High-protein and high-alpha-linolenic acid walnut composite milk and green preparation method thereof

Abstract

The invention belongs to the technical field of food biotechnology and functional food processing, and particularly relates to high-protein and high-alpha-linolenic acid walnut composite milk and a green preparation method thereof. The high-protein and high-alpha-linolenic-acid walnut composite milk is obtained by taking microwave-detoxified and non-degummed flaxseeds and microwave-cooked and non-peeled walnuts as raw materials, optimizing the conditions such as the addition amount of the flaxseeds, the material-liquid ratio, the soaking time and the grinding time and combining an ultrasonic coupling enzymolysis technology. The preparation method is green and environment-friendly, the product stability can be maintained without an exogenous additive, and the prepared walnut composite common plant milk is safe, nutritional, delicious, purely natural and suitable for popularization and application.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology and functional food processing technology, and particularly relates to a high-protein, high-α-linolenic acid walnut composite milk and an environmentally friendly preparation method thereof. Background Art

[0002] While animal-based protein boasts high biological value, its production system faces the challenge of being resource-intensive. Animal husbandry contributes 14.5% of global greenhouse gas emissions and consumes 9% of freshwater resources. Against this backdrop, plant-based protein offers significant advantages as a sustainable alternative: its production process has a 40-60% lower carbon footprint than animal protein, and its resource utilization efficiency is 3-5 times higher. From a nutritional perspective, plant-based protein is not only low in cholesterol (<5mg / 100g) and saturated fatty acids (<1g / 100g), but also rich in dietary fiber (2-15g / 100g) and phytochemicals (such as polyphenols and flavonoids). Plant-based beverages offer a particularly valuable alternative for those with special nutritional needs. Epidemiological surveys show that the prevalence of lactase deficiency in adults in Asia, the Middle East, and sub-Saharan Africa is as high as 80%-100%, significantly higher than in European and American populations (<20%). This hereditary lactose metabolism disorder has promoted the intergenerational inheritance of plant-based dairy products. For example, China's traditional soy milk consumption has reached 6.2 kg per person per year, and the compound growth rate remains at 8.3%.

[0003] The current plant-based beverage market is experiencing explosive growth. In 2024, the global market size reached US$40.6 billion, of which nut beverages accounted for 28.3%. Using walnuts as raw materials, research and development of beverage products with a smooth taste, rich flavor, rich nutrition, and easy absorption can fully utilize the nutritional and health value of walnuts, and is conducive to better development and utilization of plant resources such as walnut protein and walnut oil, which are widely suitable for infants, teenagers and middle-aged and elderly people to drink. However, at present, walnut-based plant milks in my country often use processes such as peeling and astringency removal, grinding, blending, and high-temperature and high-pressure sterilization. The stability of its O / W water-in-oil system requires a large reliance on emulsifiers and stabilizers. Peeling and astringency removal makes the process costly and the environmental burden heavy, and it also loses phytonutrients such as flavonoids. The walnut plant milk obtained by this process has a low nutritional value (protein content of about 0.5%) and can no longer meet consumers' demand for nutritious and healthy plant milk. As a new type of functional raw material, flaxseed is not only rich in the essential fatty acid α-linolenic acid (ALA) required by the human body, but also rich in natural colloidal macromolecular flaxseed gum. Therefore, the special nutritional and physical and chemical properties of flaxseed are expected to improve the stability of the walnut milk system. However, there is still a large research gap in its addition ratio and processing technology optimization. Summary of the Invention

[0004] In view of this, in order to solve the problems existing in the prior art, the present invention discloses a green preparation technology for walnut compound milk with high nutrient content, which does not require the addition of exogenous stabilizers or peeling treatment. The present invention utilizes flaxseed compounding and low-consumption ultrasound-coupled bioenzymatic hydrolysis technology to regulate the physical and chemical self-stability of plant milk, change the particle size distribution and droplet surface charge distribution, and significantly reduce the bitterness and sourness of unpeeled walnut milk. This method is green, energy-saving and water-saving, and significantly improves the nutrition (nutrient content, certain essential amino acid content), stability (centrifugal sedimentation rate, particle size, potential, microstructure), and health attributes (protein bioavailability) of walnut compound milk, thus realizing a method for preparing a high-protein, high-α-linolenic acid walnut compound milk without peeling and de-bittering.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention discloses an environmentally friendly preparation method for a high-protein, high-α-linolenic acid walnut composite milk. The method uses walnuts and flax seeds as raw materials, and adds 10%-50% flax seeds based on the dry matter weight ratio. The raw materials are firstly subjected to microwave pretreatment, and then soaked for 0.5-2.5 hours at a solid-liquid ratio of 1:(7-11), followed by colloid grinding for 6-14 minutes, and solid-liquid separation to obtain the high-protein, high-α-linolenic acid walnut composite milk.

[0007] Furthermore, the microwave pretreatment method includes: adjusting the water content of flax seeds to 13% without degumming, standing at 4°C for 8-12 hours, and then subjecting the flax seeds to microwave treatment to achieve the effect of detoxification and fragrance production; and subjecting raw walnuts to microwave treatment directly without peeling, to achieve the effect of cooking and enhancing fragrance.

[0008] It is worth noting that the peeling and astringency removal process is often used in the traditional preparation process of walnut-based plant milk, which not only causes process cost height, heavy environmental burden, but also causes the loss of plant nutrients such as flavonoids. And, considering the stabilization of O / W walnut-based plant milk oil-in-water system, it is necessary to rely on emulsifiers and stabilizers in a large amount. Considering that linseed is rich in water-soluble polysaccharides (such as arabinoxylan), has excellent thickening and emulsifying ability, alternative synthetic colloids (such as CMC, xanthan gum), form stable interfacial film, prevent oil droplets from gathering, the present invention is compounded and coordinated with the optimization of composite milk preparation process by linseed and walnut, replaces exogenous additives with natural ingredients. After the two are compounded, protein composition is complementary (linseed protein is rich in sulfur-containing amino acids, and walnut protein contains hydrophobic region), and can form a denser interfacial network structure by thermal effect, and then promotes emulsion stability. In addition, the lignans in flaxseed combine with walnut tannins to effectively reduce the perception of astringency while retaining the total polyphenol content; flaxseed is rich in α-linolenic acid (ALA), which forms a fatty acid balance with the linoleic acid (LA) in walnuts, thereby enhancing the nutritional value of the product.

[0009] Moreover, flaxseed husks are rich in unique natural plant gum - flaxseed gum, which is a natural emulsifier and stabilizer. It plays an important role in stabilizing oil-in-water emulsions and has good water retention and thickening capabilities. However, when the flaxseed gum content is relatively high, the flaxseed gum in the flaxseed plant milk and the other phases of the emulsion undergo "exclusion flocculation", while the fluidity of the system decreases and solid properties appear. Or because the content of polysaccharide macromolecules such as flaxseed gum is relatively low, "bridging flocculation" is caused, and the construction of a self-stabilizing system for flaxseed plant milk cannot be achieved. Therefore, in the preparation process of flaxseed plant milk, it is necessary to control the degree of flaxseed degumming. The present invention utilizes the naturally rich emulsifying components (such as walnut protein and phospholipids) in walnut milk to form a dense membrane structure through interfacial co-adsorption with flaxseed gum, thereby inhibiting flocculation. At the same time, the two construct a composite network through hydrogen bonds or electrostatic interactions, dynamically balancing the colloid concentration and avoiding the common fluidity loss or bridging flocculation problems of a single system. This technology solves the pain points of the traditional process of complex degumming steps and high energy consumption. It achieves self-stabilization through component complementarity, eliminates the risk of fat floating or protein sedimentation, and significantly improves product uniformity and shelf life. Compared with the limitation of single flaxseed milk that requires precise gel control, the compound system broadens the colloid tolerance range and does not require the addition of artificial emulsifiers, which is in line with the clean label trend. In addition, retaining the flaxseed gum completely retains active ingredients such as dietary fiber, enhancing nutritional functions, while reducing wastewater discharge and promoting green production. This innovation breaks through process limitations with the synergistic effect of natural components, and has the triple advantages of simplifying the process, improving quality and reducing costs.

[0010] Furthermore, the flaxseed is microwaved at 600-800W for 6-12 minutes, and the walnut is microwaved at 700-900W for 6-10 minutes.

[0011] It is worth noting that microwave cooking of walnuts and flaxseeds not only enhances the flavor, but also improves the total phenolic and lignan quality scores of the composite milk. In addition, it can remove cyanogenic glycosides from flaxseeds, ensuring the safety of the composite milk. Microwave cooking of flaxseeds has the following significant advantages over other cooking techniques (such as baking, steaming or frying): (1) Fast heating speed: Microwaves directly act on the water molecules and polar molecules in flaxseeds, achieving simultaneous heating inside and outside, significantly shortening the cooking time (usually only a few minutes), while traditional baking or frying may take longer. (2) Reduced loss of heat-sensitive components: Microwave heating time is short, which can better preserve the ω-3 fatty acids (α-linolenic acid), antioxidants (such as lignans) and vitamins (such as B vitamins) in flaxseeds, avoiding oxidative degradation caused by long-term high temperature. (3) Reduced protein denaturation: Compared with high-temperature baking, microwave treatment has less damage to protein structure. (4) Maintaining color and flavor: Microwave-treated flaxseeds have a more uniform color, avoiding the darkening or bitterness caused by traditional methods. (5) Low energy consumption: Microwaves consume less energy than traditional ovens or woks. (6) No open flames or hot surfaces: Reduces fire risks and creates a safer operating environment.

[0012] Furthermore, after colloid grinding, ultrasound-coupled enzymatic hydrolysis is performed, the enzyme is immediately inactivated, and solid-liquid separation is performed to obtain a walnut compound milk with high protein and high α-linolenic acid.

[0013] It is worth noting that in order to improve the dissolution rate of plant nutrients in plant milk, enzyme-assisted extraction is considered to be a green and environmentally friendly method. It can increase the extraction rate of nutrients such as proteins and polyphenols in plant matrices and improve their functional properties by modifying protein structure. However, traditional enzymatic hydrolysis has limitations, such as insufficient contact between substrate and enzyme due to uneven stirring, reduced enzyme activity, protein aggregation and irregular protein structure. In order to solve these shortcomings, it is urgent to couple new processing technologies. Ultrasound, as a non-thermal physical treatment technology, can generate cavitation bubbles that violently implode on the surface of the raw material, forming high shear force and mechanical energy. This effect may produce a synergistic effect with green enzymatic hydrolysis to improve the nutritional quality and sensory flavor of walnut milk.

[0014] Furthermore, the ultrasound-coupled enzymatic hydrolysis method includes: subjecting the mixed system after colloid grinding to ultrasound at 100-500W for 4-12 minutes, and then adding 5000-25000U of enzyme and performing enzymatic hydrolysis at 50°C for 0.5-2.5 hours.

[0015] It is worth noting that the ultrasonic-coupled enzymatic hydrolysis technology significantly improves the physical stability, flavor characteristics, nutritional value and bioavailability of the compound milk compared to the traditional preparation technology. Ultrasonic-coupled enzymatic hydrolysis technology achieves multi-dimensional optimization for the special technical bottlenecks of walnut-flaxseed compound milk (such as the difficulty in dissolving walnut skin polyphenol-protein complexes, the high viscosity of flaxseed gum hindering dispersion, and low nutrient release rate) through the synergistic effect of ultrasonic physical effects and enzymatic hydrolysis reactions. First, the ultrasonic cavitation effect (instantaneous high pressure >100MPa and microjet) efficiently breaks up the dense lignin-polyphenol complex structure in the walnut skin and the β-1,4 glycosidic bond network of flaxseed gum, reducing the particle size of the raw material from 21.13μm to 15.45μm, while activating cellulase / pectinase to target the degradation of the colloid, releasing the encapsulated protein and unsaturated fatty acids, and forming a stable interface film (TSI 2.02) with the enzymatic protein through nano-fat globules (<200nm), significantly inhibiting floating oil. Secondly, targeting walnut tannins and flaxseed oxidative off-flavors, the localized thermal effect of ultrasound (50-60°C) inactivates lipoxygenase, reducing the production of grassy aldehydes. It also accelerates the targeted degradation of astringent polyphenols by tannins into low-molecular-weight phenolic acids, synergizing with the Maillard reaction to produce pyrazines (sweet aroma) and phenylethanol (honey aroma), masking any residual bitterness. Furthermore, ultrasonic microfluidization microcracks the flaxseed cell wall (containing pectin and hemicellulose), enabling targeted hydrolysis by pectinase, releasing more omega-3 (α-linolenic acid) and optimizing the omega-6 / 3 ratio to 2.12:1. Furthermore, ultrasonic pretreatment unfolds proteins, exposing trypsin sites and improving enzymatic hydrolysis efficiency. This results in a 20.5% increase in protein content (to 1.76g / 100g), a 21% increase in essential amino acids, a 75.54% fatty acid release rate, and a protein bioavailability of 67.36%. This technology systematically solves the problems of poor dispersion stability, flavor deterioration and nutrient lock-in of walnut-flaxseed compound milk through the three-in-one mechanism of "physical wall breaking-targeted enzymatic hydrolysis-interface reconstruction", and is both innovative and practical.

[0016] Furthermore, the enzyme includes one of flavor protease, bromelain, papain, alkaline protease, acid protease, neutral protease, aminopeptidase, α-amylase, pectinase, lipase, and cellulase.

[0017] Furthermore, the enzyme is bromelain.

[0018] It's worth noting that bromelain offers multiple advantages in plant-based beverages. As a natural digestive enzyme, it breaks down plant proteins, reducing gastrointestinal burden and enhancing the digestibility of compound milks. Furthermore, by hydrolyzing suspended proteins and colloids, it effectively improves compound milk stability, reduces precipitation, and extends shelf life. Its enzymatic hydrolysis also releases active ingredients such as polyphenols and flavonoids, enhancing nutrient absorption, and breaks down astringent substances like tannins, improving taste. Furthermore, bromelain's antimicrobial properties can inhibit the growth of some bacteria, reducing the use of chemical preservatives and aligning with the clean label trend. Its wide pH adaptability and low-temperature activity make it suitable for a variety of plant-based beverage systems.

[0019] Furthermore, the enzyme inactivation treatment method is 70-90°C water bath for 5-15 minutes.

[0020] It is worth noting that the present invention destroys the active site of the enzyme through enzyme inactivation treatment, causing it to lose its catalytic activity and terminate the enzymatic hydrolysis reaction; on the other hand, the thermal effect generated during the enzyme inactivation treatment can form a denser interface network structure, effectively improving the emulsion stability of the walnut compound milk.

[0021] The second object of the present invention is to provide a high-protein, high-α-linolenic acid walnut composite milk obtained by the green preparation method as described above.

[0022] The invention discloses a high-protein, high-alpha-linolenic acid walnut compound milk, wherein the protein content is greater than or equal to 1.46 g / 100 g, the alpha-linolenic acid content is greater than or equal to 23%, and the ratio of linoleic acid to linolenic acid is in the range of 2:1 to 3:1.

[0023] It is worth noting that the protein content of commercially available walnut milk is mostly around 0.5g / 100g, while the protein content of the walnut composite milk disclosed in the present invention reaches 1.46g / 100g. In addition, generally speaking, the ALA content of walnut milk accounts for about 10%-15% of the total fatty acids, but the present invention obtains the walnut composite milk by optimizing the properties of the raw materials and the processing technology, and the ALA content accounts for up to 23%. The ratio of Omega-6 (linoleic acid) to Omega-3 (linolenic acid) is about 2.12:1, which is within the recommended ideal health range (1:1 to 4:1), and the nutritional value is much higher than similar products on the market.

[0024] Compared with the prior art, the present invention utilizes the physical and chemical properties of flaxseed itself to improve the quality of walnut milk, which has a higher α-linolenic acid content than commercially available walnut plant milk, and further increases the content of walnut protein and essential amino acids for the human body through ultrasonic coupling enzymatic hydrolysis technology, thereby obtaining a preparation method for walnut composite milk that does not require peeling and de-bittering. This method is green in process. Based on the reconstruction of the emulsification system, the polysaccharides in flaxseed gum form a dense network structure that can maintain the system and limit the Brownian motion of the emulsion droplets. Therefore, there is no need to add exogenous artificially synthesized emulsification, stabilization and other additives to maintain the long-term storage stability of the product. In addition, it has been proved through in vitro digestion experiments that the plant milk disclosed in the present invention has obvious functional advantages in improving protein bioavailability and fatty acid release rate compared with other commercially available plant milks, and the prepared walnut permanent plant milk is safe, nutritious and delicious, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0026] Figure 1 The effects of process conditions on the total solid content and centrifugal sedimentation rate of compound milk: a) flaxseed addition amount, b) grinding time, c) soaking time, d) solid-liquid ratio.

[0027] Figure 2 The effect of solid-liquid ratio on the particle size potential of composite emulsion. a) Particle size and potential, b) Particle size distribution.

[0028] Figure 3 This is the effect of the interaction between various factors in the process conditions on the centrifugal sedimentation rate of compound plant milk.

[0029] Figure 4 This is the effect of the interaction between various factors in the process conditions on the total solid content of compound plant milk.

[0030] Figure 5 Effects of different ratios on the stability of compound emulsions: a) viscosity, b) particle size distribution, c) total solids content, d) TSI, and e) CLSM.

[0031] Figure 6 This is the process flow chart for preparing compound plant milk by ultrasound-coupled enzymatic hydrolysis.

[0032] Figure 7 Effects of different proteases on compound milk: a) centrifugal sedimentation rate, b) protein increase rate.

[0033] Figure 8 The effects of different enzyme resources on compound milk: a) centrifugal sedimentation rate, b) protein increase rate, c) particle size and potential, d) viscosity.

[0034] Figure 9 Effects of ultrasound and enzymatic hydrolysis on the total solid content of compound milk, a) ultrasound time, b) ultrasound power, c) enzyme addition amount, b) enzymatic hydrolysis time.

[0035] Figure 10 This study investigates the effects of the interaction between various factors in ultrasound-coupled enzymatic hydrolysis on the total solid content of compound plant milk.

[0036] Figure 11 Effects of ultrasound-coupled enzymatic hydrolysis on the edible quality of compound milk, a) particle size distribution, b) TSI, c) viscosity, and b) electronic tongue.

[0037] Figure 12 The effect of ultrasound-coupled enzymatic hydrolysis on the microstructure of composite milk.

[0038] Figure 13 Effects of ultrasound-coupled enzymatic hydrolysis on the nutritional quality of compound milk: a) protein increase rate, b) protein digestibility, and c) amino acid composition.

[0039] Figure 14 The fatty acid composition of walnut-flaxseed composite plant milk. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0041] The term "embodiment" is used herein specifically to describe any embodiment as "exemplary," and should not be construed as superior or preferable to other embodiments. Performance indicators in the embodiments of this application were tested using conventional testing methods in the art, unless otherwise specified. It should be understood that the terms used in this application are intended solely to describe specific implementations and are not intended to limit the disclosure herein.

[0042] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0043] In order to better illustrate the content of this application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that this application can be implemented without certain specific details. In the examples, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of this application.

[0044] Under the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of this application.

[0045] The present invention discloses a high-protein, high-α-linolenic acid walnut compound milk and an environmentally friendly preparation method thereof, belonging to the fields of food biotechnology and functional food processing technology. The present invention uses microwave-detoxified flaxseeds that do not require degumming and microwave-cooked walnuts that do not require peeling as raw materials. By optimizing the amount of flaxseed added, the material-liquid ratio, the soaking time, the grinding time and other conditions, and combining ultrasonic-coupled enzymatic hydrolysis technology, a high-protein, high-α-linolenic acid walnut compound milk is obtained. The preparation method is environmentally friendly and can maintain product stability without the need for exogenous additives. The prepared walnut compound permanent plant milk is safe, nutritious, delicious, and purely natural, making it suitable for promotion and application.

[0046] For a better understanding of the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content are also considered to fall within the scope of protection of the present invention.

[0047] Example 1

[0048] First, fresh and clean flax seeds were selected, and their moisture content was adjusted to 13%. The flax seeds were placed in a 4°C refrigerator and allowed to stand for 8-12 hours to fully absorb the moisture. The flax seeds were then subjected to a 720W microwave treatment in a closed microwave rapid extraction system for 9 minutes. Then, raw walnuts with paper skins were directly subjected to an 800W microwave treatment in a closed microwave rapid extraction system for 8 minutes without peeling to obtain cooked walnuts for later use.

[0049] Then, the microwave-pretreated flax seeds and walnuts are mixed in a certain proportion, soaked for a certain time according to a certain solid-liquid ratio, colloidally ground, and filtered through a 200-mesh filter bag at room temperature to obtain a walnut composite milk with high protein and high α-linolenic acid.

[0050] Among them, the flaxseed addition amount (dry matter mass ratio) was set to 10%, 20%, 30%, 40%, and 50%, respectively; the soaking time was 0h, 0.5h, 1h, 1.5h, and 2h, respectively; the colloid grinding time was 6min, 8min, 10min, 12min, and 14min, respectively; and the solid-liquid ratio was 1:7, 1:8, 1:9, 1:10, and 1:11, respectively.

[0051] Example 2

[0052] First, fresh and clean flax seeds were selected, and their moisture content was adjusted to 13%. The seeds were placed in a 4°C refrigerator and allowed to stand for 8-12 hours to fully absorb the moisture. The seeds were then subjected to a 720W microwave treatment in a closed microwave rapid extraction system for 9 minutes. Then, raw walnuts with paper skins were directly subjected to an 800W microwave treatment in a closed microwave rapid extraction system for 8 minutes without peeling to obtain cooked walnuts for later use.

[0053] Then, microwave-pretreated flax seeds and microwave-pretreated walnuts are prepared according to a dry weight mass ratio, that is, the flax seeds are added in an amount of 10%-50%, and the mixture is soaked (1.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours) according to a solid-liquid ratio of (1:7, 1:8, 1:9, 1:10, 1:11) and then ground for 6-10 minutes. The obtained unfiltered composite system is subjected to ultrasonic treatment and then enzymatic hydrolysis. After the enzymatic hydrolysis, the mixture is immediately bathed in an 80°C water bath for 10 minutes to inactivate the enzyme. After the enzyme is inactivated, the mixture is cooled and then filtered through a 200-mesh filter bag to obtain a walnut composite milk with high protein and high α-linolenic acid.

[0054] Among them, the enzyme screening condition optimization experiment was carried out with an enzyme addition amount of 10,000 U, an enzymatic hydrolysis temperature of 50°C, and an enzymatic hydrolysis time of 1 h. The selected enzymes were: flavor protease (Yuanye s10153), bromelain (Dongheng Huadao), papain (FDG-2201), alkaline protease (AGCS-110), neutral protease (Xiasheng), and aminopeptidase (FDG-2251). In addition, considering the enzymatic hydrolysis effects of carbohydrase and lipase on walnut compound milk, α-amylase (Dongheng Huadao), pectinase (FDG-2259), lipase (FDG-4109), and cellulase (FDG-4107) were added.

[0055] In addition, the ultrasonic power (100W, 200W, 300W, 400W, 500W), ultrasonic time (4min, 6min, 8min, 10min, 12min), enzyme addition amount (5000U, 10000U, 15000U, 20000U, 25000U), and enzymatic hydrolysis time (0.5h, 1h, 1.5h, 2h, 2.5h) were set respectively.

[0056] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the properties and application performance of the high-protein, high-α-linolenic acid walnut composite milk and the green preparation method thereof disclosed in the present invention are further illustrated by the following comparative examples and experimental examples. However, they should not be understood as limiting the present invention. The method properties obtained by other measurement experiments conducted by technicians in this field based on the above-mentioned invention content and the applications based on the above-mentioned properties are also considered to fall within the scope of protection of the present invention.

[0057] Comparative Example 1

[0058] The walnut composite milk prepared by setting the flaxseed addition amount to 40% and the solid-liquid ratio to 1:9, soaking for 1 hour, grinding for 10 minutes, and solid-liquid separation as disclosed in Example 1 is marked as the untreated group (C);

[0059] On this basis, after colloid grinding, the unfiltered mixed system was ultrasonicated at 300W for 6 minutes, and the walnut compound milk obtained by solid-liquid separation was labeled as the ultrasonic group (U); after colloid grinding, the unfiltered mixed system was added with 10,000U of enzyme and enzymatically hydrolyzed in a 50°C water bath for 1 hour, and then inactivated by water bath in an 80°C water bath for 10 minutes. The walnut compound milk obtained by solid-liquid separation was labeled as the enzymatic hydrolysis group (E);

[0060] The flaxseed addition amount was set to 40% and the solid-liquid ratio was set to 1:9 as disclosed in Example 2. The mixture was soaked for 1 hour and ground for 10 minutes. The unfiltered mixed system was ultrasonicated at 300W for 6 minutes, and then 10,000U of enzyme was added and enzymatically hydrolyzed in a 50°C water bath for 1 hour. Finally, the walnut composite milk obtained by solid-liquid separation was marked as ultrasonically coupled enzymatic hydrolysis group (U+E).

[0061] Compared with the untreated group, enzymatic hydrolysis group, and ultrasonic group, the ultrasound-coupled enzymatic hydrolysis group significantly improved the physical stability, flavor characteristics, nutritional value, and bioavailability of the composite milk, as shown in Table 1. The ultrasound-coupled enzymatic hydrolysis group treatment reduced the average particle size of the composite milk from 21.13 μm to 15.45 μm, with a more uniform particle size distribution, the lowest stability index (TSI) (2.02), and significantly reduced the oil floating phenomenon. The ultrasound-coupled enzymatic hydrolysis group treatment also improved the flavor of the composite milk, reduced the bitterness of burnt and oxalic acid, and generated sweet and honey aroma substances. In terms of nutrition, the protein content increased from 1.46 g / 100 g to 1.76 g / 100 g after ultrasound-coupled enzymatic hydrolysis treatment, and the essential amino acids increased significantly. The proportion of unsaturated fatty acids reached 89.62%, and the ratio of Omega-6 to Omega-3 was 2.12:1. The fatty acid release rate of the ultrasound-coupled enzymatic hydrolysis group reached 75.54%, and the protein bioavailability reached 67.36%, significantly improving the nutritional value and bioavailability of the compound milk.

[0062] Table 1 Performance indicators of composite milk obtained by different treatment methods

[0063]

[0064]

[0065] Experimental Example 1

[0066] The centrifugal sedimentation rate and total solid content of the walnut composite milk obtained in Example 1 were measured to determine the walnut-flaxseed composite plant milk emulsification system, and the parameters were optimized to reduce the centrifugal sedimentation rate and increase the total solid content.

[0067] Measurement methods include:

[0068] (1) Determination of centrifugal sedimentation rate

[0069] Shake the compound milk sample thoroughly to mix evenly. Take an appropriate amount of compound milk into a centrifuge tube, with the total mass being m1 and the mass of the centrifuge tube being m0. Centrifuge at 5000 rpm for 10 minutes, remove the supernatant, and dry. The total mass after drying is recorded as m2. Calculate the mass of each sample from at least three measurements. The formula is as follows:

[0070]

[0071] (2) Determination of total solids content

[0072] Shake the composite milk sample thoroughly to mix evenly. Accurately weigh 10g of the sample into a weighing dish containing sea sand of known constant weight. Evaporate on a water bath until no noticeable fluid is present. Wipe off any moisture adhering to the weighing dish and place in a constant temperature oven at 105°C to a constant weight. The total solids content is calculated as follows:

[0073]

[0074] Among them, M2 is the mass of the total solids and the weighing dish containing sea sand; M1 is the mass of the constant weight weighing dish containing sea sand, unit is g.

[0075] Assuming that the flaxseed addition amount is 40%, the soaking time is 1h, and the grinding time is 10min, a single-factor experiment is conducted as the optimal conditions. Each group of experiments changes the single-factor conditions studied according to the above settings. To ensure the accuracy of the data results, each group conducts three repeated experiments to measure the centrifugal sedimentation rate and total solid content.

[0076] The Box-Behnken design principle was adopted and the three-factor three-level response surface analysis method was used to study the effects of flaxseed addition, soaking time and grinding time on the centrifugal sedimentation rate and total solid content of compound milk. A prediction model was established using the quadratic polynomial regression equation.

[0077] Results and Analysis:

[0078] (1) Single-factor experimental results on the effect of process conditions on the centrifugal sedimentation rate and total matter content of compound milk

[0079] according to Figure 1It shows that with the increase of flax seed content and grinding time, the total solid content increases, but the centrifugal sedimentation rate will first decrease and then increase. When the flax seed addition reaches 40% and the grinding time is 10 minutes, the total solid content increases more slowly and the centrifugal sedimentation rate is the lowest. With the increase of soaking time, the total solid first increases and then decreases, but the centrifugal sedimentation rate first decreases and then increases. When the soaking time is 1 hour, the total solid content is the highest and the centrifugal sedimentation rate is the lowest. The total solid content and the centrifugal sedimentation rate show a monotonically decreasing trend with the increase of soaking water. According to Figure 2 The results show that since there is no optimal solid-liquid ratio and no interaction with the other three factors, which ultimately affects the fitting effect of the response surface, the solid-liquid ratio is not considered in the response surface experiment. When the solid-liquid ratio is 1:9, the absolute value of the potential is the largest and the particle size is the smallest, so the solid-liquid ratio of 1:9 is selected for the response surface experiment.

[0080] (2) Response surface optimization

[0081] Based on the results of the single-factor experiment, the present invention uses the amount of flaxseed added (A), soaking time (B), and grinding time (C) as independent variables and the centrifugal sedimentation rate (R1) as the response value to perform a response surface analysis. Table 2 shows the variance analysis of the response surface results of the process conditions on the centrifugal sedimentation rate. The F value of the model is 44.28, and the p value is <0.0001, indicating that the model difference is highly significant and statistically significant. The correlation coefficient R 2 It is 0.9827, indicating that there is a significant linear relationship between the centrifugal sedimentation rate and all independent variables. The correction coefficient is 0.9605, indicating that the model can explain 96.05% of the variability of the experimental data. The lack of fit term is not significant, indicating that the model has a good degree of fit and can reflect the reliability of the experimental results. CV represents the test precision. The CV of this model is less than 10%, indicating that the experimental repeatability is good. From the size of the F value, it can be seen that the influence of each factor is: A (flax seed addition amount) > C (grinding time) > B (soaking time). The flax seed addition amount, soaking time and grinding time all have a significant effect on the centrifugal sedimentation rate (p < 0.05). According to Figure 3 The response surface methodology yields contour lines and three-dimensional spatial plots of the interaction effects of various factors. The flatter the surface and the closer the contour lines are to a circle, the smaller the impact on the dependent variable. Therefore, the plot shows that the order of magnitude of the interaction effect on the centrifugal sedimentation rate is B (soaking time) + C (grinding time) > A (flaxseed addition) + C (grinding time) > A (flaxseed addition) + B (soaking time). Using Design-Expert software, a quadratic polynomial regression equation was obtained for the relationship between flaxseed addition (A), soaking time (B), grinding time (C), and centrifugal sedimentation rate (R1):

[0082] R1=5.74+1.06*A+0.4248*B+0.4318*C+0.1360*AB-2983*AC-

[0083] 0.8723*BC+1.16*A 2 +1.32*B 2 +1.84*C 2

[0084] Table 2 Variance analysis of the response surface quadratic model of process conditions on centrifugal sedimentation rate

[0085]

[0086]

[0087] Note: p<0.001 is highly significant, indicated by ***; p<0.01 is extremely significant, indicated by **; p<0.05 is significantly different, indicated by *.

[0088] Table 3 shows the variance analysis of the response surface results of the process conditions on the total solid content. The model differences of the total solid content are also highly significant. The linear relationship between all independent variables is significant. The model can explain 97.21% of the variability of the experimental data. The lack of fit term is not significant, and the model has a good degree of fit. The CV of this model is <10%, indicating that the experimental repeatability is good. From the size of the F value, it can be seen that the degree of influence of each factor is: C (grinding time) > A (flaxseed addition amount) > B (soaking time), and the flaxseed addition amount, soaking time and grinding time all have a significant effect on the total solid content (p < 0.05). According to Figure 4 Response surface methodology revealed that the order of influence of the interaction factors on total solids content was A (flaxseed addition) and B (soaking time) > A (flaxseed addition) and C (grinding time) > B (soaking time) and C (grinding time). Using Design-Expert software, a quadratic polynomial regression equation was obtained for flaxseed addition (A), soaking time (B), grinding time (C), and total solids content (R²):

[0089] R2=8.84-0.2198*A+0.1268*B-3569*C+0.2107*AB+0.1276*AC+0.0666*BC-0.64*A 2 -

[0090] 0.5737*B 2 +2.15*C 2

[0091] Table 3 Variance analysis of the response surface quadratic model of process conditions on total solid content

[0092]

[0093]

[0094] Note: p<0.001 is highly significant, indicated by ***; p<0.01 is extremely significant, indicated by **; p<0.05 is significantly different, indicated by *.

[0095] Through response surface methodology analysis and optimization, the optimal process conditions fitted according to the equation are: flax seed addition amount is 38.062%, soaking time is 1.028h, grinding time is 9.377min, the centrifugal sedimentation rate is: 5.643%; the total solid content is: 8.923%. Considering the operability of the experiment, the experiment was repeated with flax seed addition amount of 40%, soaking time of 1h, and grinding time of 10min. The experimental values are consistent with the predicted values, indicating that the model has high accuracy. It is feasible to use the response surface analysis method to evaluate the centrifugal sedimentation rate and total solid content of compound milk.

[0096] (3) Physical and chemical analysis

[0097] The physicochemical properties of compound milk with different ingredient ratios were analyzed according to the process conditions optimized by response surface methodology. Figure 5 As shown in the appearance image, when the flaxseed addition reaches 40%, delamination and flocculation are less noticeable after 10 days. Particle size results indicate a reduction in particle size and a more uniform distribution. This is likely due to the small amount of flaxseed gum reducing protein aggregation in the milk, allowing fine particles to remain uniformly suspended in the emulsion for a longer period of time. TSI analysis shows that the TSI value decreases with increasing flaxseed content, indicating that the composite emulsion becomes more stable with increasing flaxseed content. This is because the polysaccharides in the flaxseed gum form a dense network structure that maintains the system and limits the Brownian motion of the emulsion droplets. Laser confocal scanning results show that walnut milk exhibits significant aggregation, which is due to its high oil content. This leads to particle aggregation or flocculation, which triggers particle migration and causes fat to float. Flaxseed milk also exhibits significant aggregation, suggesting that the larger particle size previously explained is due to the formation of flaxseed protein aggregates. Therefore, the present invention's compounding of flaxseed and walnut milk significantly reduces particle aggregation, with the lowest level of aggregation observed when the flaxseed addition reaches 40%.

[0098] Experimental Example 2

[0099] The protein increase rate, centrifugal sedimentation rate, viscosity, particle size, potential, and sensory properties of the walnut composite milk obtained in Example 2 were measured to select the processing enzyme. The total solid content of the walnut composite milk obtained in Example 2 was measured to evaluate the ultrasonic coupled enzymatic hydrolysis conditions, and finally its edible quality was comprehensively evaluated.

[0100] Measurement methods include:

[0101] (1) Protein increase rate

[0102] The protein content of the composite plant milk was determined using an automated Kjeldahl nitrogen analyzer. The procedure was as follows: 5 g of flaxseed plant milk was weighed to the nearest 0.001 g and transferred into a dry digestion tube. 10 mL of concentrated sulfuric acid and one Kjeldahl nitrogen tablet were added. The flaxseed plant milk was then digested using a graphite digester in Curve 1 mode. After digestion, the automated Kjeldahl nitrogen analyzer performed automated liquid addition, distillation, titration, and data recording.

[0103]

[0104] Wherein, M1 is the protein content of the control group, unit (g / 100g); M2 is the protein content of the enzymatic hydrolysis, unit (g / 100g).

[0105] (2) Determination of protein digestibility

[0106] a. Simulated oral stage: 10 mL diluted sample + 8 mL SSF + 50 μL CaCl2 + 1.95 mL ultrapure water. Adjust pH to 7. Incubate in a shaker at 37°C, 150 rpm, and continuous agitation for 5 minutes.

[0107] b. Simulated gastric stage: 20 mL of the above oral experimental solution + 16 mL of SGF (adjust pH to 3 with 1 M HCL) + 1 mL of pepsin + 10 μL of CaCl2 + (2.99-V HCL ) mL ultrapure water) and incubated on a shaker at 37°C and 150 r / min for 2 h.

[0108] c. Simulated small intestine stage: 20 mL gastric experimental solution + 8.5 mL SIF + 5 mL pancreatin + 2.5 mL water + 40 μL CaCl2 (adjust pH to 7 with 1 M NaOH) + (3.96-V HCL ) mL ultrapure water was added and incubated on a shaker at 37°C and 150 rpm for 2 h to terminate the reaction. The intestinal digest was centrifuged (10,000 rpm for 30 min) and the micellar layer (middle layer) was collected for protein analysis.

[0109] d. Protein content determination: The protein content and undigested protein content in compound milk (milk) were determined using the Kjeldahl method.

[0110]

[0111] (3) Total solids content

[0112] The method is the same as that of Experimental Example 1.

[0113] (4) Enzyme screening experiment

[0114] Existing enzymes were selected and applied to the prepared composite plant milk. Screening experiments were conducted using an enzyme dosage of 100,000 units, a hydrolysis temperature of 50°C, and a hydrolysis time of 2 hours. By comparing their effects on protein increase, centrifugal sedimentation rate, and sensory properties, a total of 28 proteases were selected for initial screening. It was found that under these conditions, the composite milk all flocculated and stratified after several hours of storage, and all had a bitter taste and an unpleasant odor.

[0115] Considering this to be due to over-enzymatic hydrolysis, subsequent experiments adjusted the enzymatic hydrolysis conditions to: 10,000 IU enzyme addition, 50°C, and 1 hour of enzymatic hydrolysis. Since most enzymes used in the screening process were the same but from different manufacturers, the enzyme with the highest protein content was selected for optimization. The selected enzymes were: flavor protease (Yuanye S10153), bromelain (Dongheng Huadao), papain (FDG-2201), alkaline protease (AGCS-110), neutral protease (Xiasheng), and aminopeptidase (FDG-2251). Furthermore, considering the enzymatic hydrolysis effects of carbohydrases and lipases on the compound milk, α-amylase (Dongheng Huadao), pectinase (FDG-2259), lipase (FDG-4109), and cellulase (FDG-4107) were added. Bromelain was selected as the processing enzyme for this compound plant milk based on protein enhancement rate, centrifugal sedimentation rate, viscosity, particle size, potential, and sensory properties.

[0116] (5) Optimization of ultrasound-coupled enzymatic hydrolysis conditions

[0117] The effects of ultrasonic power (100W, 200W, 300W, 400W, 500W), ultrasonic time (4min, 6min, 8min, 10min, 12min), enzyme addition amount (5000U, 10000U, 15000U, 20000U, 25000U) and enzymatic hydrolysis time (0.5h, 1h, 1.5h, 2h, 2.5h) on the total solid content of compound milk were investigated respectively.

[0118] Assuming that the ultrasonic power is 300W, the ultrasonic time is 6min, the enzyme addition amount is 10000U, and the enzymatic hydrolysis time is 1h as the optimal conditions for a single-factor experiment, each group of experiments changes the single-factor conditions studied according to the above settings. To ensure the accuracy of the data results, each group conducts three repeated experiments to determine the total solid content.

[0119] The Box-Behnken design principle was adopted and the four-factor three-level response surface analysis method was used to study the effects of ultrasonic power, ultrasonic time, enzyme addition and enzymatic hydrolysis time on the total solid content of compound milk. A prediction model was established using the quadratic polynomial regression equation.

[0120] Results and Analysis:

[0121] (1) Screening enzyme experiment analysis

[0122] The compound milk was enzymatically hydrolyzed (100,000 U; 50°C; enzymatic hydrolysis for 2 h). Figure 8 A total of 28 proteases were selected for initial screening. Alkaline protease was found to increase protein levels in the compound milk after hydrolysis, with alkaline protease (Pangbo Bio, AGCS-110) achieving over 35%, followed by aminopeptidase (FDG-2251), neutral protease (Xiasheng), bromelain (Dongheng Huadao), flavor protease (Yuanye s10153), and papain (FDG-2201), all reaching over 20%. However, the protein content of the compound milk after hydrolysis with acidic proteases was lower than that of the other hydrolysis groups. This may be because the optimal hydrolysis pH for acidic proteases is between 2 and 4, resulting in poor hydrolysis. Centrifugal sedimentation rates revealed that, with the exception of papain, which showed no significant decrease in centrifugal sedimentation rates after hydrolysis, the rates of the hydrolysis groups were lower than those of the non-hydrolyzed groups, suggesting a potential improvement in stability after hydrolysis. However, sensory and visual evaluation revealed that the hydrolyzed groups had a bitter and off-flavored taste and a particularly poor mouthfeel. Almost all of the hydrolyzed groups exhibited poor appearance compared to the non-hydrolyzed groups, with easy stratification and flocculation. This is due to excessive hydrolysis.

[0123] Therefore, the enzymatic hydrolysis conditions were adjusted in subsequent experiments to: enzyme addition amount of 10,000 U; 50°C; enzymatic hydrolysis for 1 hour. Since most of the enzymes used in the screening process were the same enzymes from different manufacturers, the model with the highest protein content was selected for condition optimization; the selected enzymes were: flavor protease (Yuanye s10153), bromelain (Dongheng Huadao), papain (FDG-2201), alkaline protease (AGCS-110), neutral protease (Xiasheng), and aminopeptidase (FDG-2251). In addition, considering the enzymatic hydrolysis effect of carbohydrase and lipase on compound milk, four new enzymes were added to the six enzymes selected above: α-amylase (Dongheng Huadao), pectinase (FDG-2259), lipase (FDG-4109), and cellulase (FDG-4107).

[0124] according to Figure 8It was shown that a total of 10 enzymes were screened under this enzymatic hydrolysis condition (10000U; 50℃; enzymatic hydrolysis for 1h). Except for papain, aminopeptidase and lipase, the centrifugal sedimentation rate of the compound milk after hydrolysis was higher than that of the control group, the other enzymatic hydrolysis groups were lower than the control group. From the results of protein improvement rate, it can be seen that the protein improvement rate of the protease hydrolysis group was higher than that of the non-protease hydrolysis group, and the improvement rate was more than 20%. The taste was good, especially bromelain and papain. Among them, only the compound milk after alkaline protease hydrolysis had a slightly bitter taste. After the compound milk was stored at low temperature (4℃) for 7 days, only the plant milk after hydrolysis by bromelain and aminopeptidase had less obvious stratification. This shows that enzymatic hydrolysis by these two enzymes helps to improve the stability of the compound milk. From the particle size and potential results, it can be seen that the absolute value of the potential of the compound milk after bromelain hydrolysis is larger than that of the non-enzymatic group, D [4,3] In summary, bromelain was selected as the best enzyme for processing compound plant milk.

[0125] (2) Single-factor experimental results on the effects of ultrasound and enzymatic hydrolysis on the total solid content of compound milk

[0126] Depend on Figure 9 The results show that the total solids content of the composite milk initially increased and then decreased with increasing ultrasound power, ultrasound time, and enzymatic hydrolysis time. The total solids content of the composite milk reached its highest level when the ultrasound power, ultrasound time, and enzymatic hydrolysis time reached 300 W, 6 minutes, and 1 hour, respectively. The total solids content of the composite milk decreased with increasing enzyme addition, and there was no significant difference in the total solids content between 5,000 and 10,000 units of enzyme. This may be due to the cavitation effect of low-frequency, short-duration ultrasound, which reduces the size of the composite milk particles. This makes them more easily permeable during filtration, thereby increasing the total solids content. However, with increasing ultrasound power and prolonged treatment time, the particles reaggregate, making it difficult for them to pass through the filter bag. Excessive enzymatic hydrolysis may also cause the particles to form large aggregates, thereby reducing the total solids content.

[0127] (3) Response surface analysis of the effects of ultrasound and enzymatic hydrolysis on the total solid content of compound milk

[0128] Based on the results of the single-factor experiment, the present invention uses ultrasonic power (A), ultrasonic time (B), enzyme addition amount (C), and enzymolysis time (D) as independent variables and total solid content (R) as the response value to perform response surface analysis. According to Table 3, the variance analysis of the response surface results is shown. The F value of the model is 10.50 and the p value is <0.0001, indicating that the difference in the model is highly significant and statistically significant. The correlation coefficient R 2It is 0.9246, indicating that there is a significant linear relationship between the total solid content and all independent variables. The correction coefficient is 0.8365, indicating that the model can explain 83.65% of the variability of the experimental data. The lack of fit term is not significant, indicating that the model has a good degree of fit and can reflect the reliability of the experimental results. CV represents the test precision. The CV of this model is less than 10%, indicating that the experimental repeatability is good. From the size of the F value, it can be seen that the influence of each factor is: C (enzyme addition) > A (ultrasonic power) > B (ultrasonic time) > D (enzymatic hydrolysis time). Ultrasonic power, ultrasonic time, enzyme addition, and enzymatic hydrolysis time all have a significant effect on the total solid content of compound milk (p < 0.05). According to Figure 10 The three-dimensional spatial plot of the interaction of various factors in the response surface methodology shows that the flatter the surface, the smaller the impact on the dependent variable. Therefore, the figure shows that the influence of the interaction on the total solids content is in the order of A (ultrasonic power) and B (ultrasonic time) > B (ultrasonic time) and D (enzyme hydrolysis time) > A (ultrasonic power) and C (enzyme addition) > C (enzyme addition) and D (enzyme hydrolysis time) > A (ultrasonic power) and D (enzyme hydrolysis time) > B (ultrasonic time) and C (enzyme addition). Using Design-Expert software for fitting, the quadratic polynomial regression equation between ultrasonic power (A), ultrasonic time (B), enzyme addition (C), enzymatic hydrolysis time (D), and total solids content (R) was obtained as follows:

[0129] R=9.24+0.1199A+1138B-0.1790C+0.0985D+0.2565AB+0.0741AC-

[0130] 0.0255AD+0.0066BC+0.2223BD+0.0639CD-0.3629A 2 -0.4088B 2 -0.2929C 2 -0.1895D 2

[0131] Table 4 Variance analysis of the response surface quadratic model for ultrasound-coupled enzymatic hydrolysis on total solid content

[0132]

[0133]

[0134] Note: p<0.001 is highly significant, indicated by ***; p<0.01 is extremely significant, indicated by **; p<0.05 is significantly different, indicated by *.

[0135] Through response surface analysis and optimization, the optimal process conditions fitted according to the equation are: ultrasonic power of 304.03W, ultrasonic time of 6.12min, enzyme addition amount of 9856.56U, and enzymatic hydrolysis time of 0.98h. Considering the actual implementation situation, the ultrasonic power is 300W, ultrasonic time is 6min, enzyme addition amount is 10000U, and enzymatic hydrolysis time is 1h.

[0136] The indicators were measured according to the optimized conditions. Figure 11 The TSI results showed that the enzymatically hydrolyzed emulsions had lower TSI values than the unhydrolyzed emulsions, falling below 3.0, indicating that the enzymatically hydrolyzed emulsions were more stable. The ultrasound-coupled enzymatic hydrolysis group achieved the best results. The ultrasound-coupled enzymatic hydrolysis group exhibited a more uniform particle size distribution than the other three groups, with fewer large particles and aggregates. The particle size distribution results suggest the reason for this improved stability. This is likely due to the high shear and mechanical energy generated by ultrasound, which induces cavitation and causes large particles in the emulsions to implode, leading to cell wall exfoliation, erosion, and particle fragmentation. This makes the emulsions more accessible to the enzyme, improving enzymatic hydrolysis and resulting in better dispersibility. Rheological results indicate that all emulsions exhibited typical shear-thinning behavior with increasing shear rate, with apparent viscosity decreasing, reflecting the characteristics of non-Newtonian fluids. This is likely due to the disruption of the emulsion network structure and the rearrangement of droplets as the shear rate increases. Electronic tongue analysis revealed that after ultrasound, enzymatic hydrolysis, and ultrasound-co-enzymatic hydrolysis, the sourness value decreased and the freshness value increased. Figure 12 The mesostructure and microstructure results show that after ultrasound-coupled enzymatic hydrolysis, the composite milk changes from a reticular structure to a dense lamellar structure with the smallest particle size, which is consistent with the previous particle size measurement results. Therefore, the stability and flavor properties of the composite milk after ultrasound-coupled enzymatic hydrolysis are significantly improved.

[0137] Depend on Figure 13The results of protein increase rate and protein digestibility show that ultrasonic coupled enzymatic hydrolysis not only has a significant effect on increasing protein content (p<0.05), but also has a good effect on protein digestibility, increasing the protein digestibility from 51.50% of the untreated group to 67.36%. In addition, the amino acid content of the compound milk after ultrasonic coupled enzymatic hydrolysis treatment has increased significantly, as shown in the results in Table 5, among which the essential amino acids required by the human body: phenylalanine, lysine, leucine, valine, and isoleucine have all increased significantly. According to the analysis of the results in Table 6, ultrasonic coupled enzymatic hydrolysis can effectively eliminate the bitter taste in walnut plant milk caused by walnut seed skin; the application of ultrasonic coupled enzymatic hydrolysis technology can effectively reduce the sour taste after storage at 4°C for 7 days, and there is a significant improvement in appearance, and the floating oil phenomenon is reduced. The content of linolenic acid in walnut milk varies depending on the brand and processing method. Generally speaking, the ALA content of walnut milk accounts for about 10%-15% of the total fatty acids, but by utilizing the properties of the raw materials and optimizing the processing technology, the ALA content in this walnut milk can reach 23%, see Figure 14 . In summary, the present invention does not require peeling to remove bitterness or adding exogenous stabilizers, the process is green, the product has good stability and flavor; compared with walnut milk on the market, the protein content and ALA ratio are high and the protein bioavailability is good; in addition, the walnut milk after ultrasound-coupled enzymatic hydrolysis has a significant increase in the essential amino acids (phenylalanine, lysine, leucine, valine, isoleucine) required by the human body.

[0138] Table 5 Effect of ultrasound-coupled enzymatic hydrolysis on the composition of essential amino acids in compound milk

[0139]

[0140] Table 6 Sensory evaluation of the appearance and bitterness of compound milk by ultrasound-coupled enzymatic hydrolysis

[0141]

[0142] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A green preparation method for high-protein, high-α-linolenic acid walnut composite milk, characterized in that: Walnuts and flax seeds are used as raw materials, and the flax seeds are added in an amount of 10%-50% based on the dry matter mass ratio. The raw materials are firstly pretreated by microwave, and then soaked for 0.5-2.5 hours according to a solid-liquid ratio of 1:(7-11), followed by colloid grinding for 6-14 minutes, and solid-liquid separation to obtain a walnut composite milk with high protein and high α-linolenic acid.

2. The green preparation method according to claim 1, characterized in that: The microwave pretreatment method comprises: adjusting the water content of flaxseed to 13% without degumming, standing at 4°C for 8-12 hours, and then subjecting the flaxseed to microwave treatment; and subjecting raw walnuts to microwave treatment directly without peeling.

3. The green preparation method according to claim 2, characterized in that: The flaxseeds are microwaved at 600-800W for 6-12 minutes, and the walnuts are microwaved at 700-900W for 6-10 minutes.

4. The green preparation method according to any one of claims 1 to 3, characterized in that: After colloid grinding, ultrasonic coupled enzymatic hydrolysis is performed, the enzyme is immediately inactivated, and solid-liquid separation is performed to obtain walnut composite milk with high protein and high α-linolenic acid.

5. The green preparation method according to claim 4, characterized in that: The ultrasound-coupled enzymatic hydrolysis method comprises: subjecting the mixed system after colloid grinding to ultrasound at 100-500W for 4-12 minutes, and then adding 5000-25000U of enzyme and performing enzymatic hydrolysis at 50°C for 0.5-2.5 hours.

6. The green preparation method according to claim 5, characterized in that: The enzyme comprises one of flavor protease, bromelain, papain, alkaline protease, acid protease, neutral protease, aminopeptidase, α-amylase, pectinase, lipase and cellulase.

7. The green preparation method according to claim 4, characterized in that: The method of the enzyme inactivation treatment is to place the mixture in a water bath at 70-90° C. for 5-15 minutes.

8. A high-protein, high-α-linolenic acid walnut composite milk obtained by the green preparation method according to any one of claims 1 to 7, characterized in that: The protein content of the high-protein, high-α-linolenic acid walnut composite milk is ≥1.46g / 100g, the α-linolenic acid content is ≥23%, and the ratio of linoleic acid to linolenic acid is in the range of 2:1 to 3:1.

Images