WO2020082754A1 - Method employing composite chitosan flocculation to prepare stevia polyphenol - Google Patents

Abstract

Disclosed are a method employing composite chitosan flocculation to prepare stevia polyphenol and stevia polyphenol prepared by the method. The method comprises: flocculating a stevia leaf water extract with a composite chitosan at room temperature to remove impurities, and adsorbing the supernatant with a macroporous polar resin, wherein the effluent is to be further used for the extraction of stevioside; eluting the resin with aqueous alcohol solutions of different concentrations, and after elution with a low-concentration aqueous alcohol solution, combining the resultant separated solution with the effluent produced before separation to obtain a combined solution, the total phenol content in the combined solution being less than 0.5% (on a dry basis), and the combined solution being reserved for extraction of stevioside; and drying the eluent produced from elution with a high-concentration aqueous alcohol solution, to give stevia polyphenol mainly composed of isochlorogenic acid.

Description

Method for preparing stevia polyphenol by composite chitosan flocculation method Technical field

The invention relates to a method for preparing stevia polyphenol by a composite chitosan flocculation method, which belongs to the technical field of plant extraction.

Background technique

Stevia is a perennial herb that originated in South America and is widely grown in Central America, South Korea, Paraguay and other places. The content of inulin is 6% -12%. The stevioside extracted with it is a low-calorie, high-sweetness natural sweetener. It is widely used in the food and pharmaceutical industries. Its sweetness is 150-300 times that of sucrose . In addition to the well-known stevioside, stevia alcohol, water or alcohol-water extracts also contain polyphenols, flavonoids, quinic acid and caffeic acid and derivatives, amino acids and fatty acids and their derivatives, and other monomers that need to be removed Ning, tannins, pigments and pectin impurities.

In stevia extract, stevioside is the main product, and stevia polyphenols are by-products in the extraction process of stevioside. When extracting stevioside, iron and calcium salts are usually used to complex and remove impurities; this not only incorporates stevia polyphenols into stevia residue, but worse, it also generates more organic and inorganic mixed solid waste. Stevia polyphenols include chlorogenic acids, flavonoids, flavonols, flavanols, isoflavones, proanthocyanidins, etc. At present, there are at least 30 kinds of stevia polyphenols with definite structure reported. Stevia polyphenols are mostly water-soluble polyphenol compounds, which have good and stable free radical scavenging ability, but also have anti-inflammatory, diuretic, anti-hypertensive, hypoglycemic, anti-tumor and anti-oxidant properties, as well as the effect of lowering blood pressure and blood fat . Product analysis often uses 9 standards such as isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, caffeic acid, luteolin, quercetin, cryptochlorogenic acid, neochlorogenic acid and chlorogenic acid. The product or 7 kinds of polyphenols were used as standard control, and the content was analyzed by HPLC; or the polyphenol was used as the standard control by the forolinic acid method, and the total phenol content was analyzed by ultraviolet.

Due to the large variety of polyphenols in Stevia and the relatively complex ingredients, it has not been listed as a new product in China's regulations; and stevia polyphenols are by-products compared to steviosides, and their performance is not yet widely known, so few special extracts of stevia Phenol process.

As mentioned above, in the current stevioside extraction process, most of the stevioside is extracted with alcoholic water and then salted out with ferrous sulfate and quick lime to remove most of the stevia polyphenols and other proteins, tannins, pectins and other impurities. And precipitated, and then further purified and separated stevioside with macroporous resin. Therefore, the existing extraction of stevia polyphenols is basically based on the stevia residue after the extraction of stevioside. For example, using stevioside residue as raw material, on the basis of a single factor test, a response surface test design is used, with DPPH free radical scavenging ability and total phenol yield in the extract as a double response value, influencing factors on the ultrasonic extraction process That is, the material-liquid ratio, ultrasonic time, and ultrasonic power, the three factors and three levels of experimental design are used to optimize the extraction process of effective antioxidant substances in the stevioside residue: material-liquid ratio 1:24 (g / mL), ultrasonic time 38min, Ultrasonic power 164W, average DPPH free radical scavenging rate is (90.47 ± 1.43)%, total phenol yield is (16.14 ± 0.36) mg / g (Hong Yilan, "Food Science", 2016, 37 (18): 52- 57).

With the increasing awareness of environmental protection, the solid waste problem caused by the salting out of ferrous sulfate and quicklime has become a major problem faced by stevia plants; various processes to reduce ferrous sulfate and quicklime came into being. On the other hand, the understanding of the properties of stevia polyphenols is increasing. Studies on the direct extraction of steviol from stevia have been continuously reported, but they all have problems of low extraction rate, complicated extraction process, poor thermal stability and low content of the obtained stevia polyphenol products. For example, CN107625801A discloses a method for extracting stevia polyphenols using ultrasonic extraction and supercritical carbon dioxide extraction.

Chitosan is a macromolecular cationic polysaccharide rich in amino groups and has been widely used in sewage treatment. In recent years, chitosan has also been used to flocculate and remove impurities such as protein, tannin, and pectin in plant extraction. For example, when extracting stevioside from stevia, flocculation to remove impurities, and when extracting chlorogenic acid from eucommia leaves and honeysuckle, flocculation to remove impurities.

For example, Li Xue et al. Used chitosan flocculation to extract stevioside. Based on the single factor test, the response surface method was used to optimize the flocculation conditions of stevia extract, and a quadratic regression model was established. It is determined that the addition process of chitosan is as follows: add 0.3mL of 1% chitosan (molecular weight is 150kDa) to the 1st and 4th extracts, add 1g stevia leaf (dry weight), flocculation temperature is 48 ℃, flocculation time For 2.9h, the retention rate of stevioside was 92.36%. Add 0.3mL of 1% chitosan (molecular weight 50kDa) solution / g stevia leaf to the second and third extraction liquid, flocculate at 31 ℃ for 1.9h, and the retention rate of stevioside is 93.61%.

As another example, the flocculation and impurity removal process of the water extract of Eucommia ulmoides leaves was optimized by the method of chitosan flocculation. The retention rate of chlorogenic acid, rutin, quercetin and kaempferol in the water extract was used as the investigation index, and the single factor experiment was used. The suitable range of the three factors of concentration ratio of water extract, flocculation temperature and chitosan dosage was preliminarily determined, and the best process was optimized by orthogonal experiments. The optimal flocculation process conditions of the obtained chitosan as a flocculant are: the medicinal material (g) -medicine concentration ratio (L) is 30: 1, the flocculation temperature is 40 ° C, and the chitosan addition amount is 0.7 g / L. Under these conditions, the retention rates of the above four components are 94.3%, 83.1%, 80.1%, and 85.4%, respectively.

As another example, using chitosan composite flocculant, the chlorogenic acid and baicalin retention rate and tannin removal rate in Shuanghuanglian water extract were used to investigate the indicators of composite flocculant preparation process, flocculant dosage and flocculation time. And the influence of chitosan performance index on flocculation effect, optimize the conditions of selective flocculation process. The optimized flocculation process conditions are: bentonite is fired at 450 ℃ for 4h and then modified with chitosan to prepare composite flocculant. The dosage of flocculant is 60g / L and the flocculation time is 24h. The best performance index of chitosan used: degree of deacetylation 95%, viscosity 60cps.

Others have combined modified chitosan with two-water phase extraction technology to extract and purify chlorogenic acid in honeysuckle. The addition amount of flocculant was 0.5g / 50mL, flocculation temperature was 46 ℃, flocculation time was 20min, pH value was 5.0, and the yield of chlorogenic acid was 5.334%. Experiments show that the modified chitosan has obvious flocculation effect. Compared with the traditional water extraction method, the yield of chlorogenic acid is 5.419%, and the loss of chlorogenic acid is less.

The above method for extracting polyphenols from the water extract of Eucommia ulmoides leaves water extract, Shuanghuanglian water extract, honeysuckle, Qingganlidan oral liquid original extract only focuses on extracting polyphenols. For the extraction of polyphenols from stevia, it is necessary to take into account the retention rate of steviosides while obtaining higher levels of polyphenols. On the other hand, the chlorogenic acid in stevia polyphenols is mainly isochlorogenic acid and chlorogenic acid, and the classification of products is helpful for performance applications and research. We have found in a large number of experiments that using the flocculant of the present invention in combination with subsequent macroporous polar resin separation, it is possible to obtain stevia polyphenols based on isochlorogenic acid.

Summary of the invention

The invention provides a method for preparing stevia polyphenols by using a composite chitosan flocculation method. The aqueous extract of stevia leaves is flocculated with composite chitosan at room temperature to remove impurities; the supernatant after removal of impurities uses polar macropores The resin is adsorbed, and the effluent is used to further extract stevioside. The resin is eluted with a gradient of aqueous alcohol solution. After the eluent of the high-concentration aqueous alcohol solution is dried, the stevia polyphenol mainly containing isochlorogenic acid is obtained, which is also the invention. One feature. The invention obtains polyphenol by-products on the basis of the original production process of stevioside, and realizes the comprehensive utilization of stevia.

The flocculant removal of stevia leaf aqueous extract with composite chitosan is to adjust the stevia leaf aqueous extract to pH 4-5 with an aqueous acetic acid solution, and then add a composite chitosan acetic acid aqueous solution with a mass concentration of 1% -2% , The added amount is 0.05-0.2mL per gram of dry stevia leaves; at 10-40 ° C, stirring at 25-30rpm for 5-10min, and standing for 30-50min to flocculate and remove impurities. Further, the supernatant after flocculation is adsorbed with polar macroporous resin, and the effluent from the adsorption process is collected for extracting stevioside; then, the adsorption column resin is first used with a volume fraction of 0.5-1BV of 10% -20% Analysis of methanol or ethanol aqueous solution, 1-1.5BV / h, the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution, and the combined solution is reserved for the extraction of stevioside; the volume fraction of 1-3BV is 60% -90% Analysis of methanol or ethanol aqueous solution, 1-1.5BV / h, collect the analysis solution, concentrate and dry the obtained analysis solution to obtain stevia polyphenol product. The composite chitosan includes quaternary ammonium salt chitosan and chitosan; the quaternary ammonium salt chitosan is hydroxypropyltrimethylammonium chloride chitosan, which accounts for quaternary ammonium salt chitosan 5% -20% of the total mass of sugar and chitosan, the degree of substitution of the quaternary ammonium salt chitosan is 90%; the molecular weight of the chitosan is 50-100KDa, and the degree of deacetylation is 70% -80% .

In one embodiment of the present invention, the flotation of stevia leaf aqueous extract with composite chitosan is to adjust the stevia leaf aqueous extract to pH 4-5 with a 1 mol / L acetic acid aqueous solution, and then add the mass The concentration of 1% aqueous solution of complex chitosan acetic acid is 0.05-0.2mL per gram of dry stevia leaves; at 20-40 ° C, stirring at 25-30 rpm for 5-10min, and standing for 40-50min to remove impurities.

In one embodiment of the present invention, the flotation of stevia leaf aqueous extract with composite chitosan is to adjust the stevia leaf aqueous extract to pH 4-5 with a 1 mol / L acetic acid aqueous solution, and then add the mass The concentration of 1% aqueous solution of complex chitosan acetic acid is 0.1-0.2mL per gram of dry stevia leaves; at 20-40 ° C, stir at 30rpm for 50min, and let stand for 40-50min to remove impurities.

In one embodiment of the present invention, the polar macroporous resin includes polystyrene resin LSA-7, XDA-8 and polyacrylic resin LX-17.

In an embodiment of the present invention, the preparation method of the aqueous solution of the complex chitosan acetic acid with a mass concentration of 1% is: a total of 1 gram of quaternary ammonium salt chitosan and chitosan is added at room temperature to 100 mL containing 1 gram In the aqueous solution of acetic acid, the mixed chitosan solution is obtained after mixing evenly.

In an embodiment of the present invention, the preparation method of the stevia leaf water extract is: mixing stevia leaves in a ratio of 15-20 BV of water to stevia leaf dry leaves, performing water extraction, and the extraction temperature is 50-60 The extraction time is 1-5h at ℃, and the stevia leaf water extract is obtained by filtration.

An embodiment of the present invention includes the following steps:

(1) Extracting stevia liquid:

Mix the stevia leaves with water and the dry leaves of stevia leaves at a ratio of 15-20BV, perform water extraction, the extraction temperature is 50-60 ° C, the extraction time is 3h, and the water extract of stevia leaves is filtered;

(2) Composite chitosan flocculation

After the stevia leaf aqueous extract is adjusted to pH 4-5 with a 1 mol / L acetic acid aqueous solution, a mass concentration of 1% complex chitosan acetic acid aqueous solution is added in an amount of 0.1-0.2 mL per gram of dry leaves; in 10-40 Stir at 30 rpm for 5 min at ℃, let stand for 40-50 min to remove impurities by flocculation; then, filter and separate through a plate and frame to obtain the supernatant of flocculation;

(3) Resin adsorption separation

The flocculated supernatant obtained in step (2) is adsorbed with polar macroporous resin, and the effluent from the adsorption process is collected for extracting stevioside; then, the adsorption column resin is first used with a volume fraction of 0.5-1BV of 10% -20% Analysis of methanol or ethanol aqueous solution, 1-1.5BV / h, the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution, and the combined solution is reserved for the extraction of stevioside; then the volume fraction of 1-3BV is 60% -90% Analysis of methanol or ethanol aqueous solution, 1-1.5BV / h, collect the analysis solution, concentrate and dry the obtained analysis solution to obtain stevia polyphenol product.

The beneficial effects of adopting the above technical solution of the present invention are:

(1) While obtaining stevia polyphenols mainly based on isochlorogenic acid, high retention of stevioside can effectively reduce production costs and achieve comprehensive utilization of stevia;

(2) Chitosan compounded with quaternary ammonium salt as a flocculant, environmental protection, less dosage, short flocculation time, high flocculation efficiency, low temperature (room temperature, 10-30 degrees), use acetic acid to adjust the pH ratio Hydrochloric acid environmental protection;

(3) The total phenol content can reach about 55% by HPLC method.

(4) The loss of stevioside is less than 2%.

BRIEF DESCRIPTION

Figure 1: HPLC chart of stevia polyphenols obtained in Example 1; the retention time (min) of different polyphenols is: neochlorogenic acid 13.627; chlorogenic acid 18.183; cryptochlorogenic acid 19.053; caffeic acid 21.043; isochlorogenic acid B 38.215; isochlorogenic acid A. 41.159; isochlorogenic acid C. 47.099.

Figure 2: HPLC chart of Stevia polyphenols obtained in Comparative Example 1.

Figure 3: HPLC chart of Stevia polyphenols obtained in Comparative Example 2.

Figure 4: HPLC chart of Stevia polyphenols obtained in Example 3.

Figure 5: HPLC chart of Stevia polyphenols obtained in Example 5.

Figure 6: HPLC chart of Stevia polyphenols obtained in Comparative Example 3.

Figure 7: HPLC chart of Stevia polyphenols obtained in Comparative Example 4.

Figure 8: HPLC chart of Stevia polyphenols obtained in Comparative Example 5.

detailed description

Detection method:

(1) Detection of the total content of stevioside (total glycoside content) and calculation of the retention rate of stevioside after flocculation:

Quantitative analysis of steviol glycosides is based on the analysis and detection method of steviol glycosides in GB2760-2014 or JECFA2016. The 9 steviol glycosides specified in GB2760-2014 are quantified to calculate the total content of 9 steviol glycosides in the sample.

The retention rate of stevioside after flocculation = 100% × total glycoside content in supernatant after flocculation × total mass of supernatant / (total glycoside content in stevia leaf water extract × total mass of water extract).

(2) Detection method of steviol content (HPLC):

Tested with 9 standards: isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, caffeic acid, luteolin, quercetin, cryptochlorogenic acid, neochlorogenic acid and chlorogenic acid Nine kinds of standards are used as standard-concentration-peak area standard curve; the corresponding polyphenol content is calculated according to the nine kinds of polyphenol standards, and the total polyphenol content is calculated.

Chromatographic conditions: The chromatographic column is Syncronis Q18 C18 LC column, 4.6x250mm. Ultraviolet detector, detection wavelength 327nm. The mobile phase is acetonitrile-0.2% phosphoric acid aqueous solution, and the flow rate is 1.0 ml / min. Injection volume: 10 μL.

(3) Detection method of steviol content (glypholic acid forolinol method): According to GB / T 31740.2-2015, using gallic acid as a control substance, the total phenolic content was determined by the forolinol method.

The present invention will be further described below with reference to specific embodiments. The resins in the following examples were all purchased from China Xi'an Lanxiao Technology New Materials Co., Ltd., and their product codes are consistent with the official product descriptions; hydroxypropyltrimethylammonium chloride chitosan was purchased from Cypress, Chongqing, China Technology Co., Ltd. or Wuhan Yuancheng Technology Development Co., Ltd., China, 90% substitution; various chitosans were purchased from Zhejiang Zhongtuan Biotechnology Co., Ltd. in China, Huaxia Chemical in Chengdu, China and Zhengzhou Wanbo Chemical Products Co., Ltd. in China; 1% The compound chitosan solution refers to a solution containing 1 gram of compound chitosan per 100 mL of 1% acetic acid aqueous solution.

Example 1

(1) Extracting stevia leaves:

Stevia leaves are extracted according to the ratio of water to dry leaves of Stevia leaves at 20BV, the extraction temperature is 50 ℃, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation

After the stevia leaf aqueous extract was adjusted to pH 4.5 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 10% of the mass of the composite chitosan, the balance was the shell Chitosan; molecular weight of chitosan is 100KDa, degree of deacetylation is 80%), the added amount is 0.15mL per gram of dry leaves. Stir at 30 rpm for 5 min at 20 ℃, and let stand for 40 min to remove impurities. Then, the flocculated supernatant was obtained by filtration and separation through a plate and frame; the steviol glycoside retention rate in the flocculated supernatant after flocculation was detected and calculated.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with LSA-7 polar macroporous resin, and the effluent from the adsorption column was collected and stored for the extraction of stevioside. Then, the adsorption column resin was first analyzed with a 10% ethanol aqueous solution of 1BV. The obtained analysis solution was combined with the effluent before analysis to obtain a combined solution. Because the polyphenol content in the combined solution is very low, it is difficult to quantify by high pressure liquid chromatography, so According to GB / T 31740.2-2015, using gallic acid as a reference substance, the total phenol content in the combined liquid was determined by the forolin method, and the total phenol content in the combined liquid was 0.2% (on a dry basis). The combined solution is used to extract stevioside. Then, it was analyzed with 2BV 80% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 58.74% (neo-chlorogenic acid 0.01%, chlorogenic acid 0.05%, cryptochlorogenic acid 0.07%, caffeic acid 0%, luteolin 0%, isochlorogenic acid B 1.45%, (Isochlorogenic acid A 39.5%, quercetin 0%, isochlorogenic acid 17.7%).

Comparative Example 1 Replace LSA-7 polar macroporous resin with LSA-10 (medium polarity macroporous resin)

(1) Extracting stevia leaves:

Stevia leaves are extracted according to the ratio of water to dry leaves of Stevia leaves at 20BV, the extraction temperature is 50 ℃, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4.5 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 10% of the mass of the composite chitosan, the balance was the shell Chitosan; molecular weight of chitosan is 100KDa, degree of deacetylation is 80%), the added amount is 0.15mL per gram of dry leaves. Stir at 30 rpm for 5 min at 20 ° C, and let stand for 60 min to remove impurities. Then, the supernatant of flocculation was obtained by filtration through a plate and frame; the retention rate of stevioside after flocculation was detected and calculated to be 98.5%.

(3) Resin adsorption separation:

Adsorb the flocculated supernatant obtained in step (2) with LSA-10 (medium polar macroporous resin, also styrene type, purchased from Xi'an Lanxiao Technology New Material Co., Ltd.) resin, collect the effluent from the adsorption column and save it Used to extract stevioside. Then, the adsorption column resin is first analyzed with a 1BV 10% ethanol aqueous solution, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid is used as a control substance, and the forolin method is used. The total phenol content in the combined solution was determined. The total phenol content in the combined solution was 0.4% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it was analyzed with 2BV 80% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 40.8% (neo-chlorogenic acid 0.2%, chlorogenic acid 0.6%, cryptochlorogenic acid 0.2%, caffeic acid 0.1%, luteolin 0%, isochlorogenic acid B 1.1%, Isochlorogenic acid A 26.2%, quercetin 0%, isochlorogenic acid C 12.4%).

Comparative Example 2

(1) Extracting stevia leaves:

Stevia leaves are extracted according to the ratio of water to dry leaves of Stevia leaves at 20BV, the extraction temperature is 50 ℃, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4.5 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 10% of the mass of the composite chitosan, the balance was the shell Chitosan; molecular weight of chitosan is 100KDa, degree of deacetylation is 80%), the added amount is 0.15mL per gram of dry leaves. Stir at 30 rpm for 5 min at 20 ° C., and leave it for 30 min to remove impurities. Then, the supernatant was obtained by filtration and separation through a plate and frame; the retention rate of steviol glycoside after flocculation was detected and calculated to be 99.7%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with LSA-7 polar macroporous resin, and the effluent from the adsorption column was collected and stored for the extraction of stevioside. Then, the adsorption column resin is first analyzed with a 1BV 10% ethanol aqueous solution, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid is used as a control substance, and the forolin method is used. The total phenol content in the combined solution was determined. The total phenol content in the combined solution was 0.4% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it was analyzed with 2BV 80% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 52.5% (neochlorogenic acid 0.004%, chlorogenic acid 0.01%, cryptochlorogenic acid 0.01%, caffeic acid 0.2%, luteolin 0.04%, isochlorogenic acid B 0.8%, (Isochlorogenic acid A 32.2%, quercetin 2.5%, isochlorogenic acid 16.7%). It can be seen that although shortening the flocculation time does not reduce the retention rate of stevioside after flocculation, it will reduce the total phenol yield.

Example 2

(1) Extracting stevia leaves:

Stevia leaves are extracted at a ratio of 15BV according to the ratio of water to dry leaves of Stevia leaves, the extraction temperature is 60 ° C, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 5 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 15% of the mass of the composite chitosan, and the balance was chitosan ; The molecular weight of chitosan is 50KDa, the degree of deacetylation is 70%), the addition amount is 0.1mL per gram of dry leaves; at 40 ℃, 30rpm stirring for 5min, and stand for 35min to flocculate and remove impurities; then, filter and separate through the plate and frame The flocculated supernatant was obtained; the steviol glycoside retention rate after flocculation was detected and calculated to be 98.4%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with XDA-8 polar macroporous resin, and the effluent from the adsorption column was collected and stored for use in extracting stevioside. Then, the adsorption column resin is first analyzed with a 0.5BV 20% ethanol aqueous solution, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid is used as a control substance, and forolin is used The total phenol content in the combined solution was determined by the method. The total phenol content in the combined solution was 0.3% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it was analyzed with 3BV 70% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 55.41% (new chlorogenic acid 0.11%, chlorogenic acid 0.37%, cryptochlorogenic acid 0.12%, caffeic acid 0.14%, luteolin 0%, isochlorogenic acid B 9.13%, Isochlorogenic acid A 26.99%, quercetin 0.06%, isochlorogenic acid C 18.48%).

Example 3

(1) Extracting stevia leaves:

Stevia leaves are extracted according to the ratio of water to dry leaves of Stevia leaves at 20BV, the extraction temperature is 60 ℃, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4.5 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 15% of the mass of the composite chitosan, the balance was the shell Glycan; chitosan molecular weight is 80KDa, deacetylation degree is 80%), the amount of addition is 0.12mL per gram of dry leaves; at 40 ℃, 30rpm stirring for 5min, stand for 50min to flocculate and remove impurities; then, through the plate frame The flocculated supernatant was obtained by filtration and separation; the steviol glycoside retention rate after flocculation was detected and calculated to be 98.8%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with XDA-8 polar macroporous resin, and the effluent from the adsorption column was collected and stored for use in extracting stevioside. Then, the adsorption column resin is first analyzed with a 0.5BV 20% ethanol aqueous solution, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid is used as a control substance, and forolin is used The total phenol content in the combined solution was determined by the method, and the total phenol content in the combined solution was 0.2% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it was analyzed with 3BV 70% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 57.5% (new chlorogenic acid 0.01%, chlorogenic acid 0.04%, cryptochlorogenic acid 0.01%, caffeic acid 0.01%, luteolin 0%, isochlorogenic acid B 0.8%, (Isochlorogenic acid A 43.3%, quercetin 0.3%, isochlorogenic acid 13.1%).

Example 4

(1) Extracting stevia leaves:

Stevia leaves are extracted according to the ratio of water to dry leaves of Stevia leaves at 20BV, the extraction temperature is 55 ℃, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounts for 20% of the mass of the composite chitosan, and the balance is chitosan ; The molecular weight of chitosan is 80KDa, the degree of deacetylation is 80%), and the added amount is 0.08mL per gram of dry leaves. Stir at 30 rpm for 5 min at 40 ℃, and let stand for 35 min to remove impurities. Then, the supernatant was obtained by filtration and separation through a plate and frame; the steviol glycoside retention rate after flocculation was detected and calculated to be 99.4%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with XDA-8 polar macroporous resin, and the effluent from the adsorption column was collected and stored for use in extracting stevioside. Then, the adsorption column resin was first analyzed with 1BV 20% ethanol aqueous solution, and the obtained analysis solution was combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, using gallic acid as a control substance, the Folin method was used The total phenol content in the combined liquid was determined. The total phenol content in the combined liquid was 0.23% (on a dry basis), and the combined liquid was left for the extraction of stevioside. Then, it was analyzed with 3BV 70% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 51.6% (new chlorogenic acid 0%, chlorogenic acid 0.2%, cryptochlorogenic acid 0%, caffeic acid 0%, luteolin 0.1%, isochlorogenic acid B 3.1%, (Isochlorogenic acid A 30.8%, quercetin 0.5%, isochlorogenic acid C 16.9%).

Example 5

(1) Extracting stevia leaves:

Stevia leaves are extracted at a ratio of 15BV according to the ratio of water to dry leaves of Stevia leaves, the extraction temperature is 60 ° C, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 15% of the mass of the composite chitosan, the balance was chitosan Sugar; the molecular weight of chitosan is 80KDa, the degree of deacetylation is 80%), and the added amount is 0.1mL per gram of dry leaves. Stir at 30 rpm, 30 rpm for 5 min, and stand for 35 min to remove impurities. Then, filter and separate through the frame to obtain the supernatant of the flocculation; detect and calculate the retention rate of steviol glycosides after flocculation is 99.1%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with LX-17 polar macroporous resin, and the effluent from the adsorption column was collected and stored for the extraction of stevioside. Then, the adsorption column resin was first analyzed with a 1BV 15% ethanol aqueous solution, and the obtained analysis solution was combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid was used as a reference substance, and the Folin method was used. The total phenol content in the combined solution was determined. The total phenol content in the combined solution was 0.26% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it is analyzed with an 85% ethanol aqueous solution of 2BV, and the obtained analysis solution is concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 54.54% (new chlorogenic acid 0%, chlorogenic acid 0%, cryptochlorogenic acid 0%, caffeic acid 0.02%, luteolin 0.04%, isochlorogenic acid B5.32%, Isochlorogenic acid A 26.78%, quercetin 3.86%, isochlorogenic acid C 18.52%).

Comparative Example 3 Replace polar macroporous resin LX-17 with LX-11 nonpolar macroporous resin

(1) Extracting stevia leaves:

Stevia leaves are extracted at a ratio of 15BV according to the ratio of water to dry leaves of Stevia leaves, the extraction temperature is 60 ° C, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4 with a 1 mol / L acetic acid aqueous solution, 1% composite chitosan acetic acid aqueous solution (hydroxypropyltrimethylammonium chloride chitosan, accounting for 15% of the mass of the composite chitosan was added , The balance is chitosan; the molecular weight of chitosan is 80KDa, the degree of deacetylation is 80%), the amount of addition is 0.2mL per gram of dry leaves; at 30 ℃, stirring at 30rpm for 5min, let stand for 60min to remove impurities; Then, the flocculated supernatant was obtained by filtration and separation through a plate and frame; the steviol glycoside retention rate after flocculation was detected and calculated to be 99.2%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with LX-11 non-polar macroporous resin (the same series of resins as LX-17, but non-polar), and the effluent from the adsorption column was collected and stored for extraction of stevioside. Then, the adsorption column resin was first analyzed with a 1BV 15% ethanol aqueous solution, and the obtained analysis solution was combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid was used as a reference substance, and the Folin method was used. The total phenol content in the combined solution was determined. The total phenol content in the combined solution was 0.5% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it is analyzed with an 85% ethanol aqueous solution of 2BV, and the obtained analysis solution is concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 21.4% (new chlorogenic acid 0.01%, chlorogenic acid 0.02%, cryptochlorogenic acid 0.01%, caffeic acid 8.7%, luteolin 0.7%, isochlorogenic acid B1.5% , Isochlorogenic acid A0.9%, quercetin 5.3%, isochlorogenic acid C4.2%).

Comparative Example 4 This comparative example uses only chitosan flocculation, other conditions are the same as in Example 3

(1) Extracting stevia leaves:

Stevia leaves are extracted at a ratio of 20 BV of water to dry leaves of Stevia leaves, the extraction temperature is 60 ° C, the extraction time is 3 hours, and the water extract of Stevia leaves is filtered.

(2) Chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4.5 with a 1 mol / L acetic acid aqueous solution, 1% chitosan acetic acid aqueous solution was added (chitosan molecular weight 80 KDa, deacetylation degree 80%, addition amount per gram of dry leaves 0.12mL; stir at 30rpm for 5min at 40 ℃, let stand for 50min to flocculate and remove impurities, the result shows that the solution is turbid and cannot be flocculated. Add 1% chitosan acetic acid aqueous solution, the amount is 0.3mL per gram of dry leaves, continue to Leave at 40 ℃ to remove impurities by flocculation, and get a clear solution after 1h. After detection and calculation, the retention rate of stevioside after flocculation is 95.2%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with XDA-8 polar macroporous resin, and the effluent from the adsorption column was collected and stored for use in extracting stevioside. Then, the adsorption column resin is first analyzed with a 0.5BV 20% ethanol aqueous solution, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid is used as a control substance, and forolin is used The total phenol content in the combined solution was determined by the method. The total phenol content in the combined solution was 0.45% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it was analyzed with 3BV 70% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 30.36% (neochlorogenic acid 0%; chlorogenic acid 0%; cryptochlorogenic acid 0%; caffeic acid 0%; luteolin 0%; isochlorogenic acid B 0.76%; Isochlorogenic acid A 13.07%; quercetin 1.27%; isochlorogenic acid C 15.25%). It can be seen that when chitosan alone is used as the flocculant, and chitosan and quaternary ammonium salt chitosan are not used in combination, the retention rate of steviol glycosides and the content of steviol will decrease.

Comparative Example 5 This comparative example uses composite chitosan flocculation outside the power requirements, other conditions are the same as in Example 3

(1) Extracting stevia leaves:

Stevia leaves are extracted according to the ratio of water to dry leaves of Stevia leaves at 20BV, the extraction temperature is 60 ℃, the extraction time is 3h, and the water extract of Stevia leaves is filtered;

(2) Composite chitosan flocculation:

After the stevia leaf aqueous extract was adjusted to pH 4.5 with a 1 mol / L acetic acid aqueous solution, a 1% composite chitosan acetic acid aqueous solution was added (quaternary ammonium salt chitosan accounted for 15% of the mass of the composite chitosan, the balance was the shell Glycan; chitosan molecular weight is 150KDa, deacetylation degree is 80%), the amount of addition is 0.12mL per gram of dry leaves; at 40 ℃, 30rpm stirring for 5min, let stand for 50min to flocculate and remove impurities; then, through the plate frame After filtration and separation, the flocculated supernatant was obtained; after detection and calculation, the retention rate of stevioside after flocculation was 90.1%.

(3) Resin adsorption separation:

The flocculated supernatant obtained in step (2) was adsorbed with XDA-8 polar macroporous resin, and the effluent from the adsorption column was collected and stored for use in extracting stevioside. Then, the adsorption column resin is first analyzed with a 0.5BV 20% ethanol aqueous solution, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution. According to GB / T 31740.2-2015, gallic acid is used as a control substance, and forolin is used The total phenol content in the combined solution was determined by the method. The total phenol content in the combined solution was 0.5% (on a dry basis), and the combined solution was left for the extraction of stevioside. Then, it was analyzed with 3BV 70% ethanol aqueous solution, and the obtained analysis solution was concentrated and dried to obtain a stevia polyphenol product. After testing and calculation, the total phenol content in the sample was 34.2% (new chlorogenic acid 0.002%; chlorogenic acid 0.01%; cryptochlorogenic acid 0.003%; caffeic acid 1.2%; luteolin 0.1%; isochlorogenic acid B 2.9%; Isochlorogenic acid A 11.6%; Quercetin 3.8%; Isochlorogenic acid C 14.6%).

It can be seen that when the molecular weight of chitosan is too high, the retention rate of steviol glycosides after flocculation is slightly reduced, and the total phenol content is greatly reduced.

Comparative Example 6

In this comparative example, chitosan with a degree of deacetylation of 60% was used, and the remaining conditions were the same as in Example 3. A cloudy solution with lumps of different sizes was obtained and could not be flocculated.

Although the present invention has been disclosed in the above as preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be defined by the claims.

Claims (10)

1. A method for preparing stevia polyphenols, characterized in that the water extract of stevia leaves is flocculated with composite chitosan to collect impurities, and the supernatant after flocculation is collected; the supernatant after flocculation is adsorbed with polar macroporous resin The effluent is used to further extract stevioside; the resin is eluted successively with a low-concentration alcohol aqueous solution and a high-concentration alcohol aqueous solution. The analysis solution obtained by elution of the low-concentration alcohol aqueous solution is combined with the effluent before analysis to obtain a combined solution. The total phenol content in the solution is less than 0.5% (on a dry basis), and the combined solution is left for extracting stevioside; the eluent after elution with an aqueous solution of a high-concentration alcohol is dried to obtain an isochlorogenic acid-based solution Stevia polyphenols.
2. The method for preparing stevia polyphenols according to claim 1, characterized in that the flocculation of the stevia leaf aqueous extract with composite chitosan is to adjust the stevia leaf aqueous extract to pH 4 with an acetic acid aqueous solution After -5, add a mass concentration of 1% -2% complex chitosan acetic acid aqueous solution, the amount of addition is 0.05-0.2mL per gram of dry stevia leaves; at 10-40 ℃, 25-30rpm stirring 5-10min, static Set 30-50min to remove impurities by flocculation.
3. The method for preparing stevia polyphenols according to claim 1 or 2, characterized in that the flotation of stevia leaf water extract with composite chitosan is to remove stevia leaf water extract with 1mol / L After adjusting the acetic acid aqueous solution to pH 4-5, add a 1% mass concentration of chitosan acetic acid aqueous solution in an amount of 0.05-0.2mL per gram of dry stevia leaves; at 20-40 ° C, stir at 25-30rpm 5- 10min, let stand for 40-50min to remove impurities by flocculation.
4. A method for preparing stevia polyphenols according to claim 3, characterized in that the flocculation of the stevia leaf water extract with composite chitosan is to remove stevia leaf water extract with 1mol / L acetic acid After the aqueous solution is adjusted to pH 4-5, add a mass concentration of 1% complex chitosan acetic acid aqueous solution, the amount of addition is 0.1-0.2mL per gram of dry stevia leaves; at 20-40 ° C, stir at 30rpm for 50min, and stand for 40- 50min to remove impurities by flocculation.
5. A method for preparing stevia polyphenols according to any one of claims 1 to 4, characterized in that the supernatant after flocculation is adsorbed with polar macroporous resin, and the effluent from the adsorption process is collected for extracting stevioside ; Then, the adsorption column resin is first analyzed with a 0.5-1BV volume fraction of 10% -20% methanol or ethanol aqueous solution, 1-1.5BV / h, and the obtained analysis solution is combined with the effluent before analysis to obtain a combined solution, combined The liquid is left for the extraction of stevioside; then it is analyzed by a methanol or ethanol aqueous solution with a volume fraction of 1-3BV of 60% -90%, 1-1.5BV / h, the analysis solution is collected, and the obtained analysis solution is concentrated and dried to obtain stevia Phenol products.
6. A method for preparing stevia polyphenols according to claim 5, characterized in that the polar macroporous resin comprises polystyrene resin, polyacrylic resin, preferably polystyrene resin LSA-7, poly Styrene resin XDA-8, polyacrylic resin LX-17.
7. A method for preparing stevia polyphenols according to any one of claims 1-6, characterized in that the composite chitosan comprises two components of quaternary ammonium salt chitosan and chitosan; the quaternary ammonium Salt chitosan is hydroxypropyltrimethylammonium chloride chitosan, which accounts for 5% -20% of the total mass of quaternary ammonium chitosan and chitosan. The degree of substitution of the quaternary ammonium chitosan is 90%; the molecular weight of the chitosan is 50-100KDa, and the degree of deacetylation is 70% -80%.
8. The method for preparing stevia polyphenol according to claim 2 or 7, characterized in that, the method for preparing the aqueous solution of complex chitosan acetic acid with a mass concentration of 1% is: combining quaternary ammonium salt chitosan and chitosan A total of 1 gram of sugar was added to 100 mL of an aqueous solution containing 1 gram of acetic acid at room temperature, mixed uniformly to obtain a composite chitosan solution.
9. A method for preparing stevia polyphenols according to any one of claims 1-8, characterized in that the method for preparing the water extract of stevia leaves is: the ratio of stevia leaves to dry leaves of stevia leaves is 15 Mix with -20BV, perform water extraction, the extraction temperature is 50-60 ℃, the extraction time is 1-5h, and filter to obtain the stevia leaf water extract.
10. Steviol based on isochlorogenic acid prepared according to any one of claims 1-9.

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