Preparation method and application of mulberry leaf extract

Abstract

The invention discloses a preparation method and application of a mulberry leaf extract, and belongs to the technical field of natural medicine extraction. The preparation method comprises the following steps: S1, crushing mulberry leaves, adding water, extracting, filtering, collecting filtrate, cooling and centrifuging the filtrate, and retaining clear liquid; s2, enabling the clear liquid to pass through macroporous adsorption resin, and collecting effluent; s3, carrying out microfiltration and concentration on the effluent, and collecting a concentrated solution; and S4, adding ethanol into the concentrated solution, standing for crystallization, and drying the crystal under reduced pressure to obtain the mulberry leaf extract. The yield of the extract prepared through the preparation method is high, the content of mulberry leaf polysaccharide in the prepared extract is high, and the mulberry leaf extract can be applied to feed for improving the body length of crucian carps, feed for improving liver health of crucian carps and feed for improving intestinal health of crucian carps.

Description

Technical Field

[0001] The invention belongs to the technical field of natural drug extraction, and specifically relates to a preparation method and application of a mulberry leaf extract. Background Art

[0002] Mulberry leaves, also known as iron fan, are leaves of the mulberry tree of the Moraceae family. Mulberry leaves are rich in polysaccharides, flavonoids, alkaloids, polyphenols and other natural active substances.

[0003] Mulberry leaf polysaccharides may directly or indirectly promote the growth of crucian carp and increase its body length by regulating the secretion of growth hormone in crucian carp. Growth hormone is essential for the growth and development of fish. It can promote protein synthesis, accelerate cell division and proliferation, and thus affect the growth rate of fish. Mulberry leaf polysaccharides have strong antioxidant and anti-inflammatory activities, can effectively remove free radicals in the body, reduce oxidative stress and inflammatory response, and have a protective effect on organs such as the liver; after the health of the liver improves, its detoxification and metabolic capacity are enhanced, which is conducive to the overall health and growth of the fish. Mulberry leaf polysaccharides have a good prebiotic effect, which can promote the growth of beneficial intestinal bacteria, inhibit the reproduction of harmful bacteria, and maintain the balance of intestinal microecology. This not only helps the digestion and absorption of food, but also increases the height and number of intestinal folds and villi, improves the absorption efficiency of nutrients, and reduces the intestinal cavity rate, thereby promoting the healthy growth of fish. Summary of the invention

[0004] The present invention overcomes the above technical problems and provides a preparation method and application of a mulberry leaf extract. The preparation method of the present invention has a high extract yield, and the prepared extract has a high content of mulberry leaf polysaccharides. The mulberry leaf extract of the present invention can be applied to feed for increasing the body length of crucian carp, feed for improving the liver health of crucian carp, and feed for improving the intestinal health of crucian carp.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The present invention provides a method for preparing a mulberry leaf extract, comprising the following steps:

[0007] S1. After crushing the mulberry leaves, add water to extract, filter and collect the filtrate, cool and centrifuge the filtrate, and retain the clear liquid;

[0008] S2. passing the clear solution through a macroporous adsorption resin and collecting the effluent;

[0009] S3. After the effluent is microfiltered and concentrated, the concentrate is collected;

[0010] S4. Add ethanol to the concentrated solution and let it stand to crystallize, then dry the crystals under reduced pressure to obtain mulberry leaf extract.

[0011] In S1, the amount of water added is 5 to 200 times the mass of the mulberry leaves, preferably 5 to 20 times.

[0012] In S1, the extraction method is leakage extraction.

[0013] Further in S1, the temperature of the leakage extraction is 75-90°C, preferably 80-85°C, for example 80°C or 85°C.

[0014] When the mass of mulberry leaves is less than 100kg, the leakage rate of the leakage extraction is 100-200L / h, preferably 145-155L / h;

[0015] When the mass of mulberry leaves is 100-500 kg, the percolation rate of the percolation extraction is 180-500 L / h, preferably 200-500 L / h;

[0016] When the mass of mulberry leaves is 500-800 kg, the percolation rate of the percolation extraction is 500-800 L / h, preferably 580-620 L / h;

[0017] When the mass of mulberry leaves is greater than 800 kg, the leakage rate of the leakage extraction is 900-1500 L / h, preferably 1000-1200 L / h.

[0018] Further in S1, the leakage extracts a leakage liquid with Brix ≥ 1%.

[0019] In S1, the filtration uses a 70-90 mesh screen; preferably, a 80 mesh screen is used.

[0020] In S1, the cooling is performed by using a plate heat exchanger.

[0021] In S1, the cooling is to 20-35°C, preferably 25-30°C, for example 25°C, 30°C.

[0022] In S1, the centrifugal speed is 2000-3000 rpm, preferably 2500 rpm.

[0023] In S1, the centrifugation time is 2 to 10 minutes, preferably 3 to 5 minutes, for example 3 minutes.

[0024] In S2, the macroporous resin is Seplite Lxd-762 and Seplite Lx-T5.

[0025] According to research, Seplite Lxd-762 macroporous resin and Seplite Lx-T5 macroporous resin have high adsorption capacity for mulberry leaf polysaccharides, while removing other small molecule impurities. By collecting the effluent, the purity of the polysaccharide can be further improved.

[0026] In S2, the macroporous adsorption resin is washed with 2 to 5 column volumes of water; preferably, it is washed with 2 to 4 column volumes of water.

[0027] In S3, the microfiltration uses a hollow fiber membrane as an RO membrane for reverse osmosis concentration;

[0028] The RO membrane has a pore size of 1 to 100 Da, preferably 20 to 60 Da, such as 50 Da. The hollow fiber membrane can efficiently separate water molecules and efficiently retain mulberry leaf polysaccharides.

[0029] In S3, the concentration is reverse osmosis concentration and vacuum concentration in sequence; preferably, the concentration process is first reverse osmosis concentration to Brix 20-25%, and then vacuum concentration to Brix 35-40%.

[0030] In S4, the concentration of the ethanol is 80-100%; preferably, the concentration of the ethanol is 100%, that is, anhydrous ethanol.

[0031] In S4, the amount of ethanol added is 4 to 8 times the volume of the concentrated solution, preferably 5 to 6 times, such as 5 times, 5.5 times or 6 times.

[0032] In S4, the standing time is 6 to 20 hours, preferably 8 to 15 hours, for example 8 hours, 10 hours, 12 hours, 15 hours.

[0033] In the present invention, the yield of the mulberry leaf extract is 5.8-6.7%, preferably 5.9-6.02%, for example 5.9%, 6.02%, 6.08% or 6.3%.

[0034] In the present invention, the content of mulberry leaf polysaccharide in the mulberry leaf extract is 78-85%, preferably 78.25-83.56%, such as 78.25%, 80.68%, 82.88% or 83.56%.

[0035] The present invention also provides the use of the mulberry leaf extract prepared by the preparation method of the mulberry leaf extract in fish feed, wherein the feed includes feed for increasing the body length of crucian carp, feed for improving the liver health of crucian carp and feed for improving the intestinal health of crucian carp.

[0036] In the present invention, the improving the liver health of crucian carp also includes improving hepatocyte cavitation.

[0037] In the present invention, the improvement of crucian carp intestinal health includes promoting the increase of crucian carp intestinal folds, increasing the height and number of villi and reducing the intestinal cavity rate.

[0038] In the present invention, the amount of the mulberry leaf extract added to the feed for increasing the body length of crucian carp is 5.5-6.5 g / kg daily diet.

[0039] In the present invention, the amount of the mulberry leaf extract added to the feed for improving the liver health of crucian carp is 5.5-6.5 g / kg daily diet.

[0040] In the present invention, the mulberry leaf extract is added in an amount of 10-15 g / kg daily diet in the feed for improving the intestinal health of crucian carp.

[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The purification and concentration process of the present invention adopts macroporous adsorption resin and reverse osmosis concentration technology to effectively remove impurities in the extract. At the same time, by accurately controlling the concentration ratio, the high purity and high activity of the polysaccharide are ensured, and the loss of active ingredients is reduced.

[0044] 2. The process improvement cost of the present invention is low, and the high efficiency of polysaccharide extraction and the controllability of the results are ensured by accurately controlling the extraction temperature, crystallization solvent and concentration degree, etc. This not only improves the extraction rate of polysaccharides, but also ensures the quality and purity of the extract.

[0045] 3. The yield of the mulberry leaf extract of the present invention is 5.8-6.7%, and the content of mulberry leaf polysaccharide in the mulberry leaf extract is 78-85%. DETAILED DESCRIPTION

[0046] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and carefully in combination with preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0047] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0048] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The range defined in this way can be inclusive or exclusive of end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60-120 and 80-110 is listed for a specific parameter, it is understood that the range of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present invention, unless otherwise specified, the numerical range "ab" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" represents that all real numbers between "0-5" have been fully listed herein, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0049] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0050] Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0051] If not otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0052] If there is no special explanation, the "include" and "comprising" mentioned in the present invention represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0053] If not specifically stated, in the present invention, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0054] Example 1

[0055] S1. Take 10kg of mulberry leaves and crush them, add 100kg of hot water at 80℃ to extract the leakage liquid with Brix≥1%, and the leakage rate is 155L / h; collect the filtrate after filtering, and use an 80-mesh screen to filter; cool the filtrate to 30℃ through a plate heat exchanger; centrifuge at 2500rpm for 3min, and take the clear liquid.

[0056] S2. The clear liquid was passed through macroporous adsorption resins Seplite Lxd-762 and Seplite Lx-T5 in sequence, and the resin was washed with 2 column volumes of water, and the effluent was collected.

[0057] S3. The effluent was microfiltered and concentrated to Brix 25% by reverse osmosis using a 50Da RO membrane (hollow fiber membrane), and then concentrated to Brix 38% under reduced pressure, and the concentrate was collected.

[0058] S4. Add 5 times of anhydrous ethanol to the concentrated solution, let it stand at room temperature for 12 hours to crystallize, and then dry the crystals under reduced pressure to obtain 0.59 kg of mulberry leaf extract (yield 5.9%). The polysaccharide content in the mulberry leaf extract is 82.88%.

[0059] Example 2

[0060] S1. Take 10kg of mulberry leaves and crush them. Add 150kg of hot water at 80℃ to extract the leachate with Brix≥1% at a leakage rate of 145L / h. Collect the filtrate after filtration using an 80-mesh sieve. Cool the filtrate to 25℃ through a plate heat exchanger. Centrifuge at 2500rpm for 3min to obtain the clear liquid.

[0061] S2. The clear liquid was passed through macroporous adsorption resins Seplite Lxd-762 and Seplite Lx-T5 in sequence, and the resin was washed with 2 column volumes of water, and the effluent was collected.

[0062] S3. The effluent was microfiltered using a 50Da RO membrane (hollow fiber membrane) for reverse osmosis concentration to Brix 20%, and then concentrated under reduced pressure to Brix 35%, and the concentrate was collected.

[0063] S4. Add 4 times of anhydrous ethanol to the concentrated solution, let it stand at room temperature for 10 hours to crystallize, and then dry the crystals under reduced pressure to obtain 0.63 kg of mulberry leaf extract (yield 6.3%). The polysaccharide content in the mulberry leaf extract is 78.25%.

[0064] Example 3

[0065] S1. Take 600kg of mulberry leaves and crush them, then add 3000kg of hot water at 85℃ to extract the leachate with Brix≥1%, and the leakage rate is 600L / h; collect the filtrate after filtration, and use an 80-mesh screen to filter; cool the filtrate to 35℃ through a plate heat exchanger; centrifuge at 2500rpm for 5min, and take the clear liquid.

[0066] S2. The clear liquid was passed through macroporous adsorption resins Seplite Lxd-762 and Seplite Lx-T5 in sequence, and the resin was washed with 3 column volumes of water, and the effluent was collected.

[0067] S3. The effluent was microfiltered and concentrated to Brix 25% by reverse osmosis using a 50Da RO membrane (hollow fiber membrane), and then concentrated to Brix 40% under reduced pressure, and the concentrate was collected.

[0068] S4. Add 5.5 times of anhydrous ethanol to the concentrated solution, let it stand at room temperature for 12 hours to crystallize, and then dry the crystals under reduced pressure to obtain 36.5 kg of mulberry leaf extract (yield 6.08%), and the polysaccharide content in the mulberry leaf extract is 80.68%.

[0069] Example 4

[0070] S1. Take 900kg of mulberry leaves and crush them, then add 4500kg of hot water at 85℃ to extract the leachate with Brix≥1%, with a leakage rate of 1200L / h; collect the filtrate after filtration, and use an 80-mesh screen to filter; cool the filtrate to 35℃ through a plate heat exchanger; centrifuge at 2500rpm for 5min, and take the clear liquid.

[0071] S2. The clear liquid was passed through macroporous adsorption resins Seplite Lxd-762 and Seplite Lx-T5 in sequence, and the resin was washed with 4 column volumes of water, and the effluent was collected.

[0072] S3. The effluent was microfiltered using a 50Da RO membrane (hollow fiber membrane) for reverse osmosis concentration to Brix 20%, and then concentrated under reduced pressure to Brix 40%, and the concentrate was collected.

[0073] S4. Add 6 times the amount of anhydrous ethanol to the concentrated solution, let it stand at room temperature for 12 hours to crystallize, and then dry the crystals under reduced pressure to obtain 54.2 kg of mulberry leaf extract (yield 6.02%), and the polysaccharide content in the mulberry leaf extract is 83.56%.

[0074] Effect Example

[0075] The mulberry leaf extract prepared in Example 4 was added to crucian carp feed, and the process was as follows:

[0076] (1) Experimental grouping: The crucian carp used in the experiment was provided by Hunan Tianjiahu Fishery Technology Co., Ltd., and 540 crucian carp (60.42±0.16g) with a healthy physique and uniform size were randomly divided into groups A, B, and C. Each group had three replicates, and each replicate group had 60 fish;

[0077] Group A (control group): fed only with the basic diet;

[0078] Group B was supplemented with 10 g of mulberry leaf extract per 1 kg of diet;

[0079] Group C added 15 kg of mulberry leaf extract per 1 kg of diet;

[0080] The daily diet includes: 50 portions of soybean cake, 10 portions of fish meal, 38.7 portions of wheat bran, 0.5 portions of bone meal, 0.5 portions of salt, 0.1 portions of Vino high-vitamin for fish, 0.1 portions of Vino mycophenolate mofetil and minerals, and 0.1 portions of Vino compound probiotics.

[0081] (2) Water quality conditions: The aquaculture water source is tap water that has been filtered, disinfected, and aerated. During the experiment, the aquaculture water temperature was 25-28°C, and the transparency was about 25-30 cm. The dissolved oxygen content in the aquaculture water was guaranteed to be 5-7 mg / L, the pH was 7.2-7.6, and the ammonia nitrogen concentration was ≤0.10 mg / L.

[0082] (3) Feeding method: Before the formal experiment, the basic diet was used for a 10-day trial. The test period for Groups A, B, and C was 60 days. The daily feed was 2-3% of the carp's weight. The carp was fed once a day at 8-9 a.m. and 17-18 p.m. The feed that was not eaten within half an hour after feeding was removed, dried, and weighed. The weight of the feed eaten after each feeding was accurately recorded.

[0083] (4) Data collection: Data collection was carried out after 60 days of the test period. The crucian carp needed to be fasted for 24 hours before measuring body length and weight. Weight gain rate = (end average weight - initial average weight) / initial average weight × 100%.

[0084]

[0085]

[0086] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a mulberry leaf extract, characterized in that: The following steps are involved: S1. After crushing the mulberry leaves, add water to extract, filter and collect the filtrate, cool and centrifuge the filtrate, and retain the clear liquid; S2. passing the clear solution through a macroporous adsorption resin and collecting the effluent; S3. After the effluent is microfiltered and concentrated, the concentrate is collected; S4. Add ethanol to the concentrated solution and let it stand to crystallize, then dry the crystals under reduced pressure to obtain mulberry leaf extract.

2. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: Satisfy at least one of the following conditions ① to ⑥: ① The amount of water added is 5 to 200 times the mass of the mulberry leaves; ② The extraction method is leakage extraction; ③ The filtration is performed using a 70-90 mesh screen; ④ cooling to 20-35° C.; ⑤ The centrifugal speed is 2000-3000rpm; ⑥The centrifugation time is 2 to 10 minutes.

3. The method for preparing the mulberry leaf extract according to claim 2, characterized in that: Satisfy at least one of the following conditions ① to ⑥: ① The temperature of the leakage extraction is 75-90°C; ② When the mass of the mulberry leaves is less than 100 kg, the seepage rate of the seepage extraction is 100 to 200 L / h; ③ When the mass of the mulberry leaves is 100-500 kg, the seepage rate of the seepage extraction is 180-500 L / h; ④ When the mass of the mulberry leaves is 500-800 kg, the seepage rate of the seepage extraction is 500-800 L / h; ⑤ When the mass of the mulberry leaves is greater than 800 kg, the seepage rate of the seepage extraction is 900 to 1500 L / h; ⑥ The leakage extracts a leakage liquid with Brix ≥ 1%.

4. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: Satisfy at least one of the following conditions ①~②: ① The macroporous resin is Seplite Lxd-762 and Seplite Lx-T5; ② After passing through the macroporous adsorption resin, the solution is washed with 2 to 5 times the column volume of water.

5. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: Satisfy at least one of the following conditions ①~②: ① The microfiltration uses a hollow fiber membrane as an RO membrane for reverse osmosis concentration; ② The concentration is reverse osmosis concentration and reduced pressure concentration in sequence.

6. The method for preparing the mulberry leaf extract according to claim 5, characterized in that: Satisfy at least one of the following conditions ①~②: ① The pore size of the RO membrane is 1 to 100 Da; ② The concentration process is to firstly concentrate to Brix 20-25% by reverse osmosis, and then concentrate to Brix 35-40% by reduced pressure.

7. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: Satisfy at least one of the following conditions ① to ③: ① The concentration of the ethanol is 80-100%; ② The amount of ethanol added is 4 to 8 times the volume of the concentrated solution; ③ The standing time is 6 to 20 hours.

8. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: Satisfy at least one of the following conditions ① to ③: ① The yield of the mulberry leaf extract is 5.8-6.7%; ② The content of mulberry leaf polysaccharide in the mulberry leaf extract is 78-85%.

9. Use of the mulberry leaf extract obtained by the preparation method of the mulberry leaf extract according to any one of claims 1 to 8 in fish feed, characterized in that: The feed includes feed for increasing the body length of crucian carp, feed for improving the liver health of crucian carp and feed for improving the intestinal health of crucian carp; Wherein, the improvement of crucian carp liver health also includes improving hepatocyte cavitation; The improvement of crucian carp's intestinal health includes promoting the increase of crucian carp's intestinal folds, increasing the height and number of villi, and reducing the intestinal cavity rate.

10. Use of the mulberry leaf extract obtained by the preparation method of the mulberry leaf extract according to claim 9 in fish feed, characterized in that: Satisfy at least one of the following conditions ① to ③: ① The amount of the mulberry leaf extract added to the feed for increasing the body length of crucian carp is 5.5-6.5 g / kg daily diet; ② The amount of the mulberry leaf extract added to the feed for improving the liver health of crucian carp is 5.5-6.5 g / kg daily diet; ③ The amount of the mulberry leaf extract added to the feed for improving the intestinal health of crucian carp is 10-15g / kg daily diet.

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