Method for extracting mulberry leaf active substance and food additive

Abstract

The present application relates to a kind of mulberry leaf active substance extraction method, the extraction method includes the following steps: A, mulberry leaf is cleaned and dried to moisture content 20-30%;B, place at-1~‑5 ℃, duration 20-30min;C, thawing to normal temperature after immersion, after fishing out, beat, leaf meat is separated from leaf meat;D, leaf meat is placed in 36-40 ℃ water and forms mixed solution, the mass ratio of leaf meat and water is 1: (25-30), yeast is added to mixed solution and nitrogen source and carbon source are added simultaneously, the amount of yeast is 5-10% of the mass of mixed solution, ultrasonic cavitation, frequency 15-35kHz, after soaking time 50-70min, resin method is used to adsorb and extract mulberry leaf alkaloid, ultrasonic cavitation time 5-10min;E, after soaking 90min and when ethanol content reaches 15-25%, then ultrasonic treatment, ultrasonic frequency 5-10kHz, processing time 10-15min, then carry out suction filtration, concentration and freeze drying, obtain mulberry leaf polyphenol, the same process under step extraction is carried out in the present application, and the extraction rate of mulberry leaf alkaloid and mulberry leaf polyphenol can be guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of mulberry leaf processing technology, specifically relating to a method for extracting active substances from mulberry leaves and a food additive. Background Technology

[0002] Mulberry leaves contain numerous active substances, such as alkaloids, polysaccharides, and polyphenols. Extraction of these active substances is a crucial research area. Current techniques mostly involve individual extraction methods, such as hot water extraction, acid / alkali solution extraction, and ethanol extraction. Recent research has also explored ultrasonic extraction and resin adsorption, requiring the selection of optimal extraction methods for different active substances. Furthermore, some researchers have attempted simultaneous extraction of multiple active substances. However, it's difficult to achieve optimal extraction conditions for all these substances simultaneously, necessitating compromises and repeated sampling and testing to guide the removal of impurities, especially alkaloids and polyphenols. As the two most important active components of mulberry leaves, ultrasonic-assisted ethanol extraction is a cost-effective and efficient method for polyphenol extraction. However, ethanol extraction of alkaloids produces significant impurities, and alkaloid purification is challenging. Therefore, further research is needed on extraction methods for active substances from mulberry leaves. Summary of the Invention

[0003] This invention relates to a method for extracting active substances from mulberry leaves and a food additive. The extraction method innovates upon existing separate and simultaneous extraction methods by employing a stepwise continuous extraction process. This method can meet the optimal extraction conditions for different active substances without requiring changes to experimental equipment. The extracted active substances are then used as a food additive.

[0004] The technical solution of this invention is:

[0005] A method for extracting active substances from mulberry leaves, the key point of which is that the extraction method includes the following steps:

[0006] A. Prepare mulberry leaves, wash them clean and dry them until the moisture content is 20-30%;

[0007] B. Place the mulberry leaves at -1 to -5℃ for 20-30 minutes.

[0008] C. Soak and thaw to room temperature, then take them out and pat them to separate the leaf pulp from the leaf flesh.

[0009] D. Place the leaf mesophyll in water at 36-40℃ to form a mixture. The mass ratio of leaf mesophyll to water is 1:(25-30). Add yeast to the mixture and simultaneously add nitrogen and carbon sources. The amount of yeast added is 5-10% of the mass of the mixture. Perform ultrasonic cavitation on the mixture at an ultrasonic frequency of 15-35kHz. After soaking the leaf mesophyll for 50-70 minutes, use the resin method to adsorb and extract mulberry leaf alkaloids. During this process, the ultrasonic cavitation time is 5-10 minutes.

[0010] E. After soaking the leaf pulp for 90 minutes and the ethanol content reaches 15-25%, the mixture is then subjected to ultrasonic treatment at a frequency of 5-10 kHz for 10-15 minutes. Subsequently, it is filtered, concentrated, and freeze-dried to obtain mulberry leaf polyphenols.

[0011] In step C, the beating of mulberry leaves is carried out using a beating device. The beating device includes a machine body with a hopper, a sliding platform inside the machine body, and a belt drive mechanism surrounding it. On both sides of the belt drive mechanism are reciprocating beating units that cooperate with the upper surface of the belt drive mechanism. The reciprocating beating unit includes symmetrically arranged vertical guide rails, vertical sliding rollers, and S-shaped deflectors. Bearings are installed at both ends of the rotating shaft of the vertical sliding rollers. The bearings cooperate with the vertical guide rails. The vertical sliding rollers form a vertical reciprocating motion by moving and disengaging their rotating shafts with the help of the S-shaped deflectors.

[0012] In step C, the surface of the vertical sliding roller is uniformly covered with circular protrusions.

[0013] The hopper output end is provided with a grooved wheel, and the belt drive mechanism is located below the grooved wheel, with a gap of 2-4 cm between the two.

[0014] In step A, the leaves are washed and dried until the moisture content is 25%; in step B, the mulberry leaves are placed at -3℃ for 25 minutes.

[0015] In step D, the leaf mesophyll is placed in water at 38°C to form a mixture, with a leaf mesophyll to water mass ratio of 1:28. The amount of yeast added is 8% of the mass of the mixture. The mixture is subjected to ultrasonic cavitation at a frequency of 25kHz. After the leaf mesophyll is soaked for 60 minutes, mulberry leaf alkaloids are extracted by resin adsorption. During this process, the ultrasonic cavitation time is 8 minutes.

[0016] In step E, after the leaf mesophyll is soaked for 90 minutes and the ethanol content reaches 22%, the mixture is subjected to ultrasonic treatment at a frequency of 8 kHz for 13 minutes.

[0017] The food additive based on the extraction method of the active substances in mulberry leaves described above is characterized in that the food additive comprises 25-40 parts by weight of mulberry leaf alkaloids, 30-55 parts by weight of mulberry leaf polyphenols, 4-8 parts by weight of vitamin C, 8-12 parts by weight of licorice powder, and 7-10 parts by weight of montmorillonite powder.

[0018] The food additives include, by weight, 32 parts of mulberry leaf alkaloids, 42 parts of mulberry leaf polyphenols, 6 parts of vitamin C, 10 parts of licorice powder, and 9 parts of montmorillonite powder.

[0019] The beneficial effects of this invention are:

[0020] 1) This invention employs a stepwise extraction process for mulberry leaf alkaloids and mulberry leaf polyphenols using the same technology. The mulberry leaf alkaloid extraction process creates an ethanol environment, providing conditions for accelerated yeast reproduction and subsequent alcohol metabolism. Simultaneously, the extraction rate of mulberry leaf alkaloids remains high under the effects of crushing pretreatment and ultrasonic treatment. In the subsequent extraction of mulberry leaf polyphenols, due to the gradual production of alcohol, the amount of polyphenols dissolved in alcohol reaches a certain level before meeting the alcohol content requirement, shortening the extraction cycle. After continuous soaking, the extraction yield is correspondingly increased under the same soaking time.

[0021] 2) In this invention, the mulberry leaves are pre-treated by beating to separate the leaf pulp and veins. This eliminates the absorption of mulberry leaf alkaloids and polyphenols by the cellulose in the veins, and also avoids the involvement of impurities in the veins in the extraction process, thus avoiding the need for their removal. The process is simplified and the operation is more convenient. In this invention, the beating device uses vertical sliding rollers with circular protrusions to press the mulberry leaves down in free fall, accompanied by rolling, to beat the pre-frozen and thawed leaf pulp and veins, causing the leaf pulp to separate from the veins. This achieves automated operation of leaf pulp separation and provides technical support for the extraction of large quantities of mulberry leaves. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the patting device in this invention.

[0023] Figure 2 This is a schematic diagram of the vertical sliding roller.

[0024] In the attached diagram, 1 is the hopper, 2 is the machine body, 3 is the sliding platform, 4 is the belt drive mechanism, 5 is the vertical guide rail, 6 is the vertical sliding roller, 601 is the rotating shaft, 602 is the circular protrusion, 7 is the S-shaped dial wheel, 8 is the bearing, and 9 is the grooved wheel. Detailed Implementation

[0025] This invention relates to a method for extracting active substances from mulberry leaves and to a food additive. The extraction method innovatively performs stepwise extraction of mulberry leaf alkaloids and mulberry leaf polyphenols under the same process, maintaining a high extraction rate for both. Through steps such as crushing, ultrasonic low-temperature extraction of mulberry leaf alkaloids, fermentation, and ethanol extraction of mulberry leaf polyphenols, an ethanol environment is created for the extraction of mulberry leaf polyphenols while simultaneously extracting mulberry leaf alkaloids. Both the extracted mulberry leaf alkaloids and mulberry leaf polyphenols can be used as raw materials for food additives. The specific operation process of this invention is illustrated through three examples and two comparative examples.

[0026] Example 1, the extraction method includes the following steps:

[0027] A. Prepare mulberry leaves, wash them clean and dry them to a moisture content of 25%. This moisture content is sufficient to meet the requirements of rapid freezing without forming an ice layer on the surface of the mulberry leaves.

[0028] B. Place mulberry leaves at -3℃ for 25 minutes. At this time, the cell walls of the mulberry leaves will rupture due to freezing.

[0029] C. Soak and thaw to room temperature. At this point, the leaf pulp of the mulberry leaves will soften. After removing them from the water, pat the leaves to separate the leaf pulp from the leaf tissue. This patting should be done using a patting device, such as... Figure 1 and Figure 2 As shown, the beating device includes a body 2 with a hopper 1. Inside the body 2 is a sliding platform 3 and a belt drive mechanism 4 surrounding it. Reciprocating beating units that mate with the upper surface of the belt drive mechanism 4 are arranged on both sides of the belt drive mechanism 4. Each reciprocating beating unit includes symmetrically arranged vertical guide rails 5, vertical sliding rollers 6, and S-shaped deflectors 7. Bearings 8 are installed at both ends of the shaft 601 of the vertical sliding rollers 6. The bearings 8 mate with the vertical guide rails 5. The vertical sliding rollers 6 are driven by the S-shaped deflectors... The wheel 7 moves and disengages from its rotating shaft 601, creating a vertical reciprocating motion. During use, mulberry leaves are placed into the hopper 1. The output end of the hopper 1 is equipped with a grooved wheel 9, and the belt drive mechanism 4 is located below the grooved wheel 9, with a 3cm gap between them. Driven by the grooved wheel 9, the leaves are fed in batches onto the upper surface of the belt drive mechanism 4. The gap is controlled to ensure the mulberry leaves are spread evenly. Subsequently, the mulberry leaves are patted and crushed by the vertical sliding roller 6. The surface of the vertical sliding roller 6 is evenly covered with circular protrusions 602, such as... Figure 1As shown, driven by the counterclockwise rotation of the S-shaped dial 7, the vertical sliding roller 6 moves upward to a certain height and then falls freely. The vertical sliding roller 6 pats the mulberry leaf, and due to the operation of the belt drive mechanism 4, the vertical sliding roller 6 briefly rolls and crushes. Then, it is lifted up by the S-shaped dial 7 to enter the next patting and crushing cycle. After the operation of multiple vertical sliding rollers 6 in the reciprocating patting unit, the leaf pulp and leaf veins in the mulberry leaf gradually separate. After being output from the belt drive mechanism 4, they enter the water, are stirred, and the leaf veins are retrieved, thus achieving the separation of leaf veins and leaf pulp.

[0030] D. Place the leaf mesophyll in water at 38℃ to form a mixture with a leaf mesophyll to water mass ratio of 1:28. Add yeast to the mixture along with nitrogen and carbon sources. The amount of yeast added is 8% of the mixture mass. The amount of carbon and nitrogen sources should be sufficient to meet the reproductive and metabolic needs of the yeast. The amount can be as large as possible, but not too small. At this stage, it is necessary for the yeast to reproduce as much as possible under aerobic conditions. Perform ultrasonic cavitation on the mixture at a frequency of 25kHz. After soaking the leaf mesophyll for 60 minutes, use resin adsorption to extract alkaloids. During this process, the ultrasonic cavitation time is 8 minutes. Combined with the cell wall disruption pretreatment of mulberry leaves, ultrasonic cavitation can help improve the extraction rate and shorten the extraction time of mulberry leaf alkaloids at a low temperature of 38℃. This 38℃ environment can also provide a suitable reproductive and metabolic temperature for yeast. Furthermore, the ultrasonic process can also improve the reproductive and metabolic level of yeast, preparing the environment for the subsequent extraction of mulberry leaf polyphenols.

[0031] E. Oxygen in the mixture is consumed and multiplied by yeast, leading to an anaerobic state. The yeast then metabolizes and produces ethanol. After soaking the leaf pulp for 90 minutes and reaching an ethanol content of 22%, it is soaked for another 30 minutes. The mixture is then subjected to ultrasonic treatment at a frequency of 8 kHz for 13 minutes. Subsequently, it is filtered, concentrated, and freeze-dried to obtain mulberry leaf polyphenols. Ultrasonic-assisted ethanol extraction of mulberry leaf polyphenols can improve the extraction rate of mulberry leaf polyphenols.

[0032] Example 2, the extraction method includes the following steps:

[0033] A. Prepare mulberry leaves, wash them clean and dry them until the moisture content is 22%;

[0034] B. Place the mulberry leaves at -4℃ for 20 minutes;

[0035] C. Soak and thaw to room temperature. At this time, the leaf pulp of the mulberry leaves will soften. After taking them out, pat the mulberry leaves to separate the leaf pulp from the leaf pulp. The patting process is the same as in Example 1.

[0036] D. Place the mesophyll in water at 35℃ to form a mixture with a mesophyll to water mass ratio of 1:26. Add yeast to the mixture and simultaneously add nitrogen and carbon sources. The amount of yeast added is 6% of the mass of the mixture. Perform ultrasonic cavitation on the mixture at an ultrasonic frequency of 20kHz. After soaking the mesophyll for 55 minutes, use the resin method to adsorb and extract alkaloids. During this process, the ultrasonic cavitation time is 6 minutes.

[0037] E. Oxygen in the mixture is consumed and multiplied by yeast. After entering an anaerobic state, the yeast metabolizes and produces ethanol. When the leaf pulp is soaked for 90 minutes and the ethanol content reaches 16%, the mixture is subjected to ultrasonic treatment at a frequency of 6 kHz for 11 minutes. Subsequently, it is filtered, concentrated and freeze-dried to obtain mulberry leaf polyphenols.

[0038] Example 3, the extraction method includes the following steps:

[0039] A. Prepare mulberry leaves, wash them clean, and then dry them until the moisture content is 28%;

[0040] B. Place the mulberry leaves at -1℃ for 28 minutes;

[0041] C. Soak and thaw to room temperature. At this time, the leaf pulp of the mulberry leaves will soften. After taking them out, pat the mulberry leaves to separate the leaf pulp from the leaf pulp. The patting process is the same as in Example 1.

[0042] D. Place the mesophyll in water at 40℃ to form a mixture with a mesophyll to water mass ratio of 1:30. Add yeast to the mixture and simultaneously add nitrogen and carbon sources. The amount of yeast added is 10% of the mass of the mixture. Perform ultrasonic cavitation on the mixture with an ultrasonic frequency of 29kHz. After soaking the mesophyll for 65 minutes, use resin adsorption to extract alkaloids. During this process, the ultrasonic cavitation time is 9 minutes.

[0043] E. Oxygen in the mixture is consumed and multiplied by yeast. After entering an anaerobic state, the yeast metabolizes and produces ethanol. When the leaf pulp is soaked for 90 minutes and the ethanol content reaches 24%, the mixture is ultrasonically treated with an ultrasonic frequency of 9 kHz for 14 minutes. Then, it is filtered, concentrated and freeze-dried to obtain mulberry leaf polyphenols.

[0044] In Comparative Example 1, mulberry leaf alkaloids and mulberry leaf polyphenols were extracted separately. Mulberry leaf alkaloids were extracted using an ultrasonic + hot water extraction method. The mass ratio of mulberry leaves to water was 1:30, the ultrasonic frequency was 25kHz, the hot water temperature was 80 degrees Celsius, and the soaking time was 60 minutes, during which ultrasonic treatment was performed for 8 minutes. After the soaking time was met, resin adsorption extraction was used to obtain mulberry leaf alkaloids.

[0045] Mulberry leaf polyphenols were extracted using an ultrasonic-ethanol extraction method. The leaves were soaked in water at a mass ratio of 1:30, a water temperature of 80℃, an ultrasonic frequency of 10kHz, an ethanol concentration of 30%, and a soaking time of 40 minutes, including 10 minutes of ultrasonication. After the soaking time was met, the leaves were filtered, concentrated, and freeze-dried to obtain mulberry leaf polyphenols.

[0046] Comparative Example 2: Mulberry leaf alkaloids and mulberry leaf polyphenols were extracted using a simultaneous extraction method. The extraction conditions were as follows: the mass ratio of mulberry leaves to water was 1:30, the water temperature was 80℃, the ultrasonic frequency was 15kHz, the ethanol concentration in the water was 15%, the soaking time was 60min, during which ultrasonication was performed for 15min. After the soaking time was met, the mixture was filtered, concentrated, and freeze-dried to obtain a mixture of mulberry leaf alkaloids and mulberry leaf polyphenols.

[0047] The above three examples and Comparative Example 2 all used 500g of mulberry leaves. Comparative Example 1 used 500g of mulberry leaves to test mulberry leaf alkaloids and mulberry leaf polyphenols respectively. Each experiment was repeated in three groups to obtain the corresponding extracts of alkaloids and polyphenols, as shown in Tables 1 and 2.

[0048] Table 1. Extract content of mulberry leaf alkaloids in each experiment.

[0049] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Experiment 1 15.5mg / g 14.2 mg / g 14.1 mg / g 15.7mg / g 10.3 mg / g Experiment 2 15.8 mg / g 14.3 mg / g 14.4 mg / g 15.7mg / g 10.8 mg / g Experiment 3 16.1 mg / g 14.6 mg / g 13.8 mg / g 15.9 mg / g 9.4 mg / g

[0050] The extraction rates of Examples 1-3 and Comparative Example 1 in Table 1 are similar. Among them, the extraction rates of Example 1 and Comparative Example 1 in Examples 1-3 using the method of the present invention are basically the same, indicating that the extraction rate of mulberry leaf alkaloids extracted by the method of the present invention is basically the same as that of the process of extracting mulberry leaf alkaloids alone. The method of ultrasonication + crushing at low temperature is realized to improve the extraction rate of mulberry leaf alkaloids. However, in Comparative Example 2, due to the simultaneous extraction of alkaloids and polyphenols, the experimental conditions and environment were adjusted, and the extraction rate of mulberry leaf alkaloids was significantly reduced. The ethanol environment has a greater impact on the mulberry leaf alkaloids.

[0051] Table 2. Extract content of mulberry leaf polyphenols in each experiment.

[0052] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Experiment 1 3.88 mg / g 3.65mg / g 3.54 mg / g 3.76 mg / g 2.32 mg / g Experiment 2 3.91 mg / g 3.33 mg / g 3.51 mg / g 3.88 mg / g 2.21 mg / g Experiment 3 3.65mg / g 3.26 mg / g 3.44 mg / g 3.73 mg / g 2.47 mg / g

[0053] Table 2 shows that the extraction rates of mulberry leaf polyphenols in Examples 1-3 and Comparative Example 1 are similar. In fact, the extraction rate of mulberry leaf polyphenols in Example 1 is higher than that in Comparative Example 1. This indicates that the extraction of polyphenols from mulberry leaves begins during the process of yeast producing alcohol. As the concentration increases, the content of polyphenols soluble in ethanol also increases. Therefore, when the soaking time is similar, the method in Example 1 can improve the extraction rate of mulberry leaf polyphenols more significantly, at least to the same level as the extraction rate of mulberry leaf polyphenols alone. The decrease in the extraction rate in Comparative Example 2 is also because the simultaneous extraction with mulberry leaf alkaloids was carried out under less than ideal experimental conditions, which significantly affected the extraction yield of mulberry leaf polyphenols.

[0054] The mulberry leaf alkaloids and polyphenols extracted using the method of this invention can be used in the production of food additives. This food additive comprises, by weight, 32 parts mulberry leaf alkaloids, 42 parts mulberry leaf polyphenols, 6 parts vitamin C (antioxidant), 10 parts licorice powder (to promote digestion and absorption), and 9 parts montmorillonite powder (desiccant). Vitamin C, as an antioxidant, exerts its antioxidant effect during storage and use, preventing the consumption of mulberry leaf polyphenols, which are also antioxidants, during this process, allowing the efficacy of mulberry leaf polyphenols to be released during consumption. The addition of licorice powder enhances gastrointestinal digestion, better aiding in the absorption rate of mulberry leaf alkaloids and polyphenols. Montmorillonite powder, used as a desiccant, provides a dry environment during storage and use, better promoting the uniformity of mulberry leaf alkaloids and polyphenols in the mixture during food processing, significantly contributing to the uniformity of food texture. This food additive is mainly used in pasta products, participating in the yeast fermentation process of pasta products.

Claims

1. A method for extracting active substances from mulberry leaves, characterized in that, The extraction method includes the following steps: A. Prepare mulberry leaves, wash them clean, and then dry them until the moisture content is 20-30%; B. Place the mulberry leaves at -1 to -5℃ for 20-30 minutes. C. Soak and thaw to room temperature, then take them out and pat them to separate the leaf pulp from the leaf flesh. D. Place the leaf mesophyll in water at 36-40℃ to form a mixture. The mass ratio of leaf mesophyll to water is 1:(25-30). Add yeast to the mixture and simultaneously add nitrogen and carbon sources. The amount of yeast added is 5-10% of the mass of the mixture. Perform ultrasonic cavitation on the mixture at an ultrasonic frequency of 15-35kHz. After soaking the leaf mesophyll for 50-70 minutes, use the resin method to adsorb and extract mulberry leaf alkaloids. During this process, the ultrasonic cavitation time is 5-10 minutes. E. Oxygen in the mixture is consumed and multiplied by yeast. After entering a hypoxic state, the yeast metabolizes and produces ethanol. After soaking the leaf pulp for 90 minutes and the ethanol content reaches 15-25%, the mixture is then subjected to ultrasonic treatment at a frequency of 5-10 kHz for 10-15 minutes. Subsequently, it is filtered, concentrated, and freeze-dried to obtain mulberry leaf polyphenols. In step C, the beating of mulberry leaves is carried out by means of a beating device. The beating device includes a machine body (2) with a hopper (1). The machine body (2) is provided with a sliding platform (3) and a belt drive mechanism (4) arranged around it. On both sides of the belt drive mechanism (4), there are reciprocating beating units that cooperate with the upper end face of the belt drive mechanism (4). The reciprocating beating unit includes a symmetrically arranged vertical guide rail (5), a vertical sliding roller (6) and an S-shaped dial (7). Bearings (8) are installed at both ends of the shaft (601) of the vertical sliding roller (6). The bearings (8) cooperate with the vertical guide rail (5). The vertical sliding roller (6) forms a vertical reciprocating motion by means of the S-shaped dial (7) to move and disengage its shaft (601). The vertical sliding roller (6) has circular protrusions (602) evenly arranged on its surface; The output end of the hopper (1) is provided with a grooved wheel (9), and the belt drive mechanism (4) is located below the grooved wheel (9), with a gap of 2-4cm between them.

2. The method for extracting active substances from mulberry leaves according to claim 1, characterized in that, In step A, the leaves are cleaned and then dried to a moisture content of 25%; in step B, the mulberry leaves are placed at -3℃ for 25 minutes.

3. The method for extracting active substances from mulberry leaves according to claim 1, characterized in that, In step D, the leaf mesophyll is placed in water at 38°C to form a mixture, with a leaf mesophyll to water mass ratio of 1:

28. The amount of yeast added is 8% of the mass of the mixture. The mixture is subjected to ultrasonic cavitation at a frequency of 25kHz. After the leaf mesophyll is soaked for 60 minutes, mulberry leaf alkaloids are extracted by resin adsorption. During this process, the ultrasonic cavitation time is 8 minutes.

4. The method for extracting active substances from mulberry leaves according to claim 1, characterized in that, In step E, after the leaf mesophyll is soaked for 90 minutes and the ethanol content reaches 22%, the mixture is subjected to ultrasonic treatment at a frequency of 8 kHz for 13 minutes.

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