CN115887530B - Lotus leaf aqueous extract with functions of controlling sugar and reducing lipid as well as preparation method and application thereof - Google Patents
Lotus leaf aqueous extract with functions of controlling sugar and reducing lipid as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a lotus leaf water extract with functions of controlling sugar and reducing lipid, a preparation method and application thereof. The preparation method adopts the processes of liquid nitrogen crushing, double enzyme cascade enzymolysis, colloid mill treatment, high-pressure homogenization, high-temperature extraction, low-temperature high-speed centrifugation, reduced-pressure concentration, freeze drying and the like to obtain the lotus leaf extract with the function of regulating the absorption of glycolipid. The lotus leaf extract has total sugar content of more than 40% and total flavone content of more than 6%, can effectively inhibit the activities of alpha-glucosidase and pancreatic lipase, adsorb cholate, inhibit the solubility of cholesterol in micelle, and has good effect of regulating the absorption of glycolipid. The invention has simple extraction process, the whole process flow can meet the food-grade requirement, and the invention can be applied to the fields of common foods, health care products and the like.
Description
Technical Field
The invention belongs to the field of lotus leaf finish machining and high value, and particularly relates to a preparation method of a lotus leaf water extract with functions of controlling sugar and reducing lipid.
Background
With the development of economic level and the improvement of living standard, the population of Chinese obesity is gradually increased year by year. The lotus leaf has long history of food and medicine, is rich in a plurality of main active ingredients with the functions of reducing blood sugar and blood fat, such as flavone, polysaccharide, alkaloid and the like, and has important practical significance in developing a lotus leaf full extract with the functions of controlling sugar and reducing blood fat.
The lotus leaf extraction technology mainly uses flavone and alkaloid which are extracted independently as target products, and if a team adopts an acidic hydrophilic organic solvent to extract and purify nuciferine and lotus leaf flavone, a method for separating nuciferine and lotus leaf flavone from lotus leaves is obtained (patent application number: CN 200710009464.9), but the method involves the use of a large amount of organic reagents such as acetone, isopropanol and the like, and has high requirements on production environment. There are also methods for extracting and purifying three products of flavone, alkaloid and polysaccharide in lotus leaf synchronously (patent application number: CN 200810107271.1) by extracting lotus leaf three times with different organic reagents, but the steps of stepwise extraction are more, the energy consumption is high, and in addition, the functional activity effect of the whole extract is difficult to be achieved by a single active substance. In recent years, physiological activity of lotus leaf polysaccharide is paid attention to gradually, and a hot water extraction method is the most classical technical means for industrially preparing lotus leaf polysaccharide extract, but the method has low yield and high energy consumption. Therefore, a research team adopts a cellulose, bromelain and pectinase compound auxiliary water extraction method to prepare lotus leaf polysaccharide with immunological activity (patent application number: CN 202010487093.0), and the content of lotus leaf polysaccharide in the extract is effectively improved by virtue of enzyme method compound enzymolysis, and meanwhile, active substances such as higher total flavone and the like are also ensured, but the impurity removing process involves the use of toxic reagents such as chloroform and the like, the polysaccharide loss rate is higher, the processing cost is high, the total sugar content in the extract is less than 40%, the total flavone content is less than 4%, and further improvement space is provided. In addition, in the compound polysaccharide with the blood lipid reducing effect, the hawthorn, the lotus leaf, the tartary buckwheat, the cassia seed, the medlar and the poria cocos are crushed and subjected to steam explosion in sequence, and the mixed polysaccharide is extracted by hot water after mixing, so that the compound polysaccharide has good blood lipid reducing effect, but the components are complex, and the main active substances are not clear. In the sweet Fuzhuan tea of lotus leaf polysaccharide and the preparation method thereof (patent application number: CN 202110071721.1), lotus leaf dry powder is subjected to enzymolysis by cellulase, concentrated, centrifuged, collected precipitate and dried lotus leaf dietary fiber is compounded with polysaccharides from other sources to finally prepare the tea drink with special dietary immunonutrition and metabolism, the technology mainly utilizes the compounding of active polysaccharides from various sources to jointly play a role, and the extraction preparation technology of plant raw materials with very abundant active substance content of lotus leaves is not deep enough.
In summary, (1) most extraction technologies are to extract flavone and alkaloid separately as target products, and few researches on polysaccharide and active substance co-extraction are concerned; (2) The lotus leaf extract (containing polysaccharide, flavone and alkaloid) which is concerned about regulating the absorption function of sugar and fat has fewer reports; (3) The common crushing and sieving are the most traditional and most classical pretreatment technology of lotus leaf extract, but the common crushing is insufficient for pretreatment of leaf raw materials with high fiber content, so that the extraction rate of active substances such as polysaccharide flavone is difficult to break through the upper limit; (4) The simple enzymatic extraction may be difficult to significantly improve the dissolution rate of polysaccharide, flavone and other substances, and can be considered to be combined with other physical methods.
Therefore, the invention adopts liquid nitrogen to fully crush lotus leaves to reduce the particle size of powder, then uses double enzyme cascade enzymolysis wall breaking to extract water-soluble polysaccharide and flavonoid compounds, uses colloid mill treatment, high-pressure homogenization and high-temperature extraction to further dissolve active ingredients, realizes the one-pot extraction of lotus leaf sugar-control lipid-lowering active flavone and polysaccharide, and the obtained lotus leaf extract contains polysaccharide and flavone and has the capacity of adjusting sugar and lipid absorption. The technology gives consideration to the advantages of high extraction efficiency of flavone and polysaccharide, good functional activity and capability of realizing continuous production of 3 dimensions, and has potential of industrialized popularization and application.
Disclosure of Invention
The invention aims to provide a preparation method of lotus leaf water extract with functions of reducing blood sugar and lipid, which takes the activity of reducing blood sugar and lipid as a guide, and obtains the lotus leaf extract with good effect of inhibiting excessive absorption of glycolipid through liquid nitrogen crushing, double enzyme cascade wall breaking, colloid mill treatment, high-pressure homogenization and high-temperature extraction treatment. The lotus leaf extract has the total sugar content of more than 40 percent and the total flavone content of more than 6 percent, and the extract has better effects of reducing blood sugar and controlling lipid through the in-vitro enzyme activity inhibition experimental model and the INFOGEST static digestion model, and has the potential of being applied to controlling the absorption of glycolipid into health foods. The invention has simple extraction process, the whole process flow can meet the food-grade requirement, and the invention can be applied to the fields of common foods, health care products and the like.
The technical scheme of the invention is as follows:
the inventionProvides a preparation method of lotus leaf water extract with the functions of controlling sugar and reducing lipid. The lotus leaf extract with good glycolipid absorption regulation function is prepared from lotus leaves serving as a raw material through the processes of liquid nitrogen crushing, double-enzyme cascade wall breaking, colloid mill treatment, high-pressure homogenization, high-temperature extraction, low-temperature high-speed centrifugation, reduced-pressure concentration, freeze drying and the like. The lotus leaf extract contains total sugar>40% total flavone content>6, has good effect of regulating glycolipid absorption, and can inhibit IC by alpha-glucosidase 50 Value of<0.2mg/mL pancreatic lipase inhibitor IC 50 Value of<2mg/mL, cholate adsorption at 20mg/mL>55%, cholesterol micelle dissolution inhibition rate>35%. The extraction process is simple, the whole process flow can meet the food-grade requirement, and the method can be applied to the fields of common foods, health care products and the like. The preparation method comprises the following steps:
(1) Crushing by liquid nitrogen: crushing dry lotus leaves into dry powder by a liquid nitrogen crushing device to obtain lotus leaf dry powder;
(2) Double enzyme cascade enzymolysis: uniformly mixing the lotus leaf dry powder obtained in the step (1) with water, regulating the pH value, adding cellulase after fully and uniformly mixing, and stirring at constant temperature for enzymolysis to obtain a suspension 1; adding pancreatin into the obtained suspension 1 after regulating the pH value, and stirring at constant temperature for enzymolysis to obtain a suspension 2;
(3) Colloid mill treatment: carrying out colloid mill treatment on the suspension 2 to obtain a suspension 3;
(4) High-pressure homogenization treatment: homogenizing the suspension 3 to obtain a suspension 4;
(5) High-temperature heating and extracting: heating and extracting the suspension 4 to obtain a suspension 5;
(6) Centrifuging and concentrating: centrifuging the suspension 5, and concentrating the supernatant under reduced pressure to obtain extract 1;
(7) And (3) freeze drying: and (3) freeze-drying the extracting solution 1 to obtain the lotus leaf extract.
Further, in the step (1), the condition of liquid nitrogen pulverization is that the pulverization is carried out at-150 to-120 ℃.
Further, in the step (2), the feed liquid ratio of the lotus leaf dry powder to the water is 1:8-1:12 g/mL; adjusting the pH value to 4.0-5.0 by using hydrochloric acid, adding cellulose with the mass of 0.75-1.25% (w/w, g/g) of the lotus leaf dry powder obtained in the step (1), stirring at the speed of 120-180 r/min, and performing enzymolysis at the temperature of 50-60 ℃ for 0.5-1.5 h. And (2) adding pancreatin with the mass of 0.75-1.25% (w/w, g/g) of the lotus leaf dry powder obtained in the step (1), adjusting the pH value to 6.5-7.5, stirring at the speed of 120-180 r/min, and performing enzymolysis at the temperature of 50-60 ℃ for 2-6 h.
Further, in the step (3), the grinding time of the colloid mill is 1-3 times, and the grinding time of each time is 4-10 min.
Further, in the step (4), the high-pressure homogenization treatment is performed, the homogenization pressure is 10-30 MPa, and the number of homogenization is 1-3.
Further, in the step (5), the high-temperature heating extraction temperature is 95-115 ℃ and the extraction time is 15-45 min.
Further, in the step (6), the temperature of the centrifugation is 4-8 ℃, the centrifugal force of the centrifugation is 6000-8000 g, and the time of the centrifugation is 15-25 min; the temperature of the reduced pressure concentration is 55-65 ℃, and the concentration solid content is 4-12 wt%.
The invention provides a preparation method of lotus leaf aqueous extract with functions of controlling sugar and reducing lipid, which is prepared by the preparation method, wherein the total sugar content of the lotus leaf aqueous extract with functions of controlling sugar and reducing lipid is 42.06-43.93%, and the total sugar recovery rate is not lower than 30%; the total flavone content is 6.15-6.36%, wherein the total flavone recovery rate is not less than 50%.
The invention also provides application of the lotus leaf extract with the function of regulating the absorption of glycolipid in preparation of foods, health products or medicines with the function of reducing blood glucose and controlling the lipid.
The invention has the following advantages and effects:
(1) The invention adopts liquid nitrogen crushing, double enzyme cascade enzymolysis, colloid mill treatment, high-pressure homogenization and high-temperature extraction, greatly promotes the dissolution of active ingredients, and can rapidly prepare the lotus leaf extract with high active ingredient content and good activity.
(2) The whole preparation process flow meets the food-grade requirement and has industrial application potential.
(3) The lotus leaf extract obtained by the invention has the total sugar content of more than 40 percent and the total flavone content of more than 6 percent, the preparation method is simple and quick, the extract has the alpha-glucosidase inhibiting activity, the pancrelipase inhibiting activity, the cholesterol micelle dissolution inhibiting activity and the cholate adsorption capacity, can effectively inhibit excessive absorption of sugar and lipid in the gastrointestinal tract, can improve the absorption of the glycolipid by multiple ways and multiple targets, and can be applied to the fields of common foods, health care products and the like.
Drawings
FIG. 1 is a bar graph of polysaccharide extraction, polysaccharide recovery and total sugar content in the extract powder for lotus leaf alcoholic extract RF1 of comparative example 1, lotus leaf return aqueous extracts RF2 and RF3 of comparative examples 2-3, lotus leaf extracts LLE1, LLE2 of comparative examples 4-5, and lotus leaf extracts LEA, LEB and LEC of examples 1-3.
FIG. 2 is a bar graph of total flavone extraction, total flavone recovery and total flavone content in the extract powder for lotus leaf alcoholic extract RF1 of comparative example 1, lotus leaf return aqueous extracts RF2 and RF3 of comparative examples 2-3, lotus leaf extracts LLE1, LLE2 of comparative examples 4-5, and lotus leaf extracts LEA, LEB and LEC of examples 1-3.
FIG. 3 is a graph showing the lotus leaf alcoholic extract RF1 of comparative example 1, lotus leaf return water extracts RF2 and RF3 of comparative examples 2-3, lotus leaf extracts LLE1, LLE2 of comparative examples 4-5, and the alpha-glucosidase inhibitory activity IC of lotus leaf extracts LEA, LEB and LEC of examples 1-3 50 A histogram of values.
FIG. 4 is a graph showing the pancreatic lipase inhibition activity IC of lotus leaf alcoholic extract RF1 of comparative example 1, lotus leaf return water extracts RF2 and RF3 of comparative examples 2-3, lotus leaf extracts LLE1, LLE2 of comparative examples 4-5, and lotus leaf extracts LEA, LEB and LEC of examples 1-3 50 A histogram of values.
FIG. 5 is a bar graph of cholate adsorption capacity and cholesterol micelle solubility inhibition capacity of lotus leaf alcoholic extract RF1 of comparative example 1, lotus leaf return aqueous extracts RF2 and RF3 of comparative examples 2-3, lotus leaf extracts LLE1, LLE2 of comparative examples 4-5, and lotus leaf extracts LEA, LEB, and LEC of examples 1-3 at a concentration of 20 mg/mL.
Detailed description of the preferred embodiments
For a better understanding of the present invention, the present invention will be further described with reference to specific examples, but the embodiments of the present invention are not limited thereto.
1. Method for determining alpha-glucosidase inhibitory activity
Taking 30 mu L of sample solution, adding 30 mu L of 0.2U/mL of alpha-glucosidase solution, incubating for 10min at 37 ℃, then adding 30 mu L p-NPG solution (10 mmol/L) for reaction for 15min, and then adding 100 mu L of Na 2 CO 3 (0.1 mol/L) the reaction was terminated, and the absorbance was measured at 405 min. The result was obtained by calculating the pancreatic lipase inhibition ratio according to the following formula, and finally calculating IC 50 Values.
2. Pancreatic lipase inhibition activity determination method
664mg of bile salts were dissolved in 100mL of simulated intestinal fluid (containing 6.8mmol/L KCl, 0.8mmol/L KH) 2 PO 4 、85mmol/L NaHCO 3 、38.4mmol/L NaCl、0.33mmol/L MgCl 2 8.4mmol/L HCl, water as solvent), 10. Mu. L0.3M CaCl was added 2 (H 2 O) 2 The solution was prepared as Buffer solution, and pancreatic lipase and p-NPB were dissolved in the Buffer solution. 50. Mu.L of sample solution was taken, 50. Mu.L of pancreatic lipase solution (2 mg/mL) was added thereto, incubated at 37℃for 10 minutes, then 2mg/mL of solution (30. Mu. L p-NPB) was added thereto for 20 minutes, and the absorbance was measured at 405 minutes. The result was obtained by calculating the pancreatic lipase inhibition ratio according to the following formula, and finally calculating IC 50 Values.
3. Method for measuring adsorption capacity of cholate
(1) Simulated oral liquid: comprises 15.1mmol/L KCl and 3.7mmol/L KH 2 PO 4 、13.6mmol/L NaHCO 3 、0.15mmol/L MgCl 2 、0.06mmol/L(NH 4 ) 2 CO 3 1.1mmol/L HCl and 1.5mmol/L CaCl 2 The solvents were all water.
(2) Simulating gastric juice: comprises 6.9mmol/L KCl and 0.9mmol/L KH 2 PO 4 、25mmol/L NaHCO 3 、47.2mmol/L NaCl、0.12mmol/L MgCl 2 、0.5mmol/L(NH 4 ) 2 CO 3 15.6mmol/L HCl and 0.15mmol/L CaCl 2 The solvents were all water.
(3) Simulation of intestinal juice: comprises 6.8mmol/L KCl and 0.8mmol/L KH 2 PO 4 、85mmol/L NaHCO 3 、38.4mmol/L NaCl、0.33mmol/L MgCl 2 8.4mmol/L HCl and 0.6mmol/L CaCl 2 The solvents were all water.
The digestion process of the samples in the info GEST model is as follows:
(1) Oral digestion: sampling, dissolving in 2mL simulated oral liquid, adjusting pH to 7.0 with deionized water and 5mol/L NaOH aqueous solution to make the final volume of oral digest be 2.5mL, and oscillating at 37deg.C for 2min.
(2) Gastric digestion: 2mL simulated gastric fluid and 7.50. Mu.L 0.3mmol/L CaCl were added to the oral digest 2 The aqueous solution was dissolved in 40mg of pepsin (final concentration 2000U/mL) with 0.17mL of simulated gastric fluid, added to the digest, and the pH was adjusted to 3.0 with deionized water and 5mol/L aqueous HCl to give a final volume of 5mL of gastric digest, and the gastric digest was shaken at constant temperature of 37℃for 2h.
(3) Small intestine digestion: 4mL simulated intestinal fluid and 10. Mu.L 0.3mmol/L CaCl were added to the stomach digest 2 In the aqueous solution, 5mg of pancreatin (final concentration 100U/mL) was dissolved in 1.25mL of simulated intestinal fluid, and then added to the digest, 66.4mg of pig bile salt was added to 0.75mL of simulated intestinal fluid, and after dissolution, the digest was added to the pH value was adjusted to 7.0 using deionized water and 5mol/L NaOH aqueous solution, so that the final volume of the digest was 10mL, and the mixture was subjected to constant temperature shaking at 37℃for 2 hours.
Preparation of cholesterol micelle solution: 3.125g of pig bile salt, 386.65mg of cholesterol and 706.15mg of oleic acid are dissolved by using 150mL of simulated intestinal fluid, ultrasonic emulsification is carried out for 2 hours until the cholesterol is completely dissolved, deionized water is used for fixing the volume to 200mL, the gel bundles are filled in a closed container, and the gel bundles are stored at 37 ℃ for 24 hours for use.
A sample of 0.2g (20 mg/mL concentration in the final 10mL digested volume) was taken as described aboveThe oral digestion and gastric digestion are simulated. Dissolving 5mg of pancreatin in 4mL of cholesterol micelle solution before digestion of simulated small intestine, and adding the mixed solution into simulated intestinal juice after uniform mixing; then 0.01mL of 0.3mmol/L CaCl is added 2 An aqueous solution; adjusting pH to 7.0 with deionized water and 5mol/L NaOH aqueous solution to give digestion volume of 10mL, oscillating at 37deg.C for 2h, ice-bathing for 15min, centrifuging at 8000g at 4deg.C for 15min, collecting supernatant, and measuring cholate content. The blank was treated in the same way as the sample group without the addition of sample. The cholate adsorption (%) was calculated according to the following formula:
wherein, A blank is the concentration of cholate in blank control group, and the unit is mmol/L; the A sample is the concentration of cholate in the sample group, and the unit is mmol/L.
4. Method for measuring cholesterol micelle solubility inhibition activity
A0.2 g sample (20 mg/mL concentration in the final 10mL digestion volume) was taken and simulated oral digestion and simulated gastric digestion were performed as described above. Dissolving 5mg of pancreatin in 4mL of cholesterol micelle solution before digestion of simulated small intestine, and adding the mixed solution into simulated intestinal juice after uniform mixing; then 0.01mL of 0.3mmol/L CaCl is added 2 An aqueous solution; adjusting pH to 7.0 with deionized water and 5mol/L NaOH aqueous solution to obtain a final volume of the digestate of 10mL, oscillating at 37deg.C for 2h, ice-bathing for 15min, centrifuging at 8000g and 4deg.C for 15min, collecting the digested supernatant, adding anhydrous methanol at a ratio of 1:1 (v/v), repeatedly extracting twice, collecting the supernatant, and measuring cholesterol content by OPA method. The blank was treated in the same way as the sample group without the addition of sample. Cholesterol solubility inhibition (%) was calculated according to the following formula:
wherein, C blank is cholesterol concentration in μg/mL in blank control group; c the concentration of cholesterol in the sample group in μg/mL.
The lotus leaf extracts obtained in examples and comparative examples were measured for alpha-glucosidase inhibitory activity, pancrelipase inhibitory activity, cholate adsorption capacity and cholesterol micelle solubility inhibitory capacity using the methods described above.
Example 1
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
(1) Crushing by liquid nitrogen: 1000g of dry lotus leaf is put into a liquid nitrogen crushing device at the temperature of-150 ℃ and crushed into dry powder to obtain lotus leaf dry powder, and 30g of dry powder is L1A.
(2) Double enzyme cascade enzymolysis: uniformly mixing the lotus leaf dry powder L1A obtained in the step (1) with deionized water according to the proportion of 1:8g/mL, adding hydrochloric acid to adjust the pH value to 4.0, fully uniformly mixing, adding cellulase (NoveXin (China) biotechnology company) with the mass of 0.75% (w/w, g/g) of the lotus leaf dry powder L1A obtained in the step (1), and stirring at the constant temperature of 50 ℃ at the rotating speed of 120r/min for enzymolysis for 0.5h to obtain a suspension S1A. And then regulating the pH value of the obtained suspension S1A to 6.5, fully and uniformly mixing, adding pancreatin (Nanning Pang Bo biological engineering Co., ltd.) with the mass of 0.75% (w/w, g/g) of the dry lotus leaf powder L1A obtained in the step (1), and stirring at a constant temperature of 50 ℃ at a rotating speed of 120r/min for enzymolysis for 2 hours to obtain the suspension S2A.
(3) Colloid mill: and (3) carrying out colloid mill treatment on the suspension S2A obtained in the step (2), and grinding for 1 time and 4 minutes to obtain a suspension S3A.
(4) Homogenizing under high pressure: homogenizing the suspension S3A obtained in the step (3) for 1 time under the pressure of 10MPa to obtain a suspension S4A.
(5) High-temperature extraction: extracting suspension S4A at 95deg.C for 15min to obtain suspension S5A.
(6) Centrifuging and concentrating: centrifuging suspension S5A at 4deg.C at 6000g for 15min, collecting supernatant, and concentrating under reduced pressure at 55deg.C until the solid content in the final concentrate is 4wt% to obtain extract E1A.
(7) And (3) freeze drying: and (3) freeze-drying the extracting solution E1A to obtain a lotus leaf extract LEA.
Example 2
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
(1) Crushing by liquid nitrogen: 1000g of dry lotus leaf is put into a liquid nitrogen crushing device at the temperature of minus 135 ℃ and crushed into dry powder to obtain lotus leaf dry powder, and 30g of dry powder is L1B-1.
(2) Enzymatic hydrolysis of cellulase: uniformly mixing the lotus leaf dry powder L1B-1 obtained in the step (1) with deionized water according to the proportion of 1:10g/mL, adding hydrochloric acid to adjust the pH to 4.5, fully uniformly mixing, adding cellulase, wherein the mass of the cellulase is 1% (w/w, g/g) of the mass of the lotus leaf dry powder L1B-1 obtained in the step (1), and stirring at a constant temperature of 55 ℃ at a rotating speed of 150r/min for enzymolysis for 1h to obtain a suspension S1B. And then regulating the pH value of the obtained suspension S1B to 7.0, fully and uniformly mixing, adding pancreatin, wherein the mass of pancreatin is 1% (w/w, g/g) of the mass of the lotus leaf dry powder L1B-1 obtained in the step (1), and stirring and hydrolyzing at a constant temperature of 55 ℃ at a rotating speed of 150r/min for 4 hours to obtain the suspension S2B.
(3) Colloid mill: and (3) carrying out colloid mill treatment on the suspension S2B obtained in the step (2), and grinding for 2 times, wherein the grinding time is 7min, so as to obtain the suspension S3B.
(4) Homogenizing under high pressure: homogenizing the suspension S3B obtained in the step (3) for 2 times under the pressure of 20MPa to obtain a suspension S4B.
(5) High-temperature extraction: extracting suspension S4B at 105deg.C for 30min to obtain suspension S5B.
(6) Centrifuging and concentrating: after the suspension S5B was centrifuged at 7000g for 20min at 6℃at high speed, the supernatant was concentrated under reduced pressure at 60℃until the solid content in the final concentrate was 8wt%, giving extract E1B.
(7) And (3) freeze drying: and (3) freeze-drying the extracting solution E1B to obtain a lotus leaf extract LEB.
Example 3
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
(1) Crushing by liquid nitrogen: 1000g of dry lotus leaf is put into a liquid nitrogen crushing device at the temperature of minus 120 ℃ and crushed into dry powder to obtain lotus leaf dry powder, and 30g of dry powder is taken as L1C.
(2) Enzymatic hydrolysis of cellulase: uniformly mixing the lotus leaf dry powder L1C obtained in the step (1) with deionized water according to the proportion of 1:12g/mL, adding hydrochloric acid to adjust the pH value to 5.0, fully uniformly mixing, adding cellulase with the mass of 1.25% (w/w, g/g) of the lotus leaf dry powder L1C obtained in the step (1), and stirring at a constant temperature at a speed of 180r/min for enzymolysis for 1.5h at 60 ℃ to obtain a suspension S1C. And then regulating the pH value of the obtained suspension S1C to 7.5, fully and uniformly mixing, adding pancreatin, wherein the mass of pancreatin is 1.25% (w/w, g/g) of the mass of the lotus leaf dry powder L1C obtained in the step (1), and stirring at a constant temperature of 60 ℃ at a rotating speed of 150r/min for enzymolysis for 6 hours to obtain the suspension S2C.
(3) Colloid mill: and (3) carrying out colloid mill treatment on the suspension S2C obtained in the step (2), and grinding for 3 times, wherein the grinding time is 10min, so as to obtain the suspension S3C.
(4) Homogenizing under high pressure: homogenizing the suspension S3C obtained in the step (3) for 3 times under the pressure of 30MPa to obtain a suspension S4C.
(5) High-temperature extraction: extracting suspension S4C at 115 deg.C for 45min to obtain suspension S5C.
(6) Centrifuging and concentrating: centrifuging suspension S5C at 8deg.C at 8000g for 25min, collecting supernatant, and concentrating under reduced pressure at 65deg.C until the solid content in the final concentrate is 12wt% to obtain extract E1C.
(7) And (3) freeze drying: and (3) freeze-drying the extracting solution E1C to obtain a lotus leaf extract LEC.
Comparative example 1
The preparation method of the lotus leaf alcohol extract with the functions of controlling sugar and reducing lipid comprises the following steps:
pulverizing 30g of dry lotus leaf, and sieving with 40 mesh sieve to obtain common pulverized dry powder LL1 of lotus leaf. Mixing LL1 and 75% ethanol water at a ratio of 1:10g/mL, reflux extracting for 1h, centrifuging at 6 deg.C and 6000g for 20min, collecting supernatant, concentrating under reduced pressure at 60 deg.C until solid content in the final concentrate is 8wt%, and lyophilizing to obtain lotus leaf ethanol extract RF1.
Comparative example 2
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
pulverizing 30g of dry lotus leaf, and sieving with 40 mesh sieve to obtain common pulverized dry powder LL1 of lotus leaf. LL1 and deionized water are uniformly mixed in a ratio of 1:10g/mL, reflux extraction is carried out for 1h, after centrifugation is carried out for 20min at a high speed of 6000g at a temperature of 6 ℃, supernatant fluid is taken and concentrated under reduced pressure at a temperature of 60 ℃ until the solid content in the final concentrated solution is 8wt%, and then the lotus leaf aqueous extract RF2 is obtained through freeze drying treatment.
Comparative example 3
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
1000g of dry lotus leaf is put into a liquid nitrogen crushing device at the temperature of minus 135 ℃ and crushed into dry powder to obtain lotus leaf liquid nitrogen crushed dry powder, and 30g of dry powder is L1B-2. The lotus leaf dry powder L1B-2 and deionized water are uniformly mixed in a ratio of 1:10g/mL, reflux extraction is carried out for 1h, high-speed centrifugation is carried out for 20min at a rotating speed of 6000g at 6 ℃, supernatant fluid is taken, reduced pressure concentration is carried out at 60 ℃ until the solid content in the final concentrated solution is 8wt%, and freeze drying treatment is carried out, thus obtaining the lotus leaf aqueous extract RF3.
Comparative example 4
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
1000g of dry lotus leaf is put into a liquid nitrogen crushing device at the temperature of minus 135 ℃ and crushed into dry powder to obtain lotus leaf liquid nitrogen crushed dry powder, and 30g of dry powder is L1B-3. Mixing lotus leaf dry powder L1B-3 with deionized water at a ratio of 1:10g/mL, adding hydrochloric acid to adjust pH to 4.5, fully mixing, adding cellulase, stirring at a constant temperature of 55 ℃ at a rotating speed of 150r/min for enzymolysis for 1h, adjusting pH to 7.0, adding pancreatin, stirring at a constant temperature of 55 ℃ at a rotating speed of 150r/min for enzymolysis for 4h, heating at a constant temperature of 55 ℃ at a rotating speed of 150r/min for extraction for 30min, centrifuging at a high speed of 7000g at a rotating speed of 6 ℃ for 20min, concentrating the supernatant at a reduced pressure at a rotating speed of 60 ℃ until the solid content in the final concentrated solution is 8wt%, and performing freeze drying treatment to obtain lotus leaf extract LLE1.
Comparative example 5
The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid specifically comprises the following steps:
1000g of dry lotus leaf is put into a liquid nitrogen crushing device at the temperature of minus 135 ℃ and crushed into dry powder to obtain lotus leaf liquid nitrogen crushed dry powder, and 30g of dry powder is L1B-4. Mixing lotus leaf dry powder L1B-4 with deionized water at a ratio of 1:10g/mL, adding hydrochloric acid to adjust pH to 4.5, adding cellulase after fully mixing, wherein the mass of the cellulase is 1% (w/w, g/g) of the mass of lotus leaf dry powder L1B-4, stirring and hydrolyzing at a constant temperature of 150r/min at 55 ℃ for 1h, adjusting pH to 7.0, adding pancreatin, stirring and hydrolyzing at a constant temperature of 150r/min at 55 ℃ for 4h, grinding for 2 times by a colloid mill, homogenizing at a high pressure for 1 time under a pressure of 20MPa after grinding for 7min, heating and extracting at 95 ℃ for 30min, centrifuging at a high speed of 7000g at a constant temperature of 6 ℃ for 20min, concentrating the supernatant at a reduced pressure at 60 ℃ until the solid content in the final concentrated solution is 8wt%, and performing freeze drying treatment to obtain lotus leaf extract LLE2.
Analysis of results
1. Analysis of total sugar content and extraction efficiency of lotus leaf extract
As can be seen from fig. 1, the extraction rate of the lotus leaf dry powder fully crushed by liquid nitrogen in comparative example 3 to total sugar is 3.54±0.08% by a classical reflux extraction method, which is 2.87 times higher than that of the lotus leaf dry powder prepared by reflux extraction after common crushing (comparative example 2), which indicates that the lotus leaf dry powder after liquid nitrogen crushing treatment is more beneficial to the dissolution of substances. Comparative examples 4-5 are extracts prepared by double enzyme cascade and double enzyme cascade-high pressure homogenization treatment technologies, and the total sugar extraction rates are 5.30+/-0.40% and 6.09+/-0.03%, respectively, which shows that under the actions of double enzyme cascade enzymolysis, colloid mill and high pressure homogenization, the effect of the traditional hot water reflux extraction technology can be exceeded under the medium temperature condition, and the total sugar extraction rate (p < 0.05) is obviously improved. The total sugar content of the prepared lotus leaf extract LEA, LEB, LEC is 42.06-43.93% by using the double enzyme cascade-high pressure homogenization-high temperature extraction technology adopted in the embodiments 1-3, wherein the total sugar extraction rate can reach 7.56% at the highest, and the recovery rate reaches 35.91%, which indicates that the effects of the embodiments cannot be achieved if double enzyme cascade enzymolysis, colloid mill, high pressure homogenization or high temperature extraction are not adopted. The preparation method can effectively improve the total sugar content in the lotus leaf extract, and has the effect of promoting the extraction efficiency of the polysaccharide component of the lotus leaf extract.
2. Analysis of total flavone content and extraction efficiency of lotus leaf extract
As can be seen from fig. 2, the extraction rate of the lotus leaf dry powder fully crushed by liquid nitrogen in comparative example 3 for total flavonoids by a classical reflux extraction method is 0.95±0.01%, which is improved by 24.2% compared with the extraction rate of total flavonoids by reflux extraction after common crushing, which indicates that the lotus leaf dry powder after liquid nitrogen crushing treatment is more favorable for dissolving out flavonoids. The extraction rate of the total flavone of the extract prepared by the double enzyme cascade and the double enzyme cascade coupling high-pressure homogenization treatment technology is 1.09+/-0.01% and 1.18+/-0.14%, respectively, which shows that the extraction rate of the total flavone can be effectively improved by an enzyme method and high-pressure homogenization (p is less than 0.05). However, by using the double enzyme cascade-high pressure homogenization-high temperature extraction technology adopted in examples 1-3, the total flavone content of the prepared lotus leaf extract LEA, LEB, LEC is improved to 6.15-6.36%, wherein the total flavone extraction rate can reach 1.46% at the highest, and the recovery rate reaches 55.85%, which indicates that the effects of the examples cannot be achieved if double enzyme cascade enzymolysis, colloid mill, high pressure homogenization or high temperature extraction are not achieved. The preparation method can effectively improve the total flavone content in the lotus leaf extract, and has promotion effect on the extraction efficiency of the flavone component of the lotus leaf extract.
3. Alpha-glucosidase inhibitory Activity of lotus leaf extract
As can be seen from fig. 3, the liquid nitrogen pulverization, the double enzyme cascade enzymolysis, the high pressure homogenization and the high temperature treatment have the effect of improving the alpha-glucosidase inhibition activity: alpha-glucosidase IC of lotus leaf dry powder water extract RF3 crushed by liquid nitrogen compared with common crushed lotus leaf dry powder water extract RF2 50 The value was reduced by 33%. The lotus leaf aqueous extract LLE1 obtained after cellulose-pancreatin double enzyme enzymolysis wall breaking treatment, and the alpha-glucosidase IC of the lotus leaf aqueous extract LLE2 obtained after double enzyme cascade enzymolysis-high pressure homogenization treatment 50 The values decrease stepwise, indicating a gradual increase in their inhibitory activity. In the embodiment, compared with the aqueous extracts RF2, RF3, LLE1 and LLE2, the alpha-glucosidase inhibitory activities of the lotus leaf extract LEA, LEB, LEC obtained by double enzyme cascade enzymolysis, high-pressure homogenization and high-temperature extraction are obviously improved, and the alpha-glucosidase IC is remarkably improved 50 The value is 0.11-0.14 mg/mL, and the strongest strength cannot be achieved without any processing step in the water extraction processThis also correlates with a greater abundance of active polysaccharide and flavonoid compounds in the examples. The lotus leaf alcohol extract (RF 1) has a stronger inhibitory activity than the α -glucosidase inhibitory activity of examples 1-3, which may be related to the higher content of flavonoids in the alcohol extract. The preparation method can effectively improve the alpha-glucosidase inhibitory activity in the lotus leaf extract, and has the effect of obviously inhibiting the excessive absorption of sugar by gastrointestinal tracts.
4. Pancreatic lipase inhibitory Activity of lotus leaf extract
As shown in FIG. 4, the lotus leaf extract samples all have better pancreatic lipase inhibition activity. Wherein, the liquid nitrogen is crushed, the enzymolysis is carried out in double enzyme cascade, the homogenization is carried out under high pressure, and the high-temperature treatment has an improvement effect on the pancreatic lipase inhibition activity: liquid nitrogen pulverized lotus leaf dry powder aqueous extract RF3 is compared with pancreatic lipase IC of common pulverized lotus leaf dry powder aqueous extract RF2 50 The value was reduced by 24%. Pancrelipase IC of lotus leaf aqueous extract LLE1 obtained by cellulose-pancreatin double enzyme enzymolysis wall breaking treatment 50 Pancreatic lipase IC of lotus leaf water extract LLE2 subjected to double enzyme cascade enzymolysis-high pressure homogenization treatment with value of 3.70mg/mL 50 The value was 3.84mg/mL, with no significant difference. However, in the examples, the pancreatic lipase IC of lotus leaf extract LEA, LEB, LEC obtained by double enzyme cascade enzymolysis, high-pressure homogenization and high-temperature extraction is compared with that of water extract LLE2 50 The value is reduced by about 45.5%, under the combined action of liquid nitrogen crushing, double enzyme cascade enzymolysis, high-pressure homogenization and high-temperature extraction, the pancreatic lipase inhibition activity can be obviously improved, and the lipase inhibition level of the embodiment can be equal to the inhibition activity of the ethanol extract RF1 (p>0.05 The preparation method can effectively improve the pancreatic lipase inhibition activity in the lotus leaf extract, and has the effect of obviously inhibiting excessive absorption of lipid by gastrointestinal tracts.
5. Analysis of cholate adsorption capacity and cholesterol micelle dissolution inhibition capacity in lotus leaf extract
As can be seen from fig. 5, all samples have a certain adsorption effect on gastrointestinal cholate based on simulated digestion process at the concentration of 20mg/mL, have an inhibition effect on the solubility of cholesterol in gastrointestinal micelles, and have consistent trend of change of the two lipid-lowering activities. Compared with the lotus leaf water extract RF2, the lotus leaf water extract LLE2 in the comparative example has 46.6 percent and 45.5 percent of absorption capacity and cholesterol micelle dissolution inhibition capacity respectively increased, which shows that the absorption inhibition effect of the lotus leaf water extract on lipid is improved to different degrees along with the crushing of liquid nitrogen, and the double enzyme cascade enzymolysis and the high pressure homogenization technology treatment are carried out. In the embodiment, the lotus leaf extract LEA, LEB, LEC prepared by liquid nitrogen crushing, double enzyme cascade enzymolysis, high-pressure homogenization and high-temperature extraction has the advantages that the cholate adsorption rate is 58.29-61.49% and the cholesterol dissolution inhibition rate is 36.41-37.96% under the concentration of 20mg/mL, and the lipid absorption inhibition effect is better than that of the comparative example 2-5, so that the strongest lipid absorption inhibition activity cannot be achieved in the absence of any processing step. In addition, the cholate adsorption capacity and cholesterol micelle dissolution inhibition capacity of the alcohol extract RF1 in the comparative example are the weakest, which shows that the activity of the water extract is obviously superior to that of the alcohol extract in the lipid-lowering evaluation system based on the simulated digestive system, and the polysaccharide in the water extract is supposed to play a corresponding role. The lotus leaf extract obtained by the preparation method has the effect of obviously inhibiting excessive absorption of lipid by gastrointestinal tracts.
The common lotus leaf crushed dry powder alcohol extract RF1, the common lotus leaf crushed dry powder water extract RF2, the liquid nitrogen crushed dry powder water extract RF3, the liquid nitrogen crushed-double enzyme cascade enzymatic hydrolysis extract LLE1 and the liquid nitrogen crushed-double enzyme cascade enzymatic hydrolysis-high pressure homogeneous extract LLE2 in the comparative example, the total sugar and total flavone content in the sample are gradually improved, and the metabolic activity of the glycolipid is also gradually improved. The lotus leaf extract LEA, LEB, LEC prepared by nitrogen crushing, double enzyme cascade enzymolysis, high-pressure homogenization and high-temperature extraction in the embodiment has the highest total sugar content and total flavone content, and the activity of reducing blood sugar and controlling lipid can also reach the highest; specifically, the lotus leaf extract has the total sugar content of more than 40%, the total flavone content of more than 6%, good glycolipid absorption regulation effect, alpha-glucosidase inhibition IC50 value of less than 0.2mg/mL, pancreatic lipase inhibition IC50 value of less than 2mg/mL, and under the concentration of 20mg/mL, cholate adsorption rate of more than 55% and cholesterol micelle dissolution inhibition rate of more than 35%. The lotus leaf dry powder belongs to a lotus leaf primary product, and compared with the common crushing mode, the liquid nitrogen crushing can enable active substances such as polysaccharide and the like to be extracted more easily, and shows a certain activity of adjusting glycolipid absorption, but after further extraction processing such as enzymolysis, homogenization and the like, the content of the active substances in the extract is higher, and the extract shows stronger activity of controlling glucose and reducing lipid. The double-enzyme cascade enzymolysis in the embodiment can extract total sugar and total flavone in raw materials, and the colloid mill treatment, high-pressure homogenization and high-temperature extraction can promote component dissolution with high efficiency, so that the extraction is more sufficient, the extraction process condition is mild, the operation is easy, and the time consumption is short. The lotus leaf extract finally obtained in the embodiment has the function of regulating the absorption activity of glycolipid and has the potential of being used as a food base material to be processed into foods and health care products.
The invention provides a preparation method of lotus leaf aqueous extract with functions of controlling sugar and reducing lipid. According to the preparation method, liquid nitrogen is adopted to crush the powder to reduce the particle size, water-soluble polysaccharide and flavonoid compounds are extracted through double-enzyme cascade enzymolysis wall breaking, a colloid mill, high-pressure homogenization and high-temperature extraction are adopted to further dissolve out active ingredients, and the target product has dual activities of regulating and controlling sugar and lipid absorption, so that required processing equipment is simple and easy to operate, meets the food processing specification, and has the potential of industrial popularization and application. The invention has obvious technical advantages and advancement.
The above examples are only preferred embodiments of the present invention, and are intended to be illustrative of the present invention, not limiting, and variations, substitutions, modifications, etc. which would be apparent to those skilled in the art without departing from the spirit of the present invention are to be construed as being within the scope of the present invention.
Claims (7)
1. The preparation method of the lotus leaf water extract with the functions of controlling sugar and reducing lipid is characterized by comprising the following steps:
(1) Crushing by liquid nitrogen: crushing dry lotus leaves into dry powder by a liquid nitrogen crushing device to obtain lotus leaf dry powder;
(2) Double enzyme cascade enzymolysis: uniformly mixing the lotus leaf dry powder obtained in the step (1) with water, regulating the pH value to 4.0-5.0, adding cellulase after fully uniformly mixing, and stirring at constant temperature for enzymolysis to obtain a suspension 1; regulating the pH value of the obtained suspension 1 to 6.5-7.5, adding pancreatin, stirring at constant temperature, and performing enzymolysis to obtain a suspension 2;
(3) Colloid mill treatment: carrying out colloid mill treatment on the suspension 2 to obtain a suspension 3;
(4) High-pressure homogenization treatment: homogenizing the suspension 3 to obtain a suspension 4;
(5) High-temperature heating and extracting: heating and extracting the suspension 4 to obtain a suspension 5;
(6) Centrifuging and concentrating: centrifuging the suspension 5, and concentrating the supernatant under reduced pressure to obtain extract 1;
(7) And (3) freeze drying: freeze-drying the extracting solution 1 to obtain a lotus leaf extract;
in the step (1), the condition of crushing the liquid nitrogen is that crushing is carried out at the temperature of-150 to-120 ℃;
in the step (2), the feed liquid ratio of the lotus leaf dry powder to the water is 1:8-1:12 g/mL;
in the step (2), the mass of the cellulase is 0.75-1.25% w/w and g/g of the lotus leaf dry powder obtained in the step (1);
in the step (2), the enzymolysis temperature is 50-60 ℃ and the enzymolysis time is 0.5-1.5 h; the mass of pancreatin is 0.75-1.25% w/w, g/g of the dry lotus leaf powder obtained in the step (1), the stirring speed is 120-180 r/min, the enzymolysis temperature is 50-60 ℃, and the enzymolysis time is 2-6 h;
in the step (5), the high-temperature heating extraction temperature is 95-115 ℃ and the extraction time is 15-45 min.
2. The method for preparing the lotus leaf aqueous extract with the functions of controlling sugar and reducing lipid according to claim 1, wherein in the step (2), the constant-temperature stirring speed is 120-180 r/min.
3. The method for preparing the lotus leaf aqueous extract with the functions of controlling sugar and reducing fat according to claim 1, wherein in the step (3), the grinding time of the colloid mill is 1-3 times, and the grinding time of each time is 4-10 min.
4. The method for preparing lotus leaf aqueous extract with glucose-control and lipid-lowering functions according to claim 1, wherein in the step (4), the high-pressure homogenization is performed at a pressure of 10-30 MPa, and the number of homogenization times is 1-3.
5. The method for preparing lotus leaf aqueous extract with glucose-control and lipid-lowering functions as claimed in claim 1, wherein in the step (5), the temperature of centrifugation is 4-8 ℃, and the centrifugal force of centrifugation is 6000-8000gCentrifuging for 15-25 min; the temperature of the reduced pressure concentration is 55-65 ℃, and the concentration solid content is 4-wt% -12 wt%.
6. The preparation method of any one of claims 1-5 is characterized in that the total sugar content of the lotus leaf water extract with the functions of controlling sugar and reducing lipid is 42.06-43.93%, and the total sugar recovery rate is not lower than 30%; the total flavone content is 6.15-6.36%, wherein the total flavone recovery rate is not less than 50%.
7. The use of the lotus leaf aqueous extract with glucose-control and lipid-lowering functions in the preparation of a glucose-lowering and lipid-lowering medicament according to claim 6.
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Non-Patent Citations (3)
| Title |
|---|
| Lotus Leaf Aqueous Extract Reduces Visceral Fat Mass and Ameliorates Insulin Resistance in HFD-Induced Obese Rats by Regulating PPARγ2 Expression;Kemin Yan等;Front. Pharmacol.;第 8-2017卷;1-10 * |
| 功能食品的超微粉碎技术;张霞;李琳;李冰;;食品工业科技;31(第11期);375-378 * |
| 动态高压微射流技术对荷叶多糖提取、结构及抗氧化活性影响的研究;寇玉;中国优秀硕士学位论文全文数据库医药卫生科技辑(第2014/03期);E057-21 * |
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