Preparation method and application of mulberry leaf extract with high stability and high activity in protein food system

Abstract

The invention discloses a preparation method and application of a mulberry leaf extract with high stability and high activity in a protein food system, and belongs to the field of food processing and nutrition and health. The mulberry leaf extract is prepared by taking mulberry leaves as a raw material through steam explosion and ball milling pretreatment, compound enzyme directional enzymolysis, ultrasonic-assisted extraction, protein-induced sedimentation combined with ultrafiltration membrane selective impurity removal, compound stabilization treatment and drying. The obtained extract is rich in 1-deoxynojirimycin (DNJ) and mulberry leaf polysaccharide, high-polymerization-degree polyphenol is remarkably removed, and high clarity and heat stability are achieved. The DNJ retention rate gt is obtained after the milk is stored at 37 DEG C for 7 days when the milk protein is applied to a milk system; and therefore, the product prepared by the method is obviously superior to the product prepared by the prior art. The extract is pure in flavor and high in biological activity, and can be widely applied to the field of healthy foods such as dairy products and protein beverages.

Description

Technical Field

[0001] This invention belongs to the field of food processing and nutrition technology, and relates to a method for preparing and applying a mulberry leaf extract with high stability and activity in protein food systems. Specifically, it relates to a method for extracting and stabilizing plant functional components, and particularly to a method for preparing and applying a mulberry leaf extract based on multi-stage selective extraction and impurity removal processes that maintains high stability and activity in protein food systems. The extract is prepared from mulberry leaves using steam flash explosion, enzymatic hydrolysis, ultrasonic extraction, protein-polyphenol composite impurity removal, ultrafiltration separation, and compound stabilization steps. It retains the key active component, primarily 1-deoxynojirimycin (DNJ), effectively avoiding precipitation or inactivation reactions with protein systems. It is suitable for adding functional components and enhancing health value in protein-rich food systems such as milk, yogurt, dairy beverages, and soy milk. Background Technology

[0002] Mulberry leaves are a common ingredient in traditional Chinese medicine's dietary therapy system, possessing excellent medicinal and edible properties. Modern research shows that mulberry leaves are rich in various active ingredients, including 1-deoxynojirimycin (DNJ), polysaccharides, and polyphenols. Among them, DNJ, due to its structural similarity to α-glucosidase transition state inhibitors, has been proven to have good physiological activities such as lowering blood sugar, regulating metabolism, and antioxidation, making it an important candidate ingredient for developing metabolic regulation functional foods.

[0003] Currently, commercially available mulberry leaf extracts are mostly used in health foods or traditional Chinese medicine compound preparations. However, there are many technical bottlenecks when applying them to protein-rich foods such as dairy products and plant protein beverages:

[0004] (1) The extraction process is outdated, resulting in serious loss of active ingredients, especially small molecule components such as DNJ, which are easily destroyed by heat, enzymes or pH.

[0005] (2) Polyphenols are structurally active and easily undergo oxidative cross-linking reactions with proteins, leading to precipitation or flocculation in the emulsion system, affecting the clarity and taste of the product, and even causing irreversible loss of active substances.

[0006] (3) The extract contains a variety of unstable components, especially in high-temperature storage or pH change environments, which are prone to browning, oxidation or stratification, which seriously limits its application as a functional factor in protein foods.

[0007] Furthermore, existing technologies rarely offer systematically designed solutions for the "stability of plant extracts in protein reactions," making it difficult to simultaneously achieve high yield, high activity retention, and good application stability within protein systems. Therefore, there is an urgent need to develop an innovative extraction method that can selectively remove components that adversely interact with proteins while preserving the main functional factors of mulberry leaves, thereby obtaining plant extracts with both high bioactivity and good application stability, thus broadening their application scenarios in the food industry. Summary of the Invention

[0008] Objective: To address the key technical problems of poor stability, easy precipitation and inactivation, and bitter taste of existing mulberry leaf extracts in protein food systems (such as milk, yogurt, and soy milk), this invention provides a highly stable and highly active mulberry leaf extract suitable for protein food systems and its preparation method. This method is based on a pure water extraction process, which is safe and reliable. Combining core technologies such as multi-stage directional extraction, protein-assisted selective impurity removal, and compound stabilization, it can effectively enrich the main functional components of mulberry leaves (such as DNJ and mulberry leaf polysaccharides) while specifically removing polyphenolic components that easily react adversely with proteins, significantly improving the compatibility and functionality of the extract in high-protein food systems.

[0009] To achieve the above objectives, the present invention provides a method for preparing mulberry leaf extract with high stability and high activity in protein food systems, comprising the following steps:

[0010] S1. The washed and dried mulberry leaves are pretreated by steam flash explosion and ball milling to obtain mulberry leaf powder;

[0011] S2. Disperse the mulberry leaf powder obtained in step S1 in reverse osmosis water, add enzyme preparation for directional enzymatic hydrolysis, and then extract with ultrasonic assistance to obtain primary extract.

[0012] S3. Add mulberry leaf protein for treatment, filter the coarse fraction, and then perform membrane separation to obtain a concentrated solution. Add a composite stabilizer for treatment and then dry to obtain mulberry leaf extract.

[0013] Optionally, in some embodiments of the present invention, the steam flash explosion treatment conditions in step S1 include: pressure 0.5-3 MPa and flash explosion time 150 s-450 s.

[0014] Optionally, in some embodiments of the present invention, in step S1, the ball milling conditions include: a ball-to-material ratio of 1:(1-8)w / w, a processing frequency of 100-500 r / min, and a processing time of 10-120 min.

[0015] Optionally, in some embodiments of the present invention, in step S2, the ratio of mulberry leaf powder to reverse osmosis water is 1:(5-10)w / w.

[0016] Optionally, in some embodiments of the present invention, the enzyme preparation includes one or more of cellulase, xylanase, β-glucanase, hemicellulase, and cellobiase.

[0017] Optionally, in some embodiments of the present invention, the amount of enzyme preparation used is 0.1%-1% (w / w) of mulberry leaf powder.

[0018] Optionally, in some embodiments of the present invention, the enzymatic hydrolysis time is 30-90 min.

[0019] Optionally, in some embodiments of the present invention, in step S2, the ultrasonic extraction step uses an ultrasonic frequency of 50-180 kHz and a processing time of 10-120 min.

[0020] Optionally, in some embodiments of the present invention, in step S3, the amount of protein added is 1%-3% (w / w) of the mulberry leaf powder.

[0021] Optionally, in some embodiments of the present invention, in step S3, the protein addition treatment refers to adding protein to induce sedimentation.

[0022] Optionally, in some embodiments of the present invention, in step S3, the protein is one or a combination of mulberry leaf protein, wheat protein, and soybean protein.

[0023] Optionally, in some embodiments of the present invention, in step S3, the coarse filtration is performed using one or a combination of pressure filtration, horizontal screw centrifugation, or butterfly centrifugation.

[0024] Optionally, in some embodiments of the present invention, in step S3, the membrane separation includes inorganic membrane separation or organic membrane separation.

[0025] Optionally, in some embodiments of the present invention, in step S3, the composite stabilizer includes β-cyclodextrin and sodium alginate, and the addition amounts are 0.1%-0.5% and 0.1%-0.3% of the mass of mulberry leaf powder, respectively.

[0026] Optionally, in some embodiments of the present invention, in step S3, the addition of the composite stabilizer treatment refers to stirring at 45-55°C for 30 minutes to fully dissolve it.

[0027] A second objective of this invention is to provide a mulberry leaf extract obtained according to any of the preparation methods described above.

[0028] Optionally, in some embodiments of the present invention, the mulberry leaf extract is a yellowish-brown powder, and a 1% aqueous solution is clear and transparent, comprising, by mass fraction, 0.5%-8% DNJ, 40-70% polysaccharides, and less than 0.2% polyphenols.

[0029] A third objective of this invention is to provide the application of any of the above-described mulberry leaf extracts in foods containing milk protein or plant protein, said foods including milk beverages, milk, yogurt, milk powder, protein bars, or meal replacement shakes.

[0030] Optionally, in some embodiments of the present invention, the amount of mulberry leaf extract added to the food containing milk protein or plant protein is 0.1% to 5% (w / w).

[0031] Optionally, in some embodiments of the present invention, the protein aggregation degree in the food is less than 20%.

[0032] Optionally, in some embodiments of the present invention, the present invention provides a method for preparing a mulberry leaf extract with high stability and high activity in a protein food system, comprising the following steps:

[0033] S1. Raw material pretreatment: The washed and dried mulberry leaves are subjected to steam flash explosion treatment and ball milling pretreatment in sequence to destroy the cell wall structure and obtain mulberry leaf powder with uniform particle size, thereby improving the release efficiency of subsequent effective ingredients.

[0034] S2. Release of active ingredients: The obtained mulberry leaf powder is dispersed in purified water, and compound enzyme preparations (such as cellulase, xylanase, hemicellulase, etc.) are added for targeted enzymatic hydrolysis. Ultrasonic extraction is used to significantly improve the extraction rate of DNJ and polysaccharide components.

[0035] S3. Selective impurity removal: A certain proportion of mulberry leaf protein is added to the extract after enzymatic hydrolysis and leaching. Through the natural affinity between protein and polyphenol, a protein-polyphenol complex is induced to form, precipitating or flocculating undesirable components that are prone to reacting with food proteins (such as high-polymer polyphenols). Subsequently, the precipitate is removed by means of pressure filtration, centrifugation, etc., and the extract is initially clarified.

[0036] S4. Fine separation and concentration: The clarified liquid is further processed through an ultrafiltration membrane system with a specific molecular weight cutoff range (2500–5000 Da) to effectively remove residual macromolecular impurities and enrich DNJ and polysaccharide target functional components to obtain mulberry leaf extract concentrate.

[0037] S5. Stabilization treatment: Add low molecular weight inclusion agents (such as β-cyclodextrin) and high molecular weight stabilizers (such as sodium alginate) to the concentrate to construct a stable system, forming intermolecular inclusion and steric barrier effects, which significantly improves the clarity, dispersibility and thermal stability of the extract in protein food systems.

[0038] S6. Drying the finished product: The stabilized liquid is spray-dried or freeze-dried to obtain the target mulberry leaf extract powder, which is convenient for storage and industrial application.

[0039] The mulberry leaf extract obtained by this invention is enriched with DNJ and polysaccharides in its composition, while significantly reducing the content of high-molecular-weight polyphenols that are detrimental to the protein system. Its main advantages are as follows:

[0040] (1) It exhibits excellent stability in protein systems: after being stored at 37°C for 60 days, the milk containing mulberry leaf extract remained homogeneous, with no precipitation or stratification, and the DNJ retention rate was higher than 95%;

[0041] (2) High retention of functional components and significant improvement in flavor: After removing tannins and polymeric polyphenols, the product has a pure flavor and no bitter or off-flavors, making it suitable for protein drinks with high taste requirements.

[0042] (3) High level of process safety and industrialization: The entire process is carried out with pure water, the process parameters are clear, the raw materials are clean, and it is suitable for large-scale food-grade production;

[0043] (4) Wide range of applications: This extract can be widely used in milk, yogurt, soy milk, protein bars, meal replacement shakes and other milk protein or plant protein foods. It is also suitable for nutritional supplements, functional beverages and other food scenarios, and has good promotion potential.

[0044] In summary, this invention constructs a mulberry leaf extract preparation technology system for food protein systems, which systematically solves traditional problems such as activity loss, protein incompatibility and poor flavor, and achieves the triple goals of high stability, high functionality and adaptability to food applications, and has important application value and industrialization prospects.

[0045] The beneficial effects of this invention are:

[0046] This invention provides a method for preparing and applying a mulberry leaf extract with high stability and high activity in protein food systems, which has the following beneficial effects:

[0047] (1) Improve the efficiency of component enrichment and activity retention: The present invention adopts a pretreatment method of steam flash explosion combined with ball milling to effectively break the cell wall and release the embedded components in the cell; combined with enzymatic hydrolysis-ultrasound synergistic extraction method, the extraction rate of functional components (especially 1-deoxynojirimycin and polysaccharides) in mulberry leaves is significantly improved; the entire extraction process is carried out in a pure water system to avoid degradation of active substances caused by organic solvents and ensure that the active components of the extract are retained to the maximum extent under mild conditions.

[0048] (2) Achieving high compatibility with protein food systems: In response to the problem that polyphenols in traditional mulberry leaf extract are prone to flocculation reaction with milk protein, this invention innovatively introduces a selective impurity removal technology of "protein-induced sedimentation + ultrafiltration separation". By adding food-grade mulberry leaf protein to form a protein-polyphenol complex and removing it, the content of high molecular weight tannins and polymeric polyphenols is significantly reduced, thus solving the problems of precipitation and turbidity of extracts in dairy products and other protein foods from the source.

[0049] (3) Stabilization strategy to improve product thermal stability and clarity: β-cyclodextrin and sodium alginate are added to the extract concentrate to construct a stable system with molecular-level inclusion and polymer barrier synergy, effectively improving the thermal stability and dispersibility of the extract in the protein system. The mulberry leaf extract prepared by the method of this invention is soluble in milk and remains homogeneous after being stored at 37°C for 60 days, without precipitation or stratification, and the DNJ retention rate is higher than 95%.

[0050] (4) The product has a pure flavor and good palatability: By precisely removing the bitter polyphenol components and impurities and pigments, the mulberry leaf extract has a significantly improved flavor, overcoming the problem of off-flavors caused by traditional extracts in food additives, meeting consumers' needs for taste and sensory quality, and is suitable for products such as dairy drinks and plant protein drinks with high flavor requirements.

[0051] (5) The process is safe and environmentally friendly, and suitable for industrial application in the food industry: the entire process uses pure water extraction and food-grade auxiliary materials. There are no organic solvent residues or chemical precipitants used in the production process, which meets the clean production standards of the modern food industry. The preparation process parameters are clear and the process is controllable, making it suitable for large-scale industrial promotion.

[0052] (6) Wide range of applications and great market potential: The extract obtained has good application stability and functionality, and can be widely used in functional foods with high protein content such as milk, yogurt, soy milk, protein bars, and meal replacement shakes. It is also suitable for nutritional supplements, health drinks, functional foods and other fields, providing a raw material basis for the development of new foods that regulate metabolism and supplement nutrition. Detailed Implementation

[0053] Currently, there is limited research on the processing techniques for mulberry leaf extract in protein food systems, addressing issues such as poor stability, easy loss of activity, and unpleasant flavor. DNJ and polysaccharides are the main active ingredients in mulberry leaves, playing a crucial role in combating glycation and other functionalities. While polyphenols also possess certain biological activity, excessive amounts of high-molecular-weight polyphenols can easily react adversely with proteins, leading to precipitation and reduced activity, thus affecting the extract's effectiveness in protein foods.

[0054] Therefore, this invention proposes a method for preparing and applying mulberry leaf extract with high stability and activity in protein food systems. Through pretreatment with steam flash explosion and ball milling, combined with targeted enzymatic hydrolysis and ultrasonic-assisted extraction, key active ingredients such as DNJ and polysaccharides are fully released. Simultaneously, a novel approach is adopted to introduce protein-assisted formation of polyphenol-protein complexes, combined with centrifugation and ultrafiltration techniques to selectively remove high-molecular-weight polyphenols and impurities detrimental to the stability of protein systems, thereby significantly improving the stability and activity retention of the extract in protein-rich foods. Finally, a composite stabilizer consisting of β-cyclodextrin and sodium alginate is used to further enhance its compatibility and sensory quality in complex matrices.

[0055] This process avoids the use of organic solvents and non-food additives, ensuring the safety and applicability of the product, meeting the stringent requirements of protein foods for functional additives, and has good prospects for industrial application.

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0057] In this embodiment of the invention, all materials were commercially available. Mulberry leaf protein, wheat protein, and soybean protein were purchased from Wuxi Langjian Biotechnology Co., Ltd., with a protein content greater than 40g / 100g. Cellulase and xylanase were purchased from Wuhan Xinhua Yang Biotechnology Co., Ltd., with enzyme activities greater than 3×10⁻⁶. 5 U / g.

[0058] It should be noted that in the following embodiments, all substances are expressed in parts by weight.

[0059] Example 1:

[0060] A method for preparing mulberry leaf extract that maintains high stability and high activity in protein food systems, comprising the following specific steps:

[0061] (1) Raw material pretreatment: Take the cleaned and dried mulberry leaves and place them in a steam flash explosion device for treatment under the conditions of 0.5 MPa pressure and 150 s flash explosion time. After flash explosion, take them out and cool them, add 500 parts of grinding balls and 500 parts of mulberry leaf powder to a ball mill, and ball mill at a frequency of 100 r / min for 10 min to obtain pretreated mulberry leaf powder with uniform particle size;

[0062] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 100 parts of mulberry leaf powder treated in step (1) were dispersed in 500 parts of reverse osmosis water, and 0.05 parts each of cellulase and xylanase were added for enzymatic hydrolysis for 30 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 50 kHz for 10 min to enhance the release of active substances and obtain the primary extract.

[0063] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) was separated by horizontal decanter centrifuge (2000 rpm), and the supernatant was collected. One part of mulberry leaf protein was added to the supernatant and stirred at 45℃ for 30 min to allow the polyphenolic components to bind with the protein and form a precipitate. The supernatant was centrifuged again (3000 rpm), the precipitate was discarded, and the supernatant was retained;

[0064] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0065] (5) Stabilizer formulation and spray drying: 0.1 parts of β-cyclodextrin and 0.1 parts of sodium alginate were added to the concentrate in sequence, and the mixture was sheared and stirred at high speed for 30 min to form a homogeneous and stable system. The solution was then spray dried to obtain mulberry leaf extract.

[0066] Example 2:

[0067] A method for preparing mulberry leaf extract with high stability and high activity in protein food systems, the specific steps of which are as follows:

[0068] (1) Raw material pretreatment: Take the cleaned and dried mulberry leaves and place them in a steam flash explosion device for treatment under the conditions of 3 MPa pressure and 450 s flash explosion time. After flash explosion, take them out and cool them, add 500 parts of grinding balls and 4000 parts of mulberry leaf powder to a ball mill, and ball mill at a frequency of 500 r / min for 120 min to obtain pretreated mulberry leaf powder with uniform particle size;

[0069] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 400 parts of mulberry leaf powder treated in step (1) were dispersed in 4000 parts of reverse osmosis water, and 0.8 parts of cellulase, 0.8 parts of xylanase, 0.8 parts of β-glucanase, 0.8 parts of hemicellulase and 0.8 parts of cellobiase were added. Enzymatic hydrolysis was carried out for 90 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 180 kHz for 120 min to enhance the release of active substances and obtain the primary extract.

[0070] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) was separated by horizontal decanter centrifuge (2000 rpm), and the supernatant was collected. 12 parts of mulberry leaf protein were added to the supernatant and stirred at 55℃ for 30 min to allow the polyphenolic components to bind with the protein and form a precipitate. The mixture was then centrifuged again by horizontal decanter centrifuge (3000 rpm), the precipitate was discarded, and the supernatant was retained.

[0071] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0072] (5) Stabilizer formulation and spray drying: 2 parts β-cyclodextrin and 1.2 parts sodium alginate were added to the concentrate in sequence, and the mixture was sheared and stirred at high speed for 30 min to form a homogeneous and stable system. The solution was then freeze-dried to obtain mulberry leaf extract.

[0073] Example 3:

[0074] A method for preparing mulberry leaf extract with high stability and high activity in protein food systems, the specific steps of which are as follows:

[0075] (1) Raw material pretreatment: Take the cleaned and dried mulberry leaves and place them in a steam flash explosion device for treatment under the conditions of 2 MPa pressure and 300 s flash explosion time. After flash explosion, take them out and cool them, add 500 parts of grinding balls and 2500 parts of mulberry leaf powder to a ball mill, and ball mill at a frequency of 300 r / min for 65 min to obtain pretreated mulberry leaf powder with uniform particle size;

[0076] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 400 parts of mulberry leaf powder treated in step (1) were dispersed in 3000 parts of reverse osmosis water, and 2 parts of cellobiase were added for enzymatic hydrolysis for 90 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 115 kHz for 65 min to enhance the release of active substances and obtain the primary extract.

[0077] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) was separated by horizontal spiral centrifuge (2000 rpm), and the supernatant was collected. Eight parts of mulberry leaf protein were added to the supernatant and stirred at 50℃ for 30 min to allow the polyphenols to bind with the protein and form a precipitate. The supernatant was centrifuged again (3000 rpm), the precipitate was discarded, and the supernatant was retained.

[0078] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0079] (5) Stabilizer formulation and spray drying: 1.2 parts of β-cyclodextrin and 0.8 parts of sodium alginate were added to the concentrate in sequence, and the mixture was sheared and stirred at high speed for 30 min to form a homogeneous and stable system. The solution was then vacuum dried to obtain mulberry leaf extract.

[0080] Comparative Example 1

[0081] The preparation method is the same as in Example 1, except that the mulberry leaf extract is prepared without steam flash explosion and ball mill pretreatment. The specific steps are as follows:

[0082] (1) Raw material pretreatment: Take mulberry leaves that have been washed and dried, and crush them to obtain mulberry leaf powder;

[0083] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 100 parts of mulberry leaf powder treated in step (1) were dispersed in 500 parts of reverse osmosis water, and 0.05 parts each of cellulase and xylanase were added for enzymatic hydrolysis for 30 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 50 kHz for 10 min to enhance the release of active substances and obtain the primary extract.

[0084] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) was separated by horizontal decanter centrifuge (2000 rpm), and the supernatant was collected. One part of mulberry leaf protein was added to the supernatant and stirred at 45℃ for 30 min to allow the polyphenolic components to bind with the protein and form a precipitate. The supernatant was centrifuged again (3000 rpm), the precipitate was discarded, and the supernatant was retained;

[0085] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0086] (5) Stabilizer formulation and spray drying: 0.1 parts of β-cyclodextrin and 0.1 parts of sodium alginate were added to the concentrate in sequence, and the mixture was sheared and stirred at high speed for 30 min to form a homogeneous and stable system. The solution was then spray dried to obtain mulberry leaf extract.

[0087] Comparative Example 2

[0088] The preparation method is the same as in Example 1, except that the mulberry leaf extract is prepared without protein-assisted impurity removal treatment. The specific steps are as follows:

[0089] (1) Raw material pretreatment: Take the cleaned and dried mulberry leaves and place them in a steam flash explosion device for treatment under the conditions of 0.5 MPa pressure and 150 s flash explosion time. After flash explosion, take them out and cool them, add 500 parts of grinding balls and 500 parts of mulberry leaf powder to a ball mill, and ball mill at a frequency of 100 r / min for 10 min to obtain pretreated mulberry leaf powder with uniform particle size;

[0090] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 100 parts of mulberry leaf powder treated in step (1) were dispersed in 500 parts of reverse osmosis water, and 0.05 parts each of cellulase and xylanase were added for enzymatic hydrolysis for 30 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 50 kHz for 10 min to enhance the release of active substances and obtain the primary extract.

[0091] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) is separated by horizontal decanter centrifuge (2000 rpm), and the supernatant is collected. The extract is then centrifuged again by horizontal decanter centrifuge (3000 rpm), the precipitate is discarded, and the supernatant is retained;

[0092] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0093] (5) Stabilizer formulation and spray drying: 0.1 parts of β-cyclodextrin and 0.1 parts of sodium alginate were added to the concentrate in sequence, and the mixture was sheared and stirred at high speed for 30 min to form a homogeneous and stable system. The solution was then spray dried to obtain mulberry leaf extract.

[0094] Comparative Example 3

[0095] The preparation method is the same as in Example 1, except that the mulberry leaf extract is prepared without stabilizing agent compounding treatment. The specific steps are as follows:

[0096] (1) Raw material pretreatment: Take the cleaned and dried mulberry leaves and place them in a steam flash explosion device for treatment under the conditions of 0.5 MPa pressure and 150 s flash explosion time. After flash explosion, take them out and cool them, add 500 parts of grinding balls and 500 parts of mulberry leaf powder to a ball mill, and ball mill at a frequency of 100 r / min for 10 min to obtain pretreated mulberry leaf powder with uniform particle size;

[0097] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 100 parts of mulberry leaf powder treated in step (1) were dispersed in 500 parts of reverse osmosis water, and 0.05 parts each of cellulase and xylanase were added for enzymatic hydrolysis for 30 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 50 kHz for 10 min to enhance the release of active substances and obtain the primary extract.

[0098] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) was separated by horizontal decanter centrifuge (2000 rpm), and the supernatant was collected. One part of mulberry leaf protein was added to the supernatant and stirred at 45℃ for 30 min to allow the polyphenolic components to bind with the protein and form a precipitate. The supernatant was centrifuged again (3000 rpm), the precipitate was discarded, and the supernatant was retained;

[0099] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0100] (5) Stabilizer compounding and spray drying: The concentrate was spray dried to obtain mulberry leaf extract.

[0101] Comparative Example 4

[0102] The preparation method is the same as in Example 1, except that the mulberry leaf extract is prepared without protein-assisted impurity removal and without stabilizer compounding. The specific steps are as follows:

[0103] (1) Raw material pretreatment: Take the cleaned and dried mulberry leaves and place them in a steam flash explosion device for treatment under the conditions of 0.5 MPa pressure and 150 s flash explosion time. After flash explosion, take them out and cool them, add 500 parts of grinding balls and 500 parts of mulberry leaf powder to a ball mill, and ball mill at a frequency of 100 r / min for 10 min to obtain pretreated mulberry leaf powder with uniform particle size;

[0104] (2) Targeted enzymatic hydrolysis and ultrasound-assisted extraction: 100 parts of mulberry leaf powder treated in step (1) were dispersed in 500 parts of reverse osmosis water, and 0.05 parts each of cellulase and xylanase were added for enzymatic hydrolysis for 30 min. After enzymatic hydrolysis, ultrasound treatment was applied at a frequency of 50 kHz for 10 min to enhance the release of active substances and obtain the primary extract.

[0105] (3) Protein-assisted impurity removal: The primary extract obtained in step (2) is separated by horizontal decanter centrifuge (2000 rpm), and the supernatant is collected. The extract is then centrifuged again by horizontal decanter centrifuge (3000 rpm), the precipitate is discarded, and the supernatant is retained;

[0106] (4) Membrane filtration and concentration: The supernatant from step (3) is passed through a 5000 Da organic membrane and a 200 nm inorganic membrane to remove macromolecular impurities and residual unstable components. The filtrate is then concentrated by evaporation to obtain a concentrated mulberry leaf extract.

[0107] (5) Stabilizer compounding and spray drying: The concentrate was spray dried to obtain mulberry leaf extract.

[0108] Characterization and comparison of results from examples and comparative examples:

[0109] 1. Determination of the content and yield of active ingredients in mulberry leaf extract

[0110] 1-Deoxynojirimycin (1-DNJ) is a characteristic component of mulberry leaf extract, and mulberry leaf polysaccharides are also excellent active ingredients in mulberry leaves.

[0111] The content of 1-DNJ in mulberry leaf extract was determined and the yield was calculated according to the method in GB / T 40642. The contents of mulberry leaf polysaccharides and mulberry leaf polyphenols in the extract were determined and the yields were calculated using the phenol-sulfuric acid method and the Folin-Ciocalteu method, respectively. The test results are shown in Tables 1 and 2.

[0112] Table 1 Comparison of active ingredient content in mulberry leaf extract

[0113]

[0114] Table 2 Comparison of extraction rates of active ingredients in mulberry leaf extract

[0115]

[0116] The test results in Tables 1 and 2 show that the content of 1-DNJ and mulberry leaf polysaccharides and the corresponding extraction rates of the mulberry leaf extracts prepared by the method of the present invention in Examples 1-3 and Comparative Examples 2-3 are significantly higher than those in Comparative Example 1. After pretreatment by steam flash explosion and ball mill, some insoluble fiber is converted into soluble polysaccharides, and the dissolution of mulberry leaf polysaccharides and 1-DNJ increases. The method of the present invention can significantly improve the content and extraction rate of active ingredients in mulberry leaf extracts.

[0117] Meanwhile, it can be found that the polyphenol content in the mulberry leaf extracts prepared in Examples 1-3 is significantly lower than that in Comparative Example 2. In this invention, after pretreating the mulberry leaf extract concentrate with mulberry leaf protein, the mulberry leaf protein and polyphenols undergo complexation, and the polyphenols in the mulberry leaf extract are pre-precipitated, which reduces the polyphenol content in the mulberry leaf extract and is beneficial to the application of mulberry leaf extract in protein systems.

[0118] 2. Stability evaluation of mulberry leaf extract in protein food systems

[0119] To verify the stability of the mulberry leaf extract of this invention in a high-protein food system, milk was used as a representative system to compare the protein aggregation behavior of the extract of this invention and a comparative extract after addition. Dynamic light scattering (DLS) was used to detect changes in protein particle size, reflecting its compatibility and dispersion stability in the system.

[0120] The mulberry leaf extracts prepared in Examples 1, 2, 3, and 4 were added to milk systems (0.5%, w / w), and the average particle sizes measured by DLS were 185 nm, 430 nm, 255 nm, and 710 nm, respectively. The particle size of the blank milk system was 165 nm. In Example 1, the mulberry leaf extract treated with mulberry leaf protein and stabilizer did not cause flocculation in the milk system, and the particle size of the milk system did not change significantly. In Comparative Example 4, the mulberry leaf extract contained a high content of polyphenols and was not combined with a stabilizer; the particle size of the milk system increased significantly, and the stable milk system was disrupted. Simultaneously, calculations of the relative aggregation degree revealed:

[0121] The mulberry leaf extract prepared in Example 1 of this invention resulted in a protein aggregation degree of 12.1%, and the system remained clear with a polydispersity index (PDI) of less than 0.2, indicating that the protein dispersion system was stable.

[0122] The mulberry leaf extract prepared in Comparative Example 2 caused a protein aggregation degree as high as 98.6%, and the system showed slight turbidity with a PDI of 0.39, indicating that the protein had a tendency to aggregate and the system stability was significantly reduced.

[0123] The mulberry leaf extract prepared in Comparative Example 3 caused a protein aggregation degree as high as 54.5%, and the system showed slight turbidity with a PDI of 0.32, indicating that the protein had a tendency to aggregate and the system stability was significantly reduced.

[0124] The mulberry leaf extract prepared in Comparative Example 4 caused a protein aggregation degree as high as 160.6%, and obvious precipitation occurred in the system. The PDI increased significantly to 0.45, indicating that the protein aggregated or even precipitated, and the stability of the system decreased significantly.

[0125] The results show that the mulberry leaf extract prepared by this invention has significant application stability in protein systems.

[0126] 3. Storage stability test of mulberry leaf extract in protein food system

[0127] The results of the accelerated storage stability test of milk after 60 days are shown in Table 3 below:

[0128] Table 3. Accelerated stability test of milk containing mulberry leaf extract after 60 days.

[0129]

[0130] Conclusion: The results in the table above clearly show that Example 1, prepared using the method of this invention, maintained excellent physical stability after being added to pure milk and undergoing a 60-day accelerated testing process, exhibiting a uniform appearance without precipitation. This indicates its excellent application stability in milk protein systems. More importantly, the retention rate of its core active ingredient, DNJ, reached as high as 92.5%, proving that the mild process and stabilization treatment of this invention effectively protected the active substance. In contrast, Comparative Example 4 showed severe flocculation and precipitation during the storage period, with significant loss of DNJ, completely failing to meet the requirements for commercial application. This comparison fully demonstrates the effectiveness, advancement, and significant application value of the technical solution of this invention.

[0131] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a mulberry leaf extract with high stability and high activity in protein food systems, characterized in that, Includes the following steps: S1. The washed and dried mulberry leaves are pretreated by steam flash explosion and ball milling to obtain mulberry leaf powder; S2. Disperse the mulberry leaf powder obtained in step S1 in reverse osmosis water, add enzyme preparation for directional enzymatic hydrolysis, and then extract with ultrasonic assistance to obtain primary extract. S3. After adding protein for treatment, filter the coarse fraction and then perform membrane separation to obtain a concentrated solution. After adding a composite stabilizer for treatment, dry the solution to obtain mulberry leaf extract.

2. The preparation method according to claim 1, characterized in that, In step S1, the conditions for handling the steam flash explosion include: pressure 0.5-3 MPa, flash explosion time 150 s-450 s; and / or In step S1, the ball milling conditions include: a ball-to-material ratio of 1:(1-8)w / w, a processing frequency of 100-500 r / min, and a processing time of 10-120 min.

3. The preparation method according to claim 1, characterized in that, In step S2, the ratio of mulberry leaf powder to reverse osmosis water is 1:(5-10) w / w, the enzyme preparation includes one or more of cellulase, xylanase, β-glucanase, hemicellulase, and cellobiase, the amount of enzyme preparation used is 0.1%-1% (w / w) of mulberry leaf powder, and the enzymatic hydrolysis time is 30-90 min; and / or In step S2, the ultrasonic extraction step uses an ultrasonic frequency of 50-180 kHz and a processing time of 10-120 min.

4. The preparation method according to claim 1, characterized in that, In step S3, the amount of protein added is 1%-3% (w / w) of the mulberry leaf powder; and / or In step S3, the protein addition treatment refers to adding protein to induce sedimentation, wherein the protein is one or a combination of mulberry leaf protein, wheat protein, and soybean protein; and / or In step S3, the coarse filtration is performed using one or a combination of pressure filtration, horizontal screw centrifugation, or disc centrifugation; and / or In step S3, the membrane separation includes inorganic membrane separation or organic membrane separation.

5. The preparation method according to claim 1, characterized in that, In step S3, the composite stabilizer includes β-cyclodextrin and sodium alginate, added at amounts of 0.1%-0.5% and 0.1%-0.3% of the mulberry leaf powder mass, respectively, and / or In step S3, the addition of the composite stabilizer treatment refers to stirring at 45-55°C for 30 minutes to ensure complete dissolution, and / or In step S3, the drying step employs spray drying, freeze drying, or vacuum drying.

6. Mulberry leaf extract, obtained by any one of the preparation methods according to claims 1-5.

7. The mulberry leaf extract according to claim 6, characterized in that, The mulberry leaf extract is a yellowish-brown powder, and its 1% aqueous solution is clear and transparent. By mass fraction, it includes 0.5%-8% DNJ, 40-70% polysaccharides, and less than 0.2% polyphenols.

8. The application of the mulberry leaf extract according to claim 6 or 7 in foods containing milk protein or plant protein, wherein the foods include milk beverages, milk, yogurt, milk powder, protein bars or meal replacement shakes.

9. The food product containing the mulberry leaf extract according to claim 8, characterized in that, In the food containing milk protein or plant protein, the amount of mulberry leaf extract added is 0.1% to 5% (w / w).

10. The food product according to claim 9, characterized in that, The protein aggregation degree in the food is less than 20%.

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