A preparation method of functional mulberry leaf oligosaccharide, prepared mulberry leaf oligosaccharide and application thereof

Abstract

The present application relates to the technical field of food science, and specifically discloses a method for preparing functional mulberry leaf oligosaccharides, prepared mulberry leaf oligosaccharides and applications. The method for preparing functional mulberry leaf oligosaccharides comprises the following steps: (1) drying, crushing and sieving the mulberry leaves; then dispersing them in an ethanol aqueous solution, ultrasonically extracting and filtering to obtain a filter residue and an extract solution one; (2) dispersing the filter residue in water, adding a composite enzyme and sodium hydrogen phosphate-sodium dihydrogen phosphate buffer for enzymolysis, filtering and obtaining an extract solution two; (3) mixing the extract solution one and the extract solution two for centrifugation to obtain a supernatant, adding the supernatant to an ionic liquid to obtain a mixed solution, fractionating and extracting, collecting the extract solution, evaporating the extract solution, washing with water and drying to obtain mulberry leaf oligosaccharides. The mulberry leaf oligosaccharides prepared by the preparation method of the present application have a high purity, which will subsequently help the application of mulberry leaf oligosaccharides.

Description

Technical Field

[0001] The present application relates to the technical field of food science, and more specifically, to a method for preparing functional mulberry leaf oligosaccharides, the prepared mulberry leaf oligosaccharides, and applications thereof. Background Art

[0002] Functional oligosaccharides are low-polymer sugars with branches or straight chains formed by dehydrating 2-7 monosaccharide molecules and connecting them through α-, β- and other glycosidic bonds. They have certain characteristics of sugars such as sweetness, viscosity and water solubility. They are often used as functional sweeteners and have a sweetness of 0.3-0.6 times that of sucrose.

[0003] Mulberry leaves are rich in nutrients such as protein, vitamins and minerals, as well as bioactive ingredients such as mulberry leaf polysaccharides, mulberry leaf flavonoids and alkaloids. They have multiple physiological functions such as lowering blood sugar, anti-inflammatory and antiviral. Among them, mulberry leaf polysaccharides have biological activities such as regulating glycolipid metabolism. However, due to the large molecular weight and viscosity of mulberry leaf polysaccharides, they have poor solubility in the body and low bioavailability. In addition, mulberry leaf polysaccharides contain a large number of non-α-1,4 glycosidic bonds, which cannot be completely degraded by human digestive juices, thus limiting the application of mulberry leaf polysaccharides.

[0004] In the prior art, oligosaccharides obtained by degrading polysaccharides, such as oligofructose and konjac oligosaccharides, have multiple functions such as regulating the structure of intestinal flora, lowering blood sugar, lowering blood lipids, and improving the gastrointestinal tract. Therefore, converting mulberry leaf polysaccharides into oligosaccharides expands the application of mulberry leaf polysaccharides. At present, the extraction of mulberry leaf oligosaccharides is usually based on traditional chemical methods such as acid hydrolysis and oxidative hydrolysis, which have the disadvantages of low purity and high cost. Summary of the invention

[0005] In order to improve the use of traditional chemical methods such as acid hydrolysis and oxidative hydrolysis, which have the problems of low purity and high cost, the present application provides a method for preparing functional mulberry leaf oligosaccharides, the prepared mulberry leaf oligosaccharides and their applications.

[0006] In the first aspect, the present application provides a method for preparing functional mulberry leaf oligosaccharides, using the following technical scheme: A method for preparing functional mulberry leaf oligosaccharides, comprising the following steps:

[0007] (1) Wash, dry, crush, sieve, and then disperse the mulberry leaves in an ethanol aqueous solution, perform ultrasonic extraction at a power of 400-500 W for 1-2 h, and filter to obtain a filter residue and an extract;

[0008] (2) dispersing the filter residue obtained in step (1) in water, adding a composite enzyme and sodium hydrogen phosphate-sodium dihydrogen phosphate buffer for enzymolysis, wherein the enzymolysis temperature is 55-60° C., the pH is 5.0-6.0, and the time is 3-4 h, and filtering to obtain an extract solution 2;

[0009] (3) The extract solution 1 of step (1) and the extract solution 2 of step (2) are mixed and centrifuged to obtain a supernatant, the supernatant is added to an ionic liquid to obtain a mixed solution, fractionated and extracted, the extract is collected, the extract is evaporated, vacuum concentrated, washed with water, and dried to obtain mulberry leaf oligosaccharides.

[0010] By adopting the above technical scheme, mulberry leaves contain mulberry leaf polysaccharides, which are mainly composed of mannose, galacturonic acid, glucose, rhamnose, glucuronic acid, galactose, arabinose, etc. The mulberry leaves are crushed and then dispersed in an ethanol aqueous solution for ultrasonic extraction. Ultrasonic waves can destroy the cell walls of the mulberry leaves and release the polysaccharides in the cells. The operation is simple and the extraction efficiency is high, so that an extract solution 1 is obtained.

[0011] The filter residue is subjected to secondary extraction using a composite enzyme. Under appropriate enzymatic hydrolysis temperature and pH conditions, the composite enzyme destroys the cell wall of the mulberry leaves, which is beneficial to the extraction of polysaccharides. The reaction conditions are mild, the efficiency is high, and impurities are easy to remove, thereby obtaining a high-purity extract solution II.

[0012] The extract one and the extract two are centrifuged to obtain a supernatant, which is then further extracted using an ionic liquid as an extractant. The ionic liquid has extremely low volatility and high thermal stability and can be extracted under high temperature and high pressure conditions, thereby improving the extraction efficiency to obtain mulberry leaf oligosaccharides. Ultrasonic extraction, enzyme extraction and ionic liquid are used to extract mulberry leaf oligosaccharides, and the purity of the obtained mulberry leaf oligosaccharides is relatively high.

[0013] Optionally, the complex enzyme consists of cellulase, pectinase and glucanase in a mass ratio of 7-10:1:5-7.

[0014] By adopting the above technical scheme, the composite enzyme is composed of cellulase, pectinase and glucanase in a certain mass ratio, and the combination of cellulase, pectinase and glucanase has a synergistic effect. Cellulase decomposes cellulose in the cell wall of mulberry leaves, promotes the rupture of the cell wall of mulberry leaves, and then extracts mulberry leaf oligosaccharides; pectinase can destroy the gelatinous and soluble pectin in mulberry leaves, reduce the viscosity of mulberry leaf extracts, and help extract mulberry leaf polysaccharides; glucanase can effectively degrade the cell wall of mulberry leaves, break the physical barrier, promote the release of nutrients in the cell wall, and then help extract mulberry leaf oligosaccharides; cellulase, pectinase and glucanase cooperate to further improve the extraction efficiency and purity of mulberry leaf oligosaccharides in the filter residue, and then obtain an extract solution 2 with better purity.

[0015] Optionally, the preparation method of the ionic liquid is as follows: 1-hexyl-3-methylimidazole bis(trifluoromethanesulfonyl)imide is dispersed in an ethanol solution to obtain an ionic liquid.

[0016] By adopting the above technical scheme, 1-hexyl-3-methylimidazole bis(trifluoromethanesulfonyl)imide has high chemical stability, low volatility, high selectivity, good physical and chemical stability, good solubility for organic and inorganic substances, and is easy to recycle. It is used as an extractant to increase the extraction rate, increase the extraction degree and increase the purity of the product in one step.

[0017] Optionally, the surface of the mulberry leaf oligosaccharide obtained in step (3) is sprayed with a modified chitosan solution.

[0018] By adopting the above technical scheme, mulberry leaf oligosaccharides have strong hygroscopicity. If they are in a humid environment, they are easy to absorb water and agglomerate, which will affect the subsequent consumption of mulberry leaf oligosaccharides. The modified chitosan solution is sprayed on the surface of mulberry leaf oligosaccharides, and the modified chitosan solution is used to coat the mulberry leaf oligosaccharides to form a film on the surface of mulberry leaf oligosaccharides. The obtained mulberry leaf oligosaccharides have good water resistance, are not easy to agglomerate in a humid environment, and extend the storage period of mulberry leaf oligosaccharides.

[0019] Optionally, the method for preparing the modified chitosan solution comprises the following steps:

[0020] (1) Dispersing chitosan in acetic acid solution, adding tea polyphenols and quercetin, stirring for 1-2 hours to obtain a mixed solution for later use;

[0021] (2) cutting and crushing the white radish, and then heating it in water at a temperature of 90-95° C. for 10-12 minutes, filtering to obtain an extract, dispersing the extract in water, adding citric acid, adjusting the pH to 2.0-3.0, extracting for 4-5 hours, evaporating and concentrating, and drying to obtain radish fiber;

[0022] (3) Adding the modified starch and the radish fiber obtained in step (2) to the mixed solution obtained in step (1), stirring at a temperature of 60-65° C. for 2-3 hours, to obtain a modified chitosan solution.

[0023] By adopting the above technical scheme, chitosan is dissolved in acetic acid solution, and tea polyphenols and quercetin are added. Tea polyphenols and quercetin both have strong antioxidant effects and can inhibit the peroxidation of substances. When added to the chitosan solution, they not only have good antioxidant and antibacterial activities, but also cooperate with chitosan to enhance the oxygen barrier, moisturizing and breathable properties of chitosan, so that the chitosan solution has higher viscosity, antibacterial and antioxidant properties.

[0024] White radish contains rich dietary fiber. Adding citric acid to extract the radish fiber in the white radish has a high extraction rate, and the obtained radish fiber has good strengthening and filling effects. The modified starch has good viscosity and film-forming properties. The modified starch, radish fiber and the chitosan solution of step (1) are mixed. The modified starch and the chitosan solution are mixed to enhance the mechanical properties of the chitosan after film formation. The radish fiber can enhance the mechanical properties of the chitosan after film formation, improve its tensile strength, bending strength and impact strength, and thus make the chitosan film have good mechanical properties, which is conducive to the storage of the chitosan film. The modified starch, radish fiber and chitosan are combined to obtain a modified chitosan with good mechanical properties and waterproofness, which is conducive to the long-term storage of the chitosan film, and thus helps to protect the mulberry leaf oligosaccharide from moisture.

[0025] Optionally, the mass ratio of the chitosan, radish fiber and modified starch is 1:0.4-0.6:0.3-0.5.

[0026] By adopting the above technical scheme, the mass ratio of chitosan, radish fiber and modified starch is further limited within a certain range, thereby improving the mechanical properties and water resistance of the modified chitosan after film formation. Chitosan itself has good antibacterial and water resistance, radish fiber has good mechanical strength, and modified starch has good viscosity and film-forming properties. The radish fiber and modified starch are mixed with chitosan, which improves the water resistance and mechanical properties of the chitosan after film formation, helps the storage of mulberry leaf oligosaccharides, makes mulberry leaf oligosaccharides less likely to agglomerate in a humid environment, and extends the storage period of mulberry leaf oligosaccharides.

[0027] Optionally, the preparation method of the modified starch comprises the following steps: dispersing the aloe extract in water, adding succinic acid, reacting at a temperature of 80-90° C. for 35-40 min, centrifuging, and drying to obtain a modified plant extract;

[0028] Disperse cassava starch in water, add modified plant extract, walnut protein peptide and konjac flour, stir at a temperature of 75-80° C. for 1-3 hours to obtain a membrane solution.

[0029] By adopting the above technical scheme, the aloe extract has good antibacterial property, the succinic acid has good antioxidant property, the succinic acid further improves the purity of the aloe extract, and the modified plant extract obtained not only has high purity but also has good antioxidant property; the modified plant extract, walnut protein peptide and konjac flour are added to the cassava starch aqueous solution, the modified plant extract improves the antioxidant and antibacterial properties of the cassava starch, the walnut protein peptide has strong reducing ability, high solubility and good antibacterial biological activity, can change the structure of the cassava starch, form a gel structure through hydrogen bonds and electrostatic interactions, and thus improve the viscosity and strength of the cassava starch; the konjac flour has the functions of thickening, gelling and starch anti-aging, can improve the viscosity of the mixed system of cassava starch, modified plant extract and walnut protein peptide, and thus improve the mechanical strength of the cassava starch, which is helpful for the subsequent modification of chitosan.

[0030] Optionally, the mass ratio of the cassava starch, the modified plant extract, the walnut protein peptide and the konjac flour is 1:0.5-0.7:0.3-0.6:0.2-0.4.

[0031] By adopting the above technical scheme, the mass ratio of cassava starch, modified plant extract, walnut protein peptide and konjac flour is further limited within a certain range, and a modified starch solution with good mechanical properties, strong antibacterial properties and excellent antioxidant properties is obtained. There is a synergistic effect between cassava starch, modified plant extract, walnut protein peptide and konjac flour. The modified plant extract improves the antibacterial and antioxidant properties of cassava starch, the walnut protein peptide improves the mechanical properties of cassava starch, and the konjac flour enhances the viscosity of the starch system, so that the cassava starch, modified plant extract and walnut protein peptide are better mixed, which is helpful to subsequently improve the corresponding properties of the modified starch, and subsequently helps to modify the chitosan solution.

[0032] In a second aspect, the present application also provides functional mulberry leaf oligosaccharides prepared by the preparation method.

[0033] In a second aspect, the present application also provides the application of the functional mulberry leaf oligosaccharides in food or medicine.

[0034] In summary, this application has the following beneficial effects:

[0035] 1. In the present application, mulberry leaves contain mulberry leaf polysaccharides, which are mainly composed of mannose, galacturonic acid, glucose, rhamnose, glucuronic acid, galactose, arabinose, etc. The mulberry leaves are crushed and then dispersed in an ethanol aqueous solution for ultrasonic extraction. Ultrasonic waves can destroy the cell walls of the mulberry leaves and release the polysaccharides in the cells. The operation is simple and the extraction efficiency is high to obtain an extract solution 1.

[0036] 2. In the present application, the filter residue is subjected to secondary extraction using a composite enzyme. Under appropriate enzymatic hydrolysis temperature and pH conditions, the composite enzyme destroys the cell wall of the mulberry leaves, which is beneficial to the extraction of polysaccharides. The reaction conditions are mild, efficient, and impurities are easily removed, thereby obtaining a high-purity extract solution 2.

[0037] 3. In the present application, extract one and extract two are centrifuged to obtain a supernatant, and then the supernatant is further extracted using an ionic liquid as an extractant. The ionic liquid has extremely low volatility and high thermal stability and can be extracted under high temperature and high pressure conditions, thereby improving the extraction efficiency to obtain mulberry leaf oligosaccharides. Ultrasonic extraction, enzyme extraction and ionic liquid are used to extract mulberry leaf oligosaccharides, and the purity of the obtained mulberry leaf oligosaccharides is relatively high. DETAILED DESCRIPTION

[0038] Preparation example of modified chitosan solution

[0039] Preparation Example 1-1

[0040] The preparation method of modified chitosan solution comprises the following steps:

[0041] (1) Disperse 1.2 kg of chitosan in 2 L of 12% acetic acid solution, add 0.3 kg of tea polyphenols and 0.2 kg of quercetin, stir for 2 h, and obtain a mixed solution for later use;

[0042] (2) 2.4 kg of white radish was cut into pieces and crushed, and then heated in water at a temperature of 95° C. for 12 minutes, filtered to obtain an extract, and the extract was dispersed in 4 L of water, citric acid was added to adjust the pH to 2.0, and the extraction was performed for 5 hours, evaporated, concentrated, and dried to obtain radish fiber;

[0043] (3) Adding the modified starch and the radish fiber obtained in step (2) to the mixed solution obtained in step (1), stirring at a temperature of 65° C. for 3 h, to obtain a modified chitosan solution.

[0044] The mass ratio of chitosan, radish fiber and modified starch is 1:0.6:0.5.

[0045] The preparation method of modified starch comprises the following steps: dispersing 1.2 kg of aloe extract in 3 L of water, adding 0.1 kg of succinic acid, reacting at a temperature of 90° C. for 40 min, centrifuging and drying to obtain a modified plant extract;

[0046] 2.5 kg of cassava starch was dispersed in 3.5 L of water, and the modified plant extract, walnut protein peptide and konjac flour were added, and the mixture was stirred at 80° C. for 3 h to obtain a membrane solution.

[0047] The mass ratio of cassava starch, modified plant extract, walnut protein peptide and konjac flour is 1:0.5:0.3:0.2.

[0048] Preparation Example 1-2

[0049] The difference from Preparation Example 1-1 is that in step (2), white radish is not added.

[0050] Preparation Example 1-3

[0051] The difference from Preparation Example 1-1 is that in step (3), no modified starch is added.

[0052] Preparation Example 1-4

[0053] The difference from Preparation Example 1-1 is that the mass ratio of chitosan, radish fiber and modified starch is 1:0.4:0.3.

[0054] Preparation Example 1-5

[0055] The difference from Preparation Example 1-1 is that the mass ratio of chitosan, radish fiber and modified starch is 1:0.1:0.8.

[0056] Preparation Example 1-6

[0057] The difference from Preparation Example 1-1 is that no aloe extract is added in the preparation method of the modified starch.

[0058] Preparation Example 1-7

[0059] The difference from Preparation Example 1-1 is that walnut protein peptide is not added in the preparation method of modified starch.

[0060] Preparation Example 1-8

[0061] The difference from Preparation Example 1-1 is that no konjac flour is added in the preparation method of the modified starch.

[0062] Preparation Example 1-9

[0063] The difference from Preparation Example 1-1 is that the mass ratio of cassava starch, modified plant extract, walnut protein peptide and konjac flour is 1:0.7:0.6:0.4.

[0064] Preparation Example 1-10

[0065] The difference from Preparation Example 1-1 is that the mass ratio of cassava starch, modified plant extract, walnut protein peptide and konjac flour is 1:0.2:0.9:0.1.

[0066] Example

[0067] Example 1

[0068] A method for preparing functional mulberry leaf oligosaccharides comprises the following steps:

[0069] (1) 1 kg of mulberry leaves were washed, dried, crushed, and passed through a 100 μm sieve; then dispersed in 8 kg of 60% ethanol aqueous solution, subjected to ultrasonic extraction at a power of 500 W for 2 h, and filtered to obtain a filter residue and an extract;

[0070] (2) dispersing the filter residue obtained in step (1) in 8 kg of water, adding 2 kg of complex enzyme and 0.6 kg of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (purchased from Beijing Biolab Technology Co., Ltd.) for enzymolysis, the enzymolysis temperature is 55° C., the pH is 6.0, the time is 3 h, and filtering to obtain extract 2;

[0071] (3) The extract 1 of step (1) and the extract 2 of step (2) are mixed and centrifuged to obtain a supernatant, the supernatant is added to an ionic liquid to obtain a mixed solution, n-hexane is used as a detergent, and fractional extraction is performed at 25° C. The extracts are collected, evaporated (to remove ethanol), and then back-extracted with n-hexane. The n-hexane solution is removed by vacuum concentration, washed with water, and dried to obtain mulberry leaf oligosaccharides.

[0072] The complex enzyme is composed of cellulase, pectinase and glucanase in a mass ratio of 10:1:5.

[0073] Preparation method of ionic liquid: 5 kg of 1-hexyl-3-methylimidazole bis(trifluoromethanesulfonyl)imide is dispersed in 30 kg of ethanol solution to obtain ionic liquid.

[0074] Example 2

[0075] A method for preparing functional mulberry leaf oligosaccharides, which differs from Example 1 in that it comprises the following steps:

[0076] (1) 1.5 kg of mulberry leaves were washed, dried, crushed, and passed through a 100 μm sieve; then dispersed in 10 kg of 60% ethanol aqueous solution, subjected to ultrasonic extraction at a power of 400 W for 1 h, and filtered to obtain a filter residue and an extract;

[0077] (2) dispersing the filter residue obtained in step (1) in 7 kg of water, adding 1.5 kg of complex enzyme and 0.5 kg of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (purchased from Beijing Biolab Technology Co., Ltd.) for enzymolysis, the enzymolysis temperature is 60° C., the pH is 5.0, the time is 4 h, and filtering to obtain extract 2;

[0078] (3) The extract 1 of step (1) and the extract 2 of step (2) are mixed and centrifuged to obtain a supernatant, the supernatant is added to an ionic liquid to obtain a mixed solution, n-hexane is used as a detergent, and fractional extraction is performed at 25° C. The extracts are collected, evaporated (to remove ethanol), and then back-extracted with n-hexane. The n-hexane solution is removed by vacuum concentration, washed with water, and dried to obtain mulberry leaf oligosaccharides.

[0079] The complex enzyme is composed of cellulase, pectinase and glucanase in a mass ratio of 7:1:7.

[0080] Example 3

[0081] A method for preparing functional mulberry leaf oligosaccharides, which differs from Example 1 in that the complex enzyme consists of cellulase, pectinase and glucanase in a mass ratio of 2:1:10.

[0082] Example 4

[0083] A method for preparing functional mulberry leaf oligosaccharides, which differs from Example 1 in that a modified chitosan solution is sprayed on the surface of the mulberry leaf oligosaccharides obtained in step (3), and the modified chitosan solution adopts the method of Preparation Example 1-1.

[0084] Example 5

[0085] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-2.

[0086] Example 6

[0087] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-3.

[0088] Example 7

[0089] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-4.

[0090] Example 8

[0091] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-5.

[0092] Example 9

[0093] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-6.

[0094] Example 10

[0095] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-7.

[0096] Embodiment 11

[0097] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-8.

[0098] Example 12

[0099] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Example 1-9.

[0100] Embodiment 13

[0101] A method for preparing functional mulberry leaf oligosaccharides, which is different from Example 4 in that the modified chitosan solution adopts Preparation Examples 1-10.

[0102] Comparative Example

[0103] Comparative Example 1

[0104] A method for preparing functional mulberry leaf oligosaccharides, which differs from Example 1 in that step (3) is not performed.

[0105] Comparative Example 2

[0106] A method for preparing functional mulberry leaf oligosaccharides, which differs from Example 1 in that ultrasonic extraction is not performed.

[0107] Comparative Example 3

[0108] A method for preparing functional mulberry leaf oligosaccharides, which differs from Example 1 in that no complex enzyme is added.

[0109] Performance testing

[0110] The functional mulberry leaf oligosaccharides prepared in Examples 1-3 and Comparative Examples 1-3 were tested for purity;

[0111] Oligosaccharide purity test: The sample to be tested was filtered with a 0.22 μm filter membrane and then used for high performance liquid chromatography analysis of oligosaccharide purity. The chromatographic conditions were as follows: the detector was an evaporative light detector, and the ELSD drift tube temperature was 45°C; the chromatographic column model was ShodexAsahipak NH2P-504E (4.6 mm×250 mm, 5 μm), and the column temperature was 30°C; the mobile phase was 75% acetonitrile, the flow rate was 1 mL / min, the injection volume was 20 μL, and dextran with a degree of polymerization of 2-10 was used as the standard.

[0112] Oligosaccharide purity (%) = (sum of peak areas of oligosaccharides with a degree of polymerization of 2-10 / sum of all peak areas) × 100. Tensile strength test of modified chitosan film: The modified chitosan solution was sprayed onto a plastic plate and dried to obtain a film with a thickness of 0.2 mm, which was cut into strips of 5 cm × 1 cm. The chitosan films prepared in Example 1 and Examples 4-13 were tested for tensile properties using a texture analyzer. The tensile speed was set to 0.5 mm / s, and the tensile strength was calculated using the following formula: T = F / (L×W)

[0113] Where: T is the tensile strength g / mm 2 ; F is the maximum tensile force g when the membrane breaks; L is the average thickness of the membrane in mm; W is the width of the membrane in mm.

[0114] Antibacterial rate test: The antibacterial activity of chitosan film against Staphylococcus aureus was tested by shaking flask culture method. Staphylococcus aureus was inoculated in LB test tube culture medium in advance. After culturing at 37°C and 150 rpm for 12 hours, 1 mL of bacterial solution was drawn and resuspended to 10% with sterile saline. 7 cfu / mL. Take 1mL of the diluted bacterial solution and add it to a conical flask containing 99mL of fresh LB liquid culture medium. Cut the chitosan membranes prepared in Example 1 and Example 4-13 into 1×1cm samples, put them into a conical flask containing the above LB culture medium, and place the conical flask in a 37°C shaker and incubate at 150rpm for 24h. In the clean bench, take the bacterial solution in the conical flask, dilute it tenfold several times, and then take 200μL of the diluted bacterial solution and evenly spread it on the PCA plate count agar. Place the PCA culture medium in a 37°C constant temperature incubator and incubate for 48h before counting the colonies. The colony count is counted using the method described in GB / T 4789.2-2016 "Determination of the total number of colonies in food microbiology inspection".

[0115] The antibacterial rate was calculated according to the following formula:

[0116] Antibacterial rate = [(W1-W2) / W1] × 100%

[0117] Where: W1 is the number of colonies in the blank control group; W2 is the number of colonies in the chitosan film sample group.

[0118] Waterproof test: The water vapor permeability of Example 1 and Examples 4-13 was tested using a water vapor permeability tester in accordance with GB-T 1037-1988 Test method for water vapor permeability of plastic films and sheets using the cup method; the test results are shown in Table 1.

[0119] Table 1 Test data of embodiments and comparative examples

[0120] Test items Purity of mulberry leaf oligosaccharide / % Example 1 99.2 Example 2 99.0 Example 3 98.1 Comparative Example 1 87.2 Comparative Example 2 88.3 Comparative Example 3 85.6

[0121] Table 2 Test data of embodiments and comparative examples

[0122]

[0123]

[0124] Combining the examples and the data in Table 1, it can be seen that the functional mulberry leaf oligosaccharides extracted in Examples 1-2 have good purity, among which the purity of Example 1 reaches 99.2%, indicating that the present application uses ultrasonic extraction, enzyme extraction and ionic liquid to extract mulberry leaf oligosaccharides, and the obtained mulberry leaf oligosaccharides have high purity.

[0125] Example 3 changes the mass ratio of cellulase, pectinase and glucanase in the composite enzyme. As can be seen from Table 1, compared with Example 1, the purity is 98.1%, indicating that the combination of cellulase, pectinase and glucanase has a synergistic effect. The combination of cellulase, pectinase and glucanase further improves the extraction efficiency and purity of mulberry leaf oligosaccharides in the filter residue, thereby obtaining an extract solution 2 with better purity, thereby improving the overall purity of mulberry leaf oligosaccharides.

[0126] Comparative Example 1 does not perform the ion extraction step of step (3). As can be seen from Table 1, compared with Example 1, the purity is 87.2%, indicating that the use of ionic liquid as an extractant has extremely low volatility and high thermal stability, and can be extracted under high temperature and high pressure conditions, thereby improving the extraction efficiency and obtaining mulberry leaf oligosaccharides with higher purity.

[0127] In comparative example 2, the ultrasonic extraction of step (1) is not performed. As can be seen from Table 1, compared with Example 1, the purity is 88.3%, indicating that the use of ultrasonic extraction can destroy the cell wall of mulberry leaves and release polysaccharides in the cells. The operation is simple and the extraction efficiency is high. An extract solution 1 is obtained, thereby improving the overall purity of oligosaccharides.

[0128] Comparative Example 3 does not perform the composite enzyme hydrolysis in step (2). As can be seen from Table 1, compared with Example 1, the purity is 85.6%, indicating that the use of the composite enzyme for secondary extraction, under appropriate enzymatic hydrolysis temperature and pH conditions, the composite enzyme destroys the mulberry leaf cell wall, which is beneficial to the extraction of polysaccharides. The reaction conditions are mild, efficient, and impurities are easy to remove, thereby obtaining a high-purity extract solution 2, thereby improving the overall purity of mulberry leaf oligosaccharides.

[0129] In Examples 4, 7 and 12, the modified chitosan solution was sprayed on the surface of the mulberry leaf oligosaccharide obtained in step (3). As shown in Table 1, compared with Example 1, the tensile strength of the prepared chitosan film was 23.4 g / mm 2 The antibacterial rate is 99.7%, and the water vapor permeability is 35.2%, indicating that the chitosan solution is sprayed on the surface of mulberry leaf oligosaccharides in this application, and the chitosan after film formation has a high antibacterial rate, tensile strength and waterproofness, which is conducive to the long-term storage of mulberry leaf oligosaccharides.

[0130] In the preparation method of the modified chitosan solution of Example 5, white radish is not added. As shown in Table 1, compared with Example 1, the tensile strength of the prepared chitosan film is 20.1 g / mm 2The antibacterial rate was 98.4% and the water vapor permeability was 38.3%, indicating that white radish is rich in dietary fiber. The obtained radish fiber can enhance the mechanical properties of chitosan after film formation, improve its tensile strength, flexural strength and impact strength, and thus make the chitosan film have better mechanical properties.

[0131] In the preparation method of the modified chitosan solution of Example 6, no modified starch is added. As shown in Table 1, compared with Example 1, the tensile strength of the prepared chitosan film is 18.2 g / mm 2 The antibacterial rate was 92.1%, and the water vapor permeability was 45.3%, indicating that the modified starch had good viscosity and film-forming properties. The modified starch, radish fiber and chitosan solution were mixed, and the modified starch was mixed with the chitosan solution to enhance the mechanical properties, antibacterial properties and waterproof properties of the chitosan after film formation.

[0132] Example 8 changes the mass ratio of chitosan, radish fiber and modified starch. It can be seen from Table 1 that compared with Example 1, the performance test results of tensile strength, antibacterial rate and water vapor permeability of the prepared chitosan film are better than those of Example 1 and Example 7, but worse than those of Examples 5-6, indicating that radish fiber has good mechanical strength, modified starch has good viscosity and film-forming properties, and radish fiber and modified starch are mixed with chitosan, which improves the waterproofness, antibacterial properties and mechanical properties of the chitosan after film formation, helps the storage of mulberry leaf oligosaccharides, makes mulberry leaf oligosaccharides less likely to agglomerate in a humid environment, and extends the storage period of mulberry leaf oligosaccharides.

[0133] In the preparation method of modified starch in Example 9, aloe extract is not added. As shown in Table 1, compared with Example 1, the tensile strength of the prepared chitosan film is 22.4 g / mm 2 The antibacterial rate was 94.3% and the water vapor permeability was 42.1%, indicating that aloe extract had good antibacterial properties. Aloe extract improved the antioxidant and antibacterial properties of cassava starch, and combined with other components, it further improved the mechanical properties, antibacterial and waterproof properties of chitosan film.

[0134] In the preparation method of modified starch in Example 10, walnut protein peptide is not added. As shown in Table 1, compared with Example 1, the tensile strength of the prepared chitosan film is 20.1 g / mm 2 The antibacterial rate was 94.8%, and the water vapor permeability was 43.4%, indicating that walnut protein peptide has strong reducing ability, high solubility, and good antibacterial biological activity. It can change the structure of cassava starch and form a gel structure through hydrogen bonds and electrostatic interactions, thereby improving the viscosity and strength of cassava starch, and subsequently improving the corresponding properties of chitosan film.

[0135] In the preparation method of modified starch in Example 11, konjac flour is not added. As shown in Table 1, compared with Example 1, the tensile strength of the prepared chitosan film is 20.7 g / mm 2 The antibacterial rate was 95.1%, and the water vapor permeability was 41.8%, indicating that konjac flour has the functions of thickening, gelling, and starch anti-aging, and can improve the viscosity of the mixed system of cassava starch, modified plant extracts and walnut protein peptides, thereby improving the mechanical strength of cassava starch, and subsequently helping the modification of chitosan.

[0136] Example 13 changes the mass ratio of cassava starch, modified plant extract, walnut protein peptide and konjac flour. As can be seen from Table 1, compared with Example 1, the performance test results of the tensile strength, antibacterial rate and water vapor permeability of the prepared chitosan film are better than those of Example 1 and Example 12, but worse than those of Examples 9-11, indicating that there is a synergistic effect between cassava starch, modified plant extract, walnut protein peptide and konjac flour, the modified plant extract improves the antibacterial and antioxidant properties of cassava starch, the walnut protein peptide improves the mechanical properties of cassava starch, and the konjac flour enhances the viscosity of the starch system, so that cassava starch, modified plant extract and walnut protein peptide are better mixed, which helps to subsequently improve the corresponding properties of the modified starch, and subsequently helps to modify the chitosan solution.

[0137] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing functional mulberry leaf oligosaccharides, characterized in that: The steps include: (1) Wash, dry, crush and sieve the mulberry leaves, disperse them in an ethanol aqueous solution, perform ultrasonic extraction at a power of 400-500 W for 1-2 h, filter and obtain a filter residue and an extract; (2) dispersing the filter residue obtained in step (1) in water, adding complex enzyme and sodium dihydrogen phosphate / sodium dihydrogen phosphate buffer for enzymatic hydrolysis at a temperature of 55-60° C., a pH of 5.0-6.0, and a time of 3-4 h, filtering to obtain extract solution 2; (3) Mixing the extract 1 of step (1) and the extract 2 of step (2) and subjecting them to centrifugal separation to obtain a supernatant, adding the supernatant to an ionic liquid to obtain a mixed solution, fractionating and extracting, collecting the extract, evaporating the extract, vacuum concentrating, washing with water, and drying to obtain mulberry leaf oligosaccharides; The complex enzyme is composed of cellulase, pectinase and glucanase in a mass ratio of 7-10:1:5-7; The preparation method of the ionic liquid comprises the following steps: dispersing 1-hexyl-3-methylimidazole bis(trifluoromethanesulfonyl)imide in an ethanol solution to obtain the ionic liquid.

2. The method for preparing functional mulberry leaf oligosaccharides according to claim 1, characterized in that: The modified chitosan solution is sprayed on the surface of the mulberry leaf oligosaccharide obtained in step (3).

3. The method for preparing functional mulberry leaf oligosaccharides according to claim 2, characterized in that: The preparation method of the modified chitosan solution comprises the following steps: (1) Disperse chitosan in acetic acid solution, add tea polyphenols and quercetin, stir for 1-2 hours to obtain a mixed solution for later use; (2) Cut the white radish into pieces and crush them, then heat them in water at a temperature of 90-95° C. for 10-12 minutes, filter them to obtain an extract, disperse the extract in water, add citric acid, adjust the pH to 2.0-3.0, extract for 4-5 hours, evaporate and concentrate, and dry them to obtain radish fiber; (3) Adding the modified starch and the radish fiber obtained in step (2) to the mixed solution obtained in step (1), stirring at a temperature of 60-65° C. for 2-3 hours, to obtain a modified chitosan solution.

4. The method for preparing functional mulberry leaf oligosaccharides according to claim 3, characterized in that: The mass ratio of the chitosan, radish fiber and modified starch is 1:0.4-0.6:0.3-0.

5.

5. The method for preparing functional mulberry leaf oligosaccharides according to claim 3, characterized in that: The preparation method of the modified starch comprises the following steps: dispersing aloe extract in water, adding succinic acid, reacting at a temperature of 80-90° C. for 35-40 minutes, centrifuging and drying to obtain a modified plant extract; Disperse cassava starch in water, add modified plant extract, walnut protein peptide and konjac flour, stir at a temperature of 75-80° C. for 1-3 hours to obtain a membrane solution.

6. The method for preparing functional mulberry leaf oligosaccharides according to claim 5, characterized in that: The mass ratio of the cassava starch, the modified plant extract, the walnut protein peptide and the konjac flour is 1:0.5-0.7:0.3-0.6:0.2-0.

4.

7. Functional mulberry leaf oligosaccharide prepared according to the preparation method according to any one of claims 5-6.

8. Use of the functional mulberry leaf oligosaccharide according to claim 7 in food.

Images