%, and mobile phase B is 17%; at 28.0 min, mobile phase A is 75%, and mobile phase B is 25%; at 35.0 min, mobile phase A is 65%, and mobile phase B is 35%; at 37.0 min, mobile phase A is 63%, and mobile phase B is 37%; at 38.0 min, mobile phase A is 0%, and mobile phase B is 100%; at 45.0 min, mobile phase A is 0%, and mobile phase B is 100%; mobile phase A and mobile phase B are both expressed in volume percentage; mobile phase A is formic acid-water solution with a formic acid volume concentration of 0.1%; mobile phase B is formic acid-acetonitrile solution with a formic acid volume concentration of 0.1%. The mobile phase flow rate was 0.5 mL / min; the column temperature was 35°C; the injection volume was 3 μL; and the UV detection wavelength was 266 nm.

[0059] (4) Take blank solvent, reference solution and test solution and analyze them according to the HPLC-UV conditions in step (3) above to obtain chromatogram. The results are shown in the figure. Figure 1 .

[0060] Depend on Figure 1It can be seen that the blank solvent does not interfere with the detection of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside. The coexisting chromatographic peaks in the chromatogram of the test solution can achieve baseline separation with ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside, indicating that the specificity of this method is good.

[0061] 2. Precision test

[0062] (1) Preparation of reference solution 1:

[0063] Determination of ellagic acid content: Accurately measure an appropriate amount of ellagic acid reference stock solution and prepare it with a diluent (an aqueous solution containing 70% methanol and 0.1% formic acid by volume) to a concentration of 18.0 μg mL -1 5mL of reference solution.

[0064] Determination of the content of three flavonoids: Accurately measure an appropriate amount of the mixed reference stock solution and dilute it with a diluent (an aqueous solution containing 70% methanol by volume) to a concentration of 85.3 μg mL -1 Kaempferol-3-O-rutinoside, 60.2 μg·mL -1 Astragaloside and 91.3 μg·mL -1 5mL of mixed reference solution of basilixin.

[0065] (2) Preparation of reference solution 2:

[0066] Determination of ellagic acid content: Accurately measure an appropriate amount of ellagic acid reference stock solution and prepare it with a diluent (an aqueous solution containing 70% methanol and 0.1% formic acid by volume) to a concentration of 2.70 μg mL -1 5mL of reference solution.

[0067] Determination of the content of three flavonoids: Accurately measure an appropriate amount of the mixed reference stock solution and dilute it with a diluent (an aqueous solution containing 70% methanol by volume) to a concentration of 14.2 μg mL -1 Kaempferol-3-O-rutinoside, 10.0 μg·mL -1 Astragaloside and 15.2 μg·mL -1 5mL of mixed reference solution of basilixin.

[0068] (3) HPLC-UV conditions:

[0069] Same as 1. Specificity test step (3) HPLC-UV conditions.

[0070] Mixed reference solution 1 and mixed reference solution 2 were injected and analyzed 6 times in succession according to the HPLC-UV conditions in step (3). The relative standard deviations (RSDs) of the peak areas of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside are shown in Table 1.

[0071] Table 1 RSD of the peak areas of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside in the mixed reference solution

[0072] Compound name Mixed reference solution 1 (RSD) Mixed reference solution 2 (RSD) Ellagic acid 0.4％ 2.5％ Kaempferol-3-O-rutinoside 0.9％ 0.9％ Astragaloside 1.0％ 0.9％ Basilin 0.9％ 1.0％

[0073] From the data in Table 1, it can be seen that the RSDs of the peak areas of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basilixin were all less than 3%, indicating that this detection method has good injection precision.

[0074] 3. Linearity, limit of quantification, and limit of detection tests

[0075] (1) Preparation of mixed reference substance stock solution:

[0076] Determination of ellagic acid content: Accurately weigh an appropriate amount of ellagic acid reference substance and prepare it with an aqueous solution containing 70% methanol by volume to a concentration of 20.0 μg mL -1 250mL of reference substance stock solution.

[0077] Determination of the content of three flavonoids: accurately weigh appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference substances, and prepare them with diluent (aqueous solution containing 70% methanol by volume) to a concentration of 406 μg mL -1 , 402 μg·mL -1 and 400 μg·mL -1 Accurately measure appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference solution and prepare them with diluent (aqueous solution containing 70% methanol by volume) to a concentration of 142 μg mL -1 , 100 μg·mL -1 and 152 μg·mL -1 Prepare 10 mL of mixed reference stock solution.

[0078] (2) Preparation of linear solution:

[0079] Determination of ellagic acid content: Accurately measure an appropriate amount of ellagic acid reference stock solution and prepare it with diluent (an aqueous solution containing 70% methanol and 0.1% formic acid by volume) to a concentration of 0.450 μg mL -1 , 0.900 μg·mL -1 , 1.80 μg·mL -1 , 2.70 μg·mL -1 , 4.50 μg·mL -1 , 9.00 μg·mL -1 , 13.5 μg·mL -1 and 18.0 μg·mL -15mL of each reference solution.

[0080] Determination of the content of three flavonoids: accurately weigh appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference substances, and prepare them with diluent (aqueous solution containing 70% methanol by volume) to a concentration of 406 μg mL -1 , 402 μg·mL -1 and 400 μg·mL -1 Accurately measure appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference solution and prepare them with diluent (aqueous solution containing 70% methanol by volume) to a concentration of 142 μg mL -1 , 100 μg·mL -1 and 152 μg·mL -1 Prepare 10 mL of mixed reference stock solution.

[0081] (3) HPLC-UV conditions:

[0082] Same as 1. Specificity test step (3) HPLC-UV conditions.

[0083] (4) The linear solution prepared in step (2) was injected and analyzed according to the above chromatographic conditions, and the peak areas (Y) of the chromatographic peaks of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basiliside were calculated to compare their concentrations (X, μg·mL) -1 ) were subjected to linear regression analysis. The relevant information of linear regression and the limit of quantification (S / N=10) and limit of detection (S / N=3) are shown in Table 2.

[0084] Table 2 Linear relationships, limits of quantification, and limits of detection of ellagic acid, kaempferol-3-O-rutinoside, astragaloside, and basidioside

[0085]

[0086] 4. Recovery test

[0087] (1) Preparation of mixed reference solution:

[0088] Determination of ellagic acid content: Accurately measure an appropriate amount of ellagic acid reference stock solution and use a diluent (an aqueous solution containing 70% methanol and 0.1% formic acid) to prepare the reference stock solution to contain 18.0 μg mL -1 Ellagic acid reference solution 1, containing 7.20 μg mL -1 Ellagic acid reference solution 2 and 2.88 μg mL -1 Ellagic acid reference solution 3.

[0089] Determination of the content of three flavonoids: Accurately measure appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference solution, and use diluent (aqueous solution containing 70% methanol by volume) to prepare the reference solution to contain 69.0 μg mL -1 Kaempferol-3-O-rutinoside, 52.2 μg·mL -1 Astragaloside and 76.1 μg·mL -1 Bassin mixed reference solution 1, containing 34.5 μg mL -1 Kaempferol-3-O-rutinoside, 26.1 μg·mL -1 Astragaloside and 38.0 μg·mL -1 Mixed reference solution 2 of basilicin, and 13.8 μg·mL -1 Kaempferol-3-O-rutinoside, 10.4 μg·mL -1 Astragaloside and 15.2 μg·mL -1 Mixed reference solution of basilixin 3.

[0090] (2) Preparation of test solution:

[0091] Same as 1. Specificity test step (2) Preparation of test solution.

[0092] (3) Preparation of test sample spiked solution:

[0093] Determination of ellagic acid content: Weigh about 50 mg of raspberry powder passed through a No. 4 sieve, accurately weigh it, and place it in a round-bottom flask. Add 9.2 mL of ellagic acid reference solution containing 184 μg. Prepare the test sample spiked solution according to the test sample solution method in step (2). Prepare 6 replicates.

[0094] Determination of the content of three flavonoid components: Weigh about 100 mg of raspberry powder passed through a No. 4 sieve, accurately weigh it, and place it in a stoppered conical flask. Add 1 mL of a mixed reference solution containing 43.0 μg of kaempferol-3-O-rutinoside, 24.7 μg of astragaloside, and 45.6 μg of basidioside. Prepare the test sample spiked solution according to the test sample solution method in step (2). Prepare 6 replicates.

[0095] (4) HPLC-UV conditions:

[0096] Same as 1. Specificity test step (3) HPLC-UV conditions.

[0097] (5) The mixed reference solution prepared in step (1), the test solution prepared in step (2), and the test spiked solution prepared in step (3) were sampled and analyzed according to the above chromatographic conditions, and the recoveries of ellagic acid, kaempferol-3-O-rutinoside, astragaloside, and basidioside were calculated. The results are shown in Table 3.

[0098] Table 3 Recovery results of four components

[0099]

[0100]

[0101] From the data in Table 3, it can be seen that the average recoveries of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basilixin were 103.7%, 97.2%, 96.9% and 97.4%, respectively, and the RSDs of the recoveries were 1.5%, 1.8%, 1.7% and 1.7%, respectively, indicating that the method of the present invention has good accuracy.

[0102] 5. Intra-day precision and inter-day precision tests

[0103] (1) Preparation of mixed reference solution:

[0104] Prepare the mixed reference solution in the same manner as in step (1) of 4. Recovery test.

[0105] (2) Preparation of test solution:

[0106] Same as 1. Specificity test step (2) Preparation of test solution.

[0107] (3) HPLC-UV conditions:

[0108] Same as 1. Specificity test step (3) HPLC-UV conditions.

[0109] (4) Intra-day precision test: Prepare 6 test sample solutions in parallel according to step (2), inject and analyze the mixed reference solution in step (1) and the test sample solution prepared in step (2) according to the above chromatographic conditions, and calculate the relative standard deviation (RSD / %) of the contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidiin.

[0110] (5) Inter-day precision test: Take the mixed reference solution in step (1) and prepare one sample solution every day according to step (2) for 6 consecutive days. Sampling and analysis are carried out according to the above chromatographic conditions, and the relative standard deviation (RSD / %) of the contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidiin is calculated.

[0111] The results of intra-day precision and inter-day precision tests are shown in Table 4:

[0112] Table 4 Relative standard deviations of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside contents

[0113] Compound name Intra-day precision (RSD) Inter-day precision (RSD) Ellagic acid 0.7％ 2.2％ Kaempferol-3-O-rutinoside 1.8％ 1.9％ Astragaloside 1.4％ 2.2％ Basilin 1.4％ 1.5％

[0114] From the data in Table 4, it can be seen that the RSDs of the contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basilicin were all less than 3%, indicating that this detection method has good intra-day precision and inter-day precision.

[0115] 6. Room temperature stability test

[0116] (1) Preparation of mixed reference solution:

[0117] Determination of ellagic acid content: Accurately measure an appropriate amount of ellagic acid reference stock solution and use a diluent (an aqueous solution containing 70% methanol and 0.1% formic acid) to prepare the reference stock solution to contain 18.0 μg mL -1 Ellagic acid high concentration reference solution and 2.88 μg·mL -1 Low concentration reference solution of ellagic acid.

[0118] Determination of the content of three flavonoid components: Accurately measure appropriate amounts of kaempferol-3-O-rutinoside, astragaloside, and basidioside reference solution, and use diluent (aqueous solution containing 70% methanol by volume) to prepare the reference solution to contain 142 μg mL -1 Kaempferol-3-O-rutinoside, 100 μg·mL -1 Astragaloside and 152 μg·mL -1 High concentration mixed reference solution of basilicin, containing 7.11 μg·mL -1 Kaempferol-3-O-rutinoside, 5.02 μg·mL -1 Astragaloside and 7.61 μg·mL -1 Low concentration mixed reference solution of basilixin.

[0119] (2) Preparation of test solution:

[0120] Same as 1. Specificity test step (2) Preparation of test solution.

[0121] (3) HPLC-UV conditions:

[0122] Same as 1. Specificity test step (3) HPLC-UV conditions.

[0123] (4) Take appropriate amounts of the mixed reference solution and the test solution, respectively, and analyze them every 2.4 hours according to the above chromatographic conditions to examine the room temperature stability of the mixed reference solution and the test solution within 24 hours.

[0124] The RSD of the peak area of ellagic acid in the reference solution and the test solution within 12 hours was less than 3%, indicating that the method for determining the content of ellagic acid within 12 hours was stable; the RSD of the peak area of the three flavonoid components within 24 hours was less than 3%, indicating that the method for determining the content of the three flavonoid components within 24 hours was stable.

[0125] 7. Determination of the content of four components in raspberry

[0126] The method for simultaneously detecting the contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside in raspberry comprises the following steps:

[0127] (1) Preparation of mixed reference solution:

[0128] Prepare the mixed reference solution in the same manner as in step (1) of 4. Recovery test.

[0129] (2) Preparation of test solution:

[0130] Same as 1. Specificity test step (2) Preparation of test solution.

[0131] (3) HPLC-UV conditions:

[0132] Same as 1. Specificity test step (3) HPLC-UV conditions.

[0133] (4) The mixed reference solution prepared in step (1) and the test solution prepared in step (2) were taken and analyzed according to the above chromatographic conditions. The contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside in the samples were calculated. The results are shown in Table 5.

[0134] Table 5 Contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside in raspberry (n=3)

[0135]

[0136]

[0137] Example 2 Single factor investigation test on the preparation method of raspberry wine

[0138] (1) Preparation of mixed reference solution:

[0139] Same as in Example 1, Part 4. Recovery Test (1) Preparation of mixed reference solution.

[0140] (2) Preparation of test solution:

[0141] Wine raspberry: Take 50g of cleaned raspberry slices, add appropriate amount of rice wine, mix well, stew, put in a frying container, fry to a certain degree, take out and let cool.

[0142] Determination of ellagic acid content: Weigh about 100 mg of raspberry sample powder passed through a No. 4 sieve, accurately weigh it, place it in a round-bottom flask, accurately add 50 mL of extraction solvent (an aqueous solution containing 70% methanol by volume), weigh the weight, heat and reflux for 1 hour, cool, weigh again, make up the lost weight with the extraction solvent, shake well, take an appropriate amount of solution, centrifuge at 12000 rpm for 10 minutes, accurately measure 3 mL of supernatant, add 3 μL of formic acid, and obtain a test solution containing 0.1% formic acid by volume.

[0143] Determination of the content of three flavonoid components: weigh about 200 mg of raspberry sample powder passed through a No. 4 sieve, accurately weigh, place in a round-bottom flask or a stoppered conical flask, accurately add 25 mL of extraction solvent (an aqueous solution containing 70% methanol by volume), weigh the weight, ultrasonicate for 2 hours, cool, weigh again, make up the lost weight with the extraction solvent, shake well, centrifuge at 5000 rpm for 30 minutes, accurately measure 20 mL of the supernatant, concentrate under reduced pressure, and re-dissolve in a 2 mL volumetric flask with the extraction solvent.

[0144] (3) HPLC-UV conditions:

[0145] Same as Example 1, Part 1. Specificity Test (3) HPLC-UV conditions.

[0146] (4) The mixed reference solution in step (1) and the test solution prepared in step (2) were sampled and analyzed according to the above chromatographic conditions. Figure 2 As shown, the contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside were calculated (Table 6).

[0147] Table 6 Contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside in wine raspberry

[0148]

[0149] Note: Compound content (mg·g -1 ) refers to the mass of the compound contained in each gram of raspberry sample powder.

[0150] According to the content information of each component in the single factor investigation results and the actual situation, the factor levels for optimizing the roasting method of raspberry wine were finally selected as the rice wine dosage of 10%, 15% and 20%, the steaming time of 1h, 1.5h and 2h, the processing time of 10min, 15min and 20min, and the processing temperature of 100℃, 125℃ and 150℃.

[0151] Example 3 Optimization of the preparation method of raspberry wine

[0152] 1. Definitive Screening Design (DSD)

[0153] (1) Experimental design

[0154] Evaluation index: The content of ellagic acid, kaempferol-3-O-rutinoside, astragalin and linaloside in raspberry was normalized by Hassan's method. The larger the value, the better for all the indexes (d max ), the calculation formula is d max =(Y i -Y min ) / (Y max -Y min ), Y min is the minimum value among the indicators, Y max The maximum value among the indicators. Overall desirability (OD) = (d1+d2+d2+…d k The results of a single-factor investigation of the influencing factors of rice wine dosage (A), steaming time (B), processing time (C), and processing temperature (D) during the raspberry wine-roasting process were combined with a deterministic screening experimental design. The factor levels are shown in Table 7, and the experimental design and results are shown in Table 8.

[0155] Table 7 Definitive Screening Design (DSD) factor levels

[0156] factor Low (-1) Chinese (0) High (+1) Yellow wine dosage A / % 10 15 20 Stifling time B / h 1 1.5 2 Preparation time C / min 10 15 20 Processing temperature D / ℃ 100 125 150

[0157] (2) Data processing

[0158] Data were processed using Design-Expert 12 software. The independent variables were rice wine dosage (A), steaming time (B), processing time (C), and processing temperature (D), and the normalized overall score (Y) of ellagic acid, kaempferol-3-O-rutinoside, astragalin, and basidiin content (Y) were used as the dependent variables. A goodness-of-fit analysis optimized the optimal fitting model: 1 / (Y) = 2.09 - 0.5242*A + 0.4953*B - 0.4242*D - 0.4648*AB + 0.3305*BC - 0.5177*BD. The model P value was 0.0020, indicating that the model was significant. The P value for the lack-of-fit term was 0.622, indicating that no lack-of-fit factors were present in this model (Table 9). The F-value indicated that the contribution of each factor to the evaluation index was A > B > D. R-Squared and Adj R-Squared are close to 1, 0.885 and 0.799 respectively. The model is reliable and the interaction is significant.

[0159] Table 8 Definitive screening design (DSD) and results

[0160]

[0161]

[0162] According to the established polynomial model, draw the response surface diagram and contour map of the influence of independent variables on the evaluation index ( Figure 3 ). Through the analysis of Design-Expert 12 software, the optimal parameter points of the model are the amount of rice wine 19.4%, the steaming time 1.8h, the frying time 10.5min, and the frying temperature 149℃.

[0163] Table 9 Regression analysis results of raspberry wine processing technology

[0164] Sources of variance sum of squares degrees of freedom mean square F-number P-value Model 9.490 6 1.580 10.270 0.002 A- yellow wine dosage 2.750 1 2.750 17.850 0.003 B-Stuffy time 2.450 1 2.450 15.940 0.004 D-Processing temperature 1.800 1 1.800 11.690 0.009 AB 1.440 1 1.440 9.360 0.016 BC 0.728 1 0.728 4.730 0.061 BD 1.790 1 1.790 11.610 0.009 Residual 1.230 8 0.154 Lack of Fit 0.890 6 0.148 0.870 0.622 Pure Error 0.341 2 0.171 Corrected Total (Cor Total) 10.720 14

[0165] 2. Confirmatory trials

[0166] Three validation experiments were conducted using the optimized process parameters using a deterministic screening experimental design (DSD). The contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside, and linaloside were determined, and weighted scores were assigned according to the method under "(1) Experimental Design" to obtain an overall OD value. The results are shown in Table 10. The average OD value of the three batches of wine-raspberry was 0.895. Compared with the single-factor experiment, the contents of each component were higher, and the deviations of the contents of the four components in the three batches of wine-roasted raspberries were small (RSD < 12%), indicating that the process is stable and controllable.

[0167] Table 10 Confirmatory test results

[0168]

[0169] Example 4: Comparison of the contents of four components in three batches of raspberry wine before and after roasting

[0170] (1) Preparation of mixed reference solution:

[0171] Same as Example 2

[0172] (3) Preparation of test solution:

[0173] As in Example 2, the same sample was extracted three times in parallel.

[0174] (3) HPLC-UV conditions:

[0175] Same as Example 2

[0176] (4) The mixed reference solution in step (1) and the test solution prepared in step (2) were sampled and analyzed according to the above chromatographic conditions, and the contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside were calculated. The results are shown in Table 11.

[0177] Table 11 Contents of ellagic acid, kaempferol-3-O-rutinoside, astragaloside and basidioside in raspberry (wine) (mg·g-1 )(n=3)

[0178]

[0179] Table 11 shows that the contents of ellagic acid and basidiin in wine raspberries were higher than those in raspberries in all three batches, indicating that the wine-roasting process effectively increases the contents of ellagic acid and basidiin in raspberries. The changes in the contents of kaempferol-3-O-rutinoside and astragalin before and after wine-roasting were not significant (two-tailed T-test, p > 0.6).

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing raspberry wine based on a deterministic screening experimental design, characterized in that: The following steps are involved: Add the cleaned raspberries into rice wine and mix well. The amount of rice wine used should be 10% to 40% of the mass of the raspberries. Let it simmer for 1 hour to 3 hours. Place it in a frying container. The cooking temperature should be 100℃ to 150℃ and the cooking time should be 10 minutes to 30 minutes. Let it cool and sieve out the ash.

2. The method for preparing wine-braised raspberries according to claim 1, wherein: The mass of raspberries is 50g, and the amount of rice wine used is 10% to 30% of the mass of the raspberries.

3. The method for preparing raspberry wine according to claim 2, wherein: The single factor investigation results in the process of raspberry wine roasting were combined with the deterministic screening experimental design method to conduct experimental analysis, and the optimal parameter of rice wine dosage was obtained to be 19.4%.

4. The method for preparing raspberry wine according to claim 1, wherein: The steaming time is 1h to 2h.

5. The method for preparing raspberry wine according to claim 4, characterized in that: The single factor investigation results of the raspberry wine roasting process were combined with the deterministic screening experimental design DSD to conduct experimental analysis, and the optimal parameter of the steaming time was obtained to be 1.8h.

6. The method for preparing raspberry wine according to claim 1, characterized in that: The processing temperature is 125℃~150℃.

7. The method for preparing raspberry wine according to claim 6, characterized in that: The single factor investigation results of the raspberry wine roasting process were experimentally analyzed with the deterministic screening experimental design DSD, and the optimal processing temperature parameter was obtained to be 149℃.

8. The method for preparing raspberry wine according to claim 1, characterized in that: The preparation time is 10 to 20 minutes.

9. The method for preparing raspberry wine according to claim 7, characterized in that: The single factor investigation results of the raspberry wine roasting process were combined with the deterministic screening experimental design DSD to conduct experimental analysis, and the optimal parameter of the processing temperature was obtained as 10.5min.

10. A method for detecting the content of a product prepared by the method for preparing raspberry wine according to any one of claims 1 to 9, characterized in that: The detection method comprises: determining the content of free ellagic acid, determining the content of kaempferol-3-O-rutinoside, astragaloside and basidioside; (1) The method for determining the free ellagic acid content comprises the following steps: 1) Preparation of reference substance stock solution Weigh the ellagic acid reference substance and prepare it with an aqueous solution containing 70% methanol to a concentration of 20.0 μg mL -1 250mL of reference substance stock solution; 2) Preparation of linear solution Ellagic acid reference stock solution was measured and prepared with an aqueous solution containing 70% methanol and 0.1% formic acid to a concentration of 0.450 μg mL -1 , 0.900 μg·mL -1 , 1.80 μg·mL -1 , 2.70 μg·mL -1 , 4.50 μg·mL -1 , 9.00 μg·mL -1 , 13.5 μg·mL -1 and 18.0 μg·mL -1 5 mL of each reference solution; 3) Preparation of test solution Weigh approximately 100 mg of raspberry powder passed through a No. 4 sieve, accurately weigh it, and place it in a round-bottom flask. Add 50 mL of an extraction solvent containing 70% methanol by volume in water, weigh it, heat it under reflux for 1 hour, let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, take an appropriate amount of the solution, centrifuge it at 12,000 rpm for 10 minutes, accurately measure 3 mL of the supernatant, and add 3 μL of formic acid to obtain a test solution containing 0.1% formic acid by volume; 4) HPLC-UV conditions The chromatographic column was a Waters CORTECS C18, 4.6×150 mm, 2.7 μm. The mobile phase A was a 0.1% formic acid-water solution, and the mobile phase B was a 0.1% formic acid-acetonitrile solution. The flow rate was 0.5 mL / min. The mobile phase elution gradient was as follows: 5% B at 0 min; 10% B at 10.0 min; 15% B at 25.0 min; 17% B at 30.0 min; 100% B at 31.0 min; and 100% B at 40.0 min. The column temperature was 35°C, and the injection volume was 3 μL. The detection wavelength of the UV detector was 254 nm. (2) The method for determining the content of kaempferol-3-O-rutinoside, astragaloside and basidioside comprises the following steps: 1) Preparation of mixed reference substance stock solution Weigh the reference substances of kaempferol-3-O-rutinoside, astragaloside and basidioside respectively, and prepare them into the concentration of 406 μg·mL with 70% methanol in water solution. -1 , 402 μg·mL -1 and 400 μg·mL -1 25 mL of each reference substance stock solution; accurately measure the reference substance stock solutions of kaempferol-3-O-rutinoside, astragaloside, and basidioside, and prepare them into concentrations of 142 μg mL with an aqueous solution containing 70% methanol by volume. -1 , 100 μg·mL -1 and 152 μg·mL -1 10 mL of mixed reference stock solution; 2) Preparation of linear solution Accurately measure the mixed reference stock solution and dilute it with an aqueous solution containing 70% methanol to a concentration of 1.42 μg mL-1 of kaempferol-3-O-rutinoside. -1 , 7.11 μg·mL -1 , 14.2 μg·mL -1 , 21.3 μg·mL -1 , 42.6 μg·mL -1 , 85.3 μg·mL -1 and 142 μg·mL -1 , astragalin concentrations were 1.00 μg·mL -1 , 5.02 μg·mL -1 , 10.0 μg·mL -1 , 15.1 μg·mL -1 , 30.1 μg·mL -1 , 60.2 μg·mL -1 and 100 μg·mL -1 and basilicin concentrations were 1.52 μg·mL -1 , 7.61 μg·mL -1 , 15.2 μg·mL -1 , 22.8 μg·mL -1 , 45.6 μg·mL -1 , 91.3 μg·mL -1 and 152 μg·mL -1 5 mL of each mixed reference solution; 3) Preparation of test solution Weigh 200 mg of raspberry sample powder passed through a No. 4 sieve, accurately weigh it, and place it in a stoppered conical flask. Add 25 mL of extraction solvent containing a 70% methanol solution by volume, weigh it, sonicate for 2 h, let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, centrifuge it at 5000 rpm for 30 min, measure 20 mL of the supernatant, concentrate it under reduced pressure, and reconstitute it with the extraction solvent in a 2 mL volumetric flask. 4) HPLC-UV conditions The chromatographic column was Waters CORTECS C18, 4.6×150 mm, 2.7 μm; the mobile phase A was an aqueous solution containing 0.1% formic acid by volume, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid by volume; the flow rate was 0.5 mL / min; the mobile phase elution gradient was: 10% B at 0 min; 15% B at 13.0 min; 17% B at 25.0 min; 25% B at 28.0 min; 35% B at 35.0 min; 35% B at 37.0 min; 37% B at 38.0 min; 100% B at 45.0 min; the column temperature was 35°C, the injection volume was 3 μL, and the detection wavelength of the UV detector was 266 nm.

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