Method for preparing functional almond milk beverage by using bio-enzyme and product thereof

Abstract

This invention discloses a method for preparing functional almond milk beverages using a bio-enzymatic process and the resulting product. The invention removes hydrocyanic acid from almonds through a coupled cooking and soaking process, significantly reducing harmful substances. It utilizes a nonionic surfactant, imprinted lipase, to enhance enzyme activity and thus improve reaction efficiency. By controlling the rate of glycerol injection into the system, glycerol is made as compatible with oils as possible, reducing the viscosity of the reaction system and preventing glycerol from adhering to the lipase surface. The yield of diglycerides is increased by using a lipase G50 to esterify monoglycerides in the glycerol hydrolysis products, reducing the generation of harmful substances in subsequent molecular distillation. Continuous enzymatic hydrolysis of almond meal with multiple enzymes restrictively degrades macromolecules in the almond meal, releasing polyphenols and generating antioxidant peptides, thereby improving the free radical scavenging ability and stability of the almond protein paste.

Description

Technical Field

[0001] This invention belongs to the field of plant-based beverages, specifically relating to a method for preparing functional almond milk beverages using a bio-enzymatic method and the resulting product. Background Technology

[0002] my country has abundant almond resources, with a sweet almond planting area exceeding 1.3 × 10⁴ hm². 2 China has a large almond production, which lays a solid foundation for the deep processing of almonds. Almonds are highly nutritious, rich in protein, fat, dietary fiber, and minerals, making them an excellent plant protein resource that can be used both as food and medicine.

[0003] Almonds have an oil content as high as 46%-68%, averaging about 60%, and are rich in oleic acid and linoleic acid. Essential amino acids account for approximately 28%-42% of the total amino acids in almonds, making them a good source of amino acids for the human body. For vegetarians, almonds can provide high-quality protein necessary for growth and development. Studies have shown that an equal amount of red meat and almonds contain almost the same amount of protein. Almonds are rich in carbohydrates, and their dietary fiber can effectively prevent colon cancer, rectal cancer, heart disease, and diabetes. Almonds are also rich in micronutrients and the mineral calcium, which can prevent osteoporosis. The calcium in a glass of milk is equivalent to that in four ounces of almonds, making almonds more nutritious than other nuts.

[0004] Almonds have a long history as a food. Currently, almonds are widely used in snack foods. They are highly nutritious and have important health benefits for the human body. They also have high medicinal value, such as expectorant, antitussive, anti-inflammatory, analgesic, immune-regulating, gastritis-preventing, and bowel-regulating effects. They can even be used as a drug to treat tumors. There have been reports in the United States that scholars have used amygdalin to treat cancer. Amygdalin produces cyanide under the action of nitrile lyase or β-glucosidase. Cyanide acts on the bcl-2 protein on the outer membrane of mitochondria, which can inhibit the absorption of cytochrome C, inhibit cellular respiration, and ultimately kill tumor cells.

[0005] The Compendium of Materia Medica lists three main effects of almonds: clearing indigestion, moistening the lungs, and dispersing stagnation. Clearing indigestion means that almonds can aid digestion and relieve constipation. Modern Practical Chinese Medicine records that "almonds, when taken internally, have a mild laxative effect and also have a nourishing effect." Elderly and weak individuals with chronic constipation can take almonds to relieve their constipation. The Essentials of Nourishing Life records that for treating almond poisoning, with diarrhea and vexation, plum juice can be used as an antidote. Another prescription uses indigo juice. Modern medical research has also found that almonds have good adjunctive therapeutic and preventative effects on diseases such as hypertension, hyperlipidemia, arteriosclerosis, coronary heart disease, and constipation.

[0006] In modern industry, almonds are widely used in the food sector, such as in noodles, candy, dairy products, tea drinks, nutritional tofu, nutritional yogurt, chocolate, plant-based protein drinks, and ice cream. Almonds have also been frequently reported as a raw material for plant-based milk. However, most studies utilize the inherent properties of almonds, obtaining almond-based plant-based protein milk through grinding, blending, and homogenization processes, without demonstrating functional benefits, resulting in products that are largely homogenous.

[0007] With societal development, chronic diseases such as obesity are becoming increasingly prevalent, leading to a stronger demand for health-functional beverages. Therefore, this invention utilizes supercritical carbon dioxide extraction to separate almond oil and almond meal. Almond meal is used as a raw material to prepare almond protein slurry. Continuous enzymatic hydrolysis is employed to degrade high-molecular-weight substances, enhancing the stability and free radical scavenging ability of the almond protein slurry. Simultaneously, enzymatic glycerol hydrolysis is used to prepare almond oil diglycerides, a functional oil that serves as the fat source for almond milk. Finally, through blending and high-pressure homogenization, a functional almond milk beverage is obtained. This almond milk is rich in diglycerides, thereby reducing the risk of fat accumulation after consumption. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0009] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0010] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing functional almond milk beverages using a bio-enzymatic method.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing functional almond milk beverages using a bio-enzymatic method, comprising,

[0012] Hydrocyanic acid in almonds is removed by a coupled cooking and soaking process, and almond oil and almond meal are obtained by low-temperature drying and supercritical carbon dioxide extraction.

[0013] Almond meal was prepared into almond protein slurry, α-amylase and cellulase were added for hydrolysis, and then protease was added for reaction to obtain enzymatically hydrolyzed almond protein slurry.

[0014] Glyceryl ester intermediates were prepared by lipase-catalyzed hydrolysis of almond oil. The addition of glycerol was carried out in three stages, and each stage was thoroughly mixed by high-pressure homogenization.

[0015] After glycerol hydrolysis, the glycerol hydrolysis product was catalyzed by the reaction of Lipase G50, a lipase with a double-imprinted catalytic conformation of pH and nonionic surfactant, with fatty acid esterification to obtain amygdalglycerol.

[0016] The functional almond milk beverage is obtained by mixing enzymatically hydrolyzed almond protein slurry, deionized water, almond diglyceride, compound emulsifier, compound stabilizer and sucrose, adjusting the pH to 6-8, homogenizing under high pressure and sterilizing.

[0017] As a preferred embodiment of the preparation method described in this invention, the hydrogen cyanide removal process includes,

[0018] Boil almonds in boiling water for 5-12 minutes, then remove the skin.

[0019] Soak the almonds in a 0.2-0.6% citric acid solution at 60-80℃ for 48-72 hours. The volume of the solution should be 3-5 times that of the almonds. Change the water every 12-24 hours. The hydrocyanic acid in the soaking solution should be less than 1 mg / L.

[0020] In a preferred embodiment of the preparation method described in this invention, the low-temperature drying and supercritical carbon dioxide extraction process involves drying the product in a vacuum constant-temperature drying oven at 45-60℃ until the moisture content reaches 3-5%, and then pulverizing it to 100-150 mesh; the supercritical carbon dioxide extraction temperature is 50-60℃; the extraction pressure is 20-30 MPa; the extraction time is 45-75 min; the separation pressure is 6-12 MPa; the separation temperature is 30-50℃; and the almond oil yield is above 90%.

[0021] As a preferred embodiment of the preparation method described in this invention, a nonionic surfactant is dissolved in isopropanol at a mass fraction of 10-80 mg / L, lipase at a mass fraction of 5-30% is added, the mixture is stirred at 50-100 rpm for 30-60 min at 25°C, and the lipase is obtained by filtration.

[0022] Excess imprinted template of lipase was washed away with a nonpolar solvent and dried in a vacuum desiccator at room temperature for 12-36 h to remove organic solvent, thus obtaining imprinted lipase.

[0023] The nonionic surfactants include Tween 20, 40, 60, 80, etc.; the lipases include Lipase R, Lipase AY-30SD, Lipase DF-15, etc.; the nonpolar solvents include n-hexane and octane.

[0024] In a preferred embodiment of the preparation method described in this invention, the glycerol hydrolysis reaction process is carried out in three stages with a glycerol pumping rate, including:

[0025] The first stage involves pumping in 10-20% glycerin at a constant rate over 1 hour;

[0026] The second stage involves pumping in 20-40% glycerin over 1 hour;

[0027] The third stage involves pumping in 40-65% glycerin over 0.5 hours;

[0028] The glycerol pumping rate is in a molar ratio of 1.5-4:1 to almond oil, and the reaction continues for 4-7.5 hours after the glycerol is pumped in; the amount of imprinted lipase added is 8-15 wt% of the weight of almond oil; the temperature is 50-70℃; and the stirring speed is 600-800 rpm.

[0029] As a preferred embodiment of the preparation method described in this invention, the double blotting process of the lipase G50 includes,

[0030] Prepare a buffer solution with a pH of 5-6, and add a nonionic surfactant to the buffer solution at a concentration of 20-80 mg / mL;

[0031] Add 10-30% Lipase G50 by mass, stir at 25°C for 30-60 min, freeze dry for 12-36 h, elute the imprint template with a nonpolar solvent, and remove the solvent by vacuum drying to obtain imprinted lipase.

[0032] In a preferred embodiment of the preparation method described in this invention, the following steps are taken: during the esterification reaction, the molar ratio of free fatty acids in almond oil to the glycerol backbone of glycerol ester is 1.5-5:1; the temperature is 30-50℃; the stirring speed is 600-800 rpm; the amount of imprinted lipase G50 added is 4-8%; the vacuum degree is 10-30 mbar; the reaction time is 8-12 h; the diglyceride content is above 90%, and the yield is above 90%.

[0033] In a preferred embodiment of the preparation method described in this invention, the enzymes used in the continuous enzymatic hydrolysis of almond meal include α-amylase, cellulase, pectinase, neutral protease, and alkaline protease; wherein the amount of α-amylase added is 1-2%, the amount of cellulase added is 0.3-0.5%, the amount of pectinase added is 0.1-0.3%, the reaction pH is 6, the temperature is 50℃, and the time is 0.5-1.5h; the amount of neutral protease added is 0.01-0.05%, the reaction pH is 7, the temperature is 50℃, and the time is 0.5-1.5h; and the amount of alkaline protease added is 0.03-0.08%, the temperature is 60℃, and the time is 0.5-1.5h.

[0034] In a preferred embodiment of the preparation method described in this invention, the following components are present by weight percentage: the enzymatically hydrolyzed almond protein slurry comprises 20-30%, the deionized water comprises 48-64%, the almond oil functional lipids comprise 8-16%, the monoglycerides comprise 0.04-0.08%, the sucrose ester comprises 0.01-0.03%, the sodium caseinate comprises 0.03-0.05%, the CMC comprises 0.04-0.08%, the sodium alginate comprises 0.02-0.06%, the xanthan gum comprises 0.03-0.05%, the pectin comprises 0.03-0.07%, and the sucrose comprises 7-10%.

[0035] Another objective of this invention is to overcome the shortcomings of the prior art and provide a functional almond milk beverage prepared by a bio-enzymatic method.

[0036] Beneficial effects of this invention:

[0037] (1) The present invention removes hydrocyanic acid from almonds by coupling cooking and soaking processes, which can greatly reduce harmful substances.

[0038] (2) In the preparation of almond oil diglycerides, the present invention utilizes a method of adding monoglycerides and adding glycerol stepwise coupled with high-pressure homogenization to increase the compatibility of glycerol with oils and improve the reaction efficiency. At the same time, the optimal catalytic conformation of Lipase G50 is fixed by pH treatment, which improves the esterification activity of Lipase G50. The lipase is then used as a catalyst to catalyze the esterification reaction between glycerol hydrolysis products and free fatty acids, so that monoglycerides are converted into diglycerides. This greatly increases the yield of diglycerides in the product and reduces the generation of harmful substances.

[0039] (3) This invention utilizes poly-α-amylase and cellulase to synergistically hydrolyze almond meal, thereby limiting the degradation of macromolecules in almond meal to release polyphenols and generate antioxidant peptides, thereby improving the free radical scavenging ability and stability of almond protein paste. Detailed Implementation

[0040] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0041] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0042] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0043] In this invention, α-amylase was purchased from Qingdao Haiweisen Biotechnology Co., Ltd., with an enzyme activity of 6000 U / g; cellulase was purchased from Qingdao Haiweisen Biotechnology Co., Ltd., with an enzyme activity of 100000 U / g; pectinase was purchased from Shandong Huaheng Bioengineering Co., Ltd., with an enzyme activity of 30000 U / g; alkaline protease was purchased from Jiangsu Maisheng Biotechnology Co., Ltd., with an enzyme activity of 50000 U / g; trypsin was purchased from Shanghai Xianghong Biotechnology Co., Ltd., with an enzyme activity of 4000 U / g; and neutral protease was purchased from Jiangsu Jiayuancheng Biotechnology Co., Ltd., with an enzyme activity of 20000 U / g.

[0044] Determination of lipase transesterification activity: The catalytic activity of lipase was evaluated by transesterification between soybean oil and medium-chain triglycerides (MCTs). The substrate was a mixture of soybean oil and MCTs at a molar ratio of 1:1 (soybean oil / MCTs), and the amount of lipase added was 4% relative to the total mass of the substrate. The catalytic reaction was carried out in a round-bottom flask at 50°C and a stirring rate of 600 rpm. After 1 h of reaction, the product was collected to assess its initial activity. The initial activity of the enzyme was assessed based on the content (%) of medium-chain triglycerides after 1 hour of reaction.

[0045] Determination of Lipase G50 esterification activity: Glycerol and oleic acid were added to a batch reactor at a molar ratio of 1:1, followed by the addition of 4% Lipase G50. The reaction was carried out at 30°C and a stirring rate of 600 rpm for 1 hour. The initial enzyme activity was assessed based on the esterification rate (%) of fatty acids in the system after 1 hour of reaction.

[0046] Polyphenol extraction: The enzymatically hydrolyzed sample and control sample were mixed thoroughly with a 1% (v / v) HCl-methanol solution at a ratio of 1:25 (m / V) and allowed to stand at 25°C for 24 h. The mixture was then centrifuged at 4000×g for 10 min, and the supernatant was collected. The precipitate was further mixed with a 1% HCl-methanol solution at a ratio of 1:25 and allowed to stand at 25°C for 24 h. The mixture was then centrifuged at 4000×g for 10 min, and the extracts were combined and evaporated to dryness at 40°C. The resulting extract was then dissolved in 3 mL of methanol and stored at -20°C for subsequent analysis.

[0047] Determination of total polyphenol content: Pipette 0.1 mL of moderately diluted sample solution into a 5 mL test tube. Then, add 0.5 mL of 0.2 mol / L Lolin-phenol reagent and 0.8 mL of 7.5% Na₂CO₃ reagent sequentially. Mix well and let stand in the dark at 25 °C for 30 min before measuring the absorbance at 765 nm. The results are expressed as the total polyphenol content per mL of almond milk equivalent to gallic acid (GAE), with units of μmol GAE / mL almond milk.

[0048] Determination of DPPH free radical scavenging rate: Mix 500 μL of moderately diluted polyphenol extract with 0.3 mL of 0.6 mmol / L DPPH-ethanol solution, shake well, and react in a 25℃ water bath in the dark for 30 min. Zero the sample with methanol and measure the absorbance at 517 nm. Prepare a standard curve using 0-2000 μmol / L Trolox solution instead of the sample. The results are expressed as the amount of Trolox equivalent to each gram of almond milk (μmol TE / mL almond milk).

[0049] Centrifugal sedimentation rate: Accurately weigh a certain amount of sample and place it in a centrifuge tube. Centrifuge at 5000 r / min for 20 min, discard the supernatant, invert the centrifuge tube containing the sediment for 30 min, accurately weigh the sediment mass, and calculate the centrifugal sedimentation rate (SR). Perform three parallel determinations for each sample and take the average value. The lower the centrifugal sedimentation rate, the better the stability of the beverage.

[0050] SR / % = (M1 / M2) × 100

[0051] In the formula: M1 is the mass of the precipitate after centrifugation, g; M2 is the mass of the sample before centrifugation, g.

[0052] Example 1

[0053] 1. Almond oil meal separation

[0054] (1) Peeling and Detoxification: Almonds contain β-glucosidases such as amygdalin, amygdalinase, and cherifolinase. Amygdalin is hydrolyzed sequentially by amygdalinase and cherifolinase into cherifolin glycoside, mandelic acid, and hydrocyanic acid. When the hydrocyanic acid content in the soaking solution exceeds 10.00 mg / L, it will cause toxicity. It can be detoxified by boiling and soaking. Selected almonds are boiled in boiling water for 5 minutes, then cooled in cold water, peeled, and soaked in 0.2% citric acid solution at 70℃ for 48 hours. The volume of the solution is 3 times that of the almonds. The water is changed every 12 hours for detoxification treatment. After soaking, the hydrocyanic acid content in the soaking solution is 0.67 mg / L, which is less than 1 mg / L.

[0055] (2) Drying: Place the detoxified almonds in a 45℃ vacuum constant temperature drying oven and dry them until the moisture content is 5%, then grind them to 100 mesh in a high-speed multi-functional pulverizer;

[0056] (3) Oil extraction: Almond oil was extracted using supercritical carbon dioxide at an extraction temperature of 55°C, an extraction pressure of 20 MPa, and an extraction time of 60 min. Separation pressure was 9 MPa and separation temperature was 50°C. Finally, almond oil and almond protein meal were obtained, with an almond oil yield of 92.4%.

[0057] 2. Preparation of functional oils

[0058] The activity of imprinted lipase was enhanced by using a nonionic surfactant. The nonionic surfactant Tween 20 was dissolved in isopropanol at a concentration of 80 mg / L and thoroughly dispersed. Lipase R (30% by mass) was then added, and the mixture was stirred at 100 rpm for 30 min at 25 °C. The lipase was then filtered to obtain the imprinted template. The surfactant-imprinted template on the immobilized lipase R was eluted with the nonpolar solvent n-hexane. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 12 h to remove the organic solvent, yielding the imprinted lipase.

[0059] The activities of imprinted and unimprinted lipases are shown in Table 1.

[0060] Table 1

[0061] Lipase Imprint LipaseR Non-imprinted LipaseR Increase in vitality (%) Enzyme activity 34.8％ 18.7％ 86.1

[0062] Diglycerides are obtained through glycerol hydrolysis. Glycerol has poor compatibility with fats and oils, and its high viscosity means that excessive glycerol addition will affect mass transfer in the system, thus impacting reaction efficiency. Furthermore, excess glycerol can adhere to the enzyme surface, affecting lipase activity. Therefore, by controlling the glycerol injection rate, glycerol is gradually consumed during the reaction, producing diglycerides and monoglycerides, promoting thorough mixing of glycerol and fats, thereby avoiding the side effects caused by excessive glycerol and improving reaction efficiency.

[0063] Almond oil was added to a batch reactor under nitrogen protection. Simultaneously, imprinted lipase R was added at a rate of 8 wt% of the almond oil weight. The temperature was raised to 50°C, and stirring was initiated at 700 rpm. Food-grade glycerol was pumped into the reactor at a molar ratio of glycerol to almond oil of 1.5:1. The glycerol was pumped in three stages: 10% glycerol was pumped in at a constant rate over 1 hour; 30% glycerol was pumped in over 1 hour; and 60% glycerol was pumped in over 0.5 hours. After the glycerol injection was complete, the reaction continued for 4 hours. The lipase was filtered to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 90°C and slowly stirred at 30 rpm for 1 hour to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for further glycerolysis reactions.

[0064] The composition of the obtained glycerides is shown in Table 2.

[0065] Table 2

[0066] type content(%) monoglycerides 49.6 diglycerides 41.7 Triglycerides 8.8

[0067] The activity of double-blotted glycerol lipase Lipase G50 was enhanced by using a pH- and non-ionic surfactant. A pH 5.0 buffer solution was prepared, and 10% (w / w) of Lipase G50 was added. The mixture was stirred at 25°C for 30 min to allow for sufficient ion exchange. The pH-blotted lipase was obtained by filtration and vacuum drying to remove moisture. Tween 40 (non-ionic surfactant) was added at a concentration of 20 mg / mL to isopropanol, mixed thoroughly, and then 10% of the pH-blotted lipase was added. The mixture was stirred at 25°C for 30 min, filtered, and the lipase was obtained. Excess template was eluted with the non-polar solvent octane, and the lipase was vacuum dried for 24 h to obtain the double-blotted lipase.

[0068] The enzyme activities of blotted and unblotted Lipase G50 are shown in Table 3.

[0069] Table 3

[0070] Lipase Dual Imprint Lipase G50 Non-imprinted Lipase G50 Increase in vitality (%) Enzyme activity 31.6％ 15.7％ 101.3

[0071] The glycerol ester mixture was added to a batch reactor, and free almond oil fatty acids were added based on the amount of glycerol ester backbone. The molar ratio of free fatty acids to glycerol ester backbone was 1.5:1. The temperature was raised to 50°C and stirring was started at 800 rpm. Double-imprinted Lipase G50 lipase was added at a rate of 4%. Vacuum was applied at a vacuum level of 20 mbar and the reaction was carried out for 10 h to obtain the glycerol ester product.

[0072] The composition of glycerides in the products after esterification is shown in Table 4.

[0073] Table 4

[0074]

[0075]

[0076] Fatty acids were removed by molecular distillation under the following conditions: distillation temperature 160℃, pressure 3Pa, condenser temperature 30℃. The resulting product contained 90.3% diglycerides, 0.6% monoglycerides, and 9.1% triglycerides. The yield of diglycerides was 90.3%, glycidyl esters were 1.35 mg / kg, and chloropropanol esters were 0.27 mg / kg.

[0077] 3. Preparation of almond protein paste

[0078] Almond meal and water were mixed at a ratio of 1:5 and milled using a colloid mill to obtain almond milk. The pH of the almond milk was adjusted to 6, and 2% α-amylase, 0.3% cellulase, and 0.1% pectinase were added. The mixture was incubated in a 50°C water bath for 1.5 hours. The pH was then adjusted to 7, and 0.03% neutral protease was added. The mixture was reacted in a 50°C water bath for 1 hour. The pH was then adjusted to 9.5, and 0.03% alkaline protease was added. The mixture was hydrolyzed in a 60°C water bath for 1 hour for restricted proteolytic hydrolysis. The almond milk was heated to 100°C to inactivate the enzymes for 15 minutes and filtered through a 200-mesh filter to obtain enzymatically hydrolyzed almond protein slurry. The polyphenol content, DPPH free radical scavenging rate, and emulsification stability of the enzymatically hydrolyzed almond protein slurry were determined.

[0079] The polyphenol content, DPPH free radical scavenging rate and centrifugal sedimentation rate in enzymatically hydrolyzed almond protein slurry are shown in Table 5.

[0080] Table 5

[0081]

[0082] 4. Preparation of Functional Almond Milk Beverages

[0083] In the preparation of functional almond milk beverage, 20% enzymatically hydrolyzed almond protein slurry, 64% soft water, and 8% almond oil functional lipids were used. A complex emulsifier was added, consisting of 0.08% monoglyceride, 0.02% sucrose ester, and 0.03% sodium caseinate. A complex stabilizer was added, consisting of 0.08% CMC, 0.02% sodium alginate, 0.03% xanthan gum, and 0.07% pectin. The sucrose content was 7.67%. High-pressure homogenization was performed twice at 30 MPa and 60℃ to ensure a uniform and stable beverage texture and a smooth, delicate taste. The resulting almond milk beverage contained not only a large amount of functional peptides but also some protein. pH value significantly affects protein solubility, sensory properties, and product appearance. The stability and sensory properties of the almond milk were evaluated by adjusting the pH. The pH values ​​were adjusted to 4-6, 6-8, and 8-10.

[0084] The effects of pH on stability and sensory characteristics are shown in Table 6.

[0085] Table 6

[0086] Quality Indicators pH = 4-6 pH = 6-8 pH = 8-10 Appearance milky milky grayish white Flavor Sour Sweet and pure Astringent taste stability Precipitation occurs Emulsification is uniform and stable Emulsification is uniform and stable

[0087] This shows that the products exhibit good stability and sensory properties at pH levels between 6 and 8.

[0088] After homogenization, the mixture was sterilized at 121°C for 20 minutes, and finally degassed in a vacuum degassing tank with a vacuum degree of 500-600 mmHg to obtain a functional almond milk beverage.

[0089] Example 2

[0090] 1. Almond oil meal separation

[0091] (1) Peeling and Detoxification: Almonds contain β-glucosidases such as amygdalin, amygdalinase, and cherifolinase. Amygdalin is hydrolyzed sequentially by amygdalinase and cherifolinase into cherifolin glycoside, mandelic acid, and hydrocyanic acid. When the hydrocyanic acid content in the soaking solution exceeds 10.00 mg / L, it will cause toxicity. It can be detoxified by boiling and soaking. Selected almonds are boiled in boiling water for 8 minutes, then cooled in cold water, peeled, and soaked in 0.4% citric acid solution at 80℃ for 60 hours. The volume of the solution is 4 times that of the almonds. The water is changed every 15 hours for detoxification treatment. After soaking, the hydrocyanic acid content in the soaking solution is 0.55 mg / L, which is less than 1 mg / L.

[0092] (2) Drying: Place the detoxified almonds in a 60℃ vacuum constant temperature drying oven and dry them until the moisture content is 3%, then grind them to 150 mesh in a high-speed multi-functional pulverizer;

[0093] (3) Oil extraction: Almond oil was extracted using supercritical carbon dioxide at an extraction temperature of 60℃, an extraction pressure of 30MPa, and an extraction time of 45min. Separation pressure was 12MPa and separation temperature was 40℃. Finally, almond oil and almond protein meal were obtained, with an almond oil yield of 94.1%.

[0094] 2. Preparation of functional oils

[0095] The activity of imprinted lipase was enhanced by using a nonionic surfactant. The nonionic surfactant Tween 40 was dissolved in isopropanol at a concentration of 40 mg / L and thoroughly dispersed. 20% (w / w) of lipase Lipase AY-30SD was added to the solution. The mixture was stirred at 50 rpm for 45 min at 25 °C, filtered to obtain the lipase, and the surfactant-imprinted template on the lipase Lipase AY-30SD was eluted with the nonpolar solvent n-hexane. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 24 h to remove the organic solvent, yielding the imprinted lipase.

[0096] The activities of imprinted and unimprinted lipases are shown in Table 7.

[0097] Table 7

[0098] Lipase Imprint LipaseAY-30SD Non-Imprinted LipaseAY-30SD Increase in vitality (%) Enzyme activity 38.5％ 23.7％ 64.4

[0099] Diglycerides are obtained through glycerol hydrolysis. Glycerol has poor compatibility with fats and oils, and its high viscosity means that excessive glycerol addition will affect mass transfer in the system, thus impacting reaction efficiency. Furthermore, excess glycerol can adhere to the enzyme surface, affecting lipase activity. Therefore, by controlling the glycerol injection rate, glycerol is gradually consumed during the reaction, producing diglycerides and monoglycerides, promoting thorough mixing of glycerol and fats, thereby avoiding the side effects caused by excessive glycerol and improving reaction efficiency.

[0100] Almond oil was added to a batch reactor under nitrogen protection. Simultaneously, imprinted lipase LipaseAY-30SD was added at a rate of 12 wt% of the almond oil weight. The temperature was raised to 60°C, and stirring was initiated at 600 rpm. Food-grade glycerol was pumped into the reactor at a molar ratio of glycerol to almond oil of 3:1. The glycerol was pumped in three stages: 20% glycerol was pumped in at a constant rate over 1 hour; 40% glycerol was pumped in over 1 hour; and 40% glycerol was pumped in over 0.5 hours. After the glycerol injection was complete, the reaction continued for 6 hours. The lipase was filtered to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 85°C and slowly stirred at 50 rpm for 0.5 hours to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for further glycerolysis reactions.

[0101] The composition of the obtained glycerides is shown in Table 8.

[0102] Table 8

[0103] type content(%) monoglycerides 53.6 diglycerides 40.5 Triglycerides 5.8

[0104] The activity of lipase G50, a pH- and nonionic surfactant, was enhanced by double-blotting. A buffer solution with a pH of 5.5 was prepared, and 20% (w / w) of Lipase G50 was added. The mixture was stirred at 25°C for 45 min to allow for sufficient ion exchange. The pH-blotted lipase was obtained by filtration and vacuum drying to remove moisture. Tween 20, a nonionic surfactant, was added to isopropanol at a concentration of 60 mg / mL. After thorough mixing, 20% of the pH-blotted lipase was added, and the mixture was stirred at 25°C for 45 min. The lipase was obtained by filtration, and excess template was eluted with the nonpolar solvent n-hexane. After vacuum drying for 36 h, the double-blotted lipase was obtained.

[0105] The enzyme activities of blotted and unblotted Lipase G50 are shown in Table 9.

[0106] Table 9

[0107] Lipase Dual Imprint Lipase G50 Non-imprinted Lipase G50 Increase in vitality (%) Enzyme activity 33.1％ 15.7％ 110.8

[0108] A mixture of glycerides was added to a batch reactor, and free almond oil fatty acids were added based on the amount of glycerides' glycerol backbone. The molar ratio of free fatty acids to glycerides' glycerol backbone was 3:1. The temperature was raised to 40°C, and stirring was started at 700 rpm. Double-imprinted Lipase G50 lipase was added at a rate of 6%. Vacuum was applied at a vacuum level of 10 mbar, and the reaction was carried out for 8 hours to obtain the glycerides product.

[0109] The composition of glycerides in the products after esterification is shown in Table 10.

[0110] Table 10

[0111] type content(%) monoglycerides 0.7 diglycerides 93.6 Triglycerides 5.7

[0112] Fatty acids were removed by molecular distillation under the following conditions: distillation temperature of 160℃, pressure of 3Pa, and condenser temperature of 30℃. The resulting product contained 93.1% diglycerides, 0.6% monoglycerides, and 6.3% triglycerides. The yield of diglycerides was 93.1%, the content of glycidyl esters was 1.38 mg / kg, and the content of chloropropanol esters was 0.25 mg / kg.

[0113] 3. Preparation of almond protein paste

[0114] Almond meal and water were mixed at a ratio of 1:8 and milled using a colloid mill to obtain almond milk. The pH of the almond milk was adjusted to 6, and 1% α-amylase, 0.5% cellulase, and 0.2% pectinase were added. The mixture was then incubated in a 50°C water bath for 1 hour. The pH was then adjusted to 7, and 0.05% neutral protease was added. The mixture was then reacted in a 50°C water bath for 0.5 hours. The pH was then adjusted to 9.5, and 0.05% alkaline protease was added. The mixture was then hydrolyzed in a 60°C water bath for 0.5 hours for restricted proteolytic hydrolysis. The almond milk was then heated to 100°C to inactivate the enzymes for 15 minutes and filtered through a 200-mesh filter to obtain enzymatically hydrolyzed almond protein slurry. The polyphenol content, DPPH free radical scavenging rate, and emulsion stability of the enzymatically hydrolyzed almond protein slurry were determined.

[0115] The polyphenol content, DPPH free radical scavenging rate and centrifugation sedimentation rate of enzymatically hydrolyzed almond protein slurry are shown in Table 11.

[0116] Table 11

[0117]

[0118]

[0119] 4. Preparation of Functional Almond Milk Beverages

[0120] In the preparation of functional almond milk beverage, 30% enzymatically hydrolyzed almond protein slurry, 48% soft water, and 12% almond oil functional lipids were used. A complex emulsifier was added, consisting of 0.06% monoglyceride, 0.01% sucrose ester, and 0.05% sodium caseinate. A complex stabilizer was added, consisting of 0.06% CMC, 0.04% sodium alginate, 0.05% xanthan gum, and 0.05% pectin. The sucrose content was 9.68%. High-pressure homogenization was performed twice at 40 MPa and 50℃ to ensure a uniform and stable beverage texture and a smooth, delicate taste. The resulting almond milk beverage contained not only a large amount of functional peptides but also some protein. pH value significantly affects protein solubility, taste, and product appearance. Adjusting the pH to 6.5 resulted in a milky white color, a pure sweetness, and uniform and stable emulsification. The product was sterilized at 121℃ for 20 minutes, and then degassed under vacuum in a degassed tank with a vacuum degree of 500-600 mmHg to obtain a functional almond milk beverage.

[0121] Example 3

[0122] 1. Almond oil meal separation

[0123] (1) Peeling and Detoxification: Almonds contain β-glucosidases such as amygdalin, amygdalinase, and cherifolinase. Amygdalin is hydrolyzed sequentially by amygdalinase and cherifolinase into cherifolin glycoside, mandelic acid, and hydrocyanic acid. When the hydrocyanic acid content in the soaking solution exceeds 10.00 mg / L, it will cause toxicity. It can be detoxified by boiling and soaking. Selected almonds are boiled in boiling water for 12 minutes, then cooled in cold water, peeled, and soaked in 0.6% citric acid solution at 60℃ for 72 hours. The volume of the solution is 5 times that of the almonds. The water is changed every 24 hours for detoxification treatment. After soaking, the hydrocyanic acid content in the soaking solution is 0.61 mg / L, which is less than 1 mg / L.

[0124] (2) Drying: Place the detoxified almonds in a 50℃ vacuum constant temperature drying oven and dry them until the moisture content is 4%, then grind them to 120 mesh in a high-speed multi-functional pulverizer;

[0125] (3) Oil extraction: Almond oil was extracted using supercritical carbon dioxide at an extraction temperature of 50°C, an extraction pressure of 25 MPa, and an extraction time of 75 min. Separation pressure was 6 MPa and separation temperature was 30°C. Finally, almond oil and almond protein meal were obtained, with an almond oil yield of 93.3%.

[0126] 2. Preparation of functional oils

[0127] The activity of imprinted lipase was enhanced using a nonionic surfactant. The nonionic surfactant Tween 80 was dissolved in isopropanol at a concentration of 10 mg / L and thoroughly dispersed. Lipase DF-15 at a concentration of 5% (w / w) was added, and the mixture was stirred at 75 rpm for 60 min at 25 °C. The mixture was then filtered to obtain the lipase. The surfactant-imprinted template on the lipase DF-15 was eluted with the nonpolar solvent octane. The lipase was then filtered and dried in a vacuum desiccator at room temperature for 36 h to remove the organic solvent, yielding the imprinted lipase.

[0128] The activities of imprinted and unimprinted lipases are shown in Table 12.

[0129] Table 12

[0130] Lipase Imprint Lipase DF-15 Non-imprinted Lipase DF-15 Increase in vitality (%) Enzyme activity 28.8％ 15.5％ 85.8

[0131] Diglycerides are obtained through glycerol hydrolysis. Glycerol has poor compatibility with fats and oils, and its high viscosity means that excessive glycerol addition will affect mass transfer in the system, thus impacting reaction efficiency. Furthermore, excess glycerol can adhere to the enzyme surface, affecting lipase activity. Therefore, by controlling the glycerol injection rate, glycerol is gradually consumed during the reaction, producing diglycerides and monoglycerides, promoting thorough mixing of glycerol and fats, thereby avoiding the side effects caused by excessive glycerol and improving reaction efficiency.

[0132] Almond oil was added to a batch reactor under nitrogen protection. Simultaneously, imprinted lipase DF-15 was added at a rate of 15 wt% of the almond oil weight. The temperature was raised to 70°C, and stirring was initiated at 800 rpm. Food-grade glycerol was pumped into the reactor at a molar ratio of 4:1 to almond oil. The glycerol was pumped in three stages: 15% glycerol was pumped in at a uniform rate over 1 hour; 20% glycerol was pumped in over 1 hour; and 65% glycerol was pumped in over 0.5 hours. After the glycerol injection was complete, the reaction continued for 7.5 hours. The lipase was filtered to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 80°C and slowly stirred at 80 rpm for 1.5 hours to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for further glycerolysis reactions.

[0133] The composition of the obtained glycerides is shown in Table 13.

[0134] Table 13

[0135] type content(%) monoglycerides 61.1 diglycerides 34.4 Triglycerides 4.5

[0136] The activity of double-blotted glycerol lipase (Lipase G50) was enhanced by using a pH- and non-ionic surfactant. A pH 6 buffer solution was prepared, and 30% (w / w) of Lipase G50 was added. The mixture was stirred at 25°C for 60 min to allow for sufficient ion exchange. The pH-blotted lipase was obtained by filtration and vacuum drying to remove moisture. Tween 80 (80 mg / mL) was added to isopropanol, mixed thoroughly, and 30% (w / w) of the pH-blotted lipase was added. The mixture was stirred at 25°C for 60 min, filtered, and the lipase was obtained. Excess template was eluted with the non-polar solvent octane, and the lipase was vacuum dried for 12 h to obtain the double-blotted lipase.

[0137] The enzyme activities of blotted and unblotted Lipase G50 are shown in Table 14.

[0138] Table 14

[0139] Lipase Dual Imprint Lipase G50 Non-imprinted Lipase G50 Increase in vitality (%) Enzyme activity 32.4％ 15.7％ 106.4

[0140] A mixture of glycerides was added to a batch reactor, and free almond oil fatty acids were added based on the amount of glycerides' glycerol backbone. The molar ratio of free fatty acids to glycerides' glycerol backbone was 5:1. The temperature was raised to 30°C, and stirring was started at 600 rpm. Double-imprinted Lipase G50 lipase was added at a rate of 8%. Vacuum was then applied at a vacuum level of 30 mbar, and the reaction was carried out for 12 hours to obtain the glycerides product.

[0141] The composition of glycerides in the products after esterification is shown in Table 15.

[0142] Table 15

[0143] type content(%) monoglycerides 1.1 diglycerides 94.5 Triglycerides 4.4

[0144] Fatty acids were removed by molecular distillation under the following conditions: distillation temperature 160℃, pressure 3Pa, condenser temperature 30℃. The resulting product contained 94.2% diglycerides, 0.9% monoglycerides, and 4.9% triglycerides. The yield of diglycerides was 94.2%, the content of glycidyl esters was 1.31 mg / kg, and the content of chloropropanol esters was 0.22 mg / kg.

[0145] 3. Preparation of almond protein paste

[0146] Almond meal and water were mixed at a ratio of 1:12 and milled using a colloid mill to obtain almond milk. The pH of the almond milk was adjusted to 6, and 1.5% α-amylase, 0.4% cellulase, and 0.3% pectinase were added. The mixture was incubated in a 50°C water bath for 0.5 hours. The pH was then adjusted to 7, and 0.01% neutral protease was added. The mixture was incubated in a 50°C water bath for 1.5 hours. The pH was then adjusted to 9.5, and 0.08% alkaline protease was added. The mixture was hydrolyzed in a 60°C water bath for 1.5 hours for restricted proteolytic hydrolysis. The almond milk was then heated to 100°C to inactivate the enzyme for 15 minutes and filtered through a 200-mesh filter to obtain enzymatically hydrolyzed almond protein slurry. The polyphenol content, DPPH free radical scavenging rate, and emulsion stability of the enzymatically hydrolyzed almond protein slurry were determined.

[0147] The polyphenol content, DPPH free radical scavenging rate and centrifugal sedimentation rate of enzymatically hydrolyzed almond protein slurry are shown in Table 16.

[0148] Table 16

[0149]

[0150] 4. Preparation of Functional Almond Milk Beverages

[0151] In the preparation of functional almond milk beverage, 25% enzymatically hydrolyzed almond protein slurry, 50% soft water, and 16% functional lipids from almond oil were used. A complex emulsifier was added, consisting of 0.04% monoglyceride, 0.03% sucrose ester, and 0.04% sodium caseinate. A complex stabilizer was added, consisting of 0.04% CMC, 0.06% sodium alginate, 0.04% xanthan gum, and 0.03% pectin. The sucrose content was 8.72%. High-pressure homogenization was performed twice at 35 MPa and 55℃ to ensure a uniform and stable beverage texture and a smooth, delicate taste. The resulting almond milk beverage contained not only a large amount of functional peptides but also some protein. pH value significantly affects protein solubility, taste, and product appearance. Adjusting the pH to 7.5 resulted in a milky white color, a pure sweetness, and uniform and stable emulsification. The product was sterilized at 121℃ for 20 minutes, and then degassed under vacuum in a degassed tank with a vacuum degree of 500-600 mmHg to obtain a functional almond milk beverage.

[0152] Comparative Example 1

[0153] Refer to Example 1 for oilseed meal separation process

[0154] 1. Almond oil meal separation

[0155] (1) Peeling and detoxification: Almonds contain β-glucosidases such as amygdalin, amygdalinase, and cherifolinase. Amygdalin is hydrolyzed sequentially by amygdalinase and cherifolinase into cherifolin glycoside, mandelic acid, and hydrocyanic acid. When the hydrocyanic acid content in the soaking solution exceeds 10.00 mg / L, it will cause toxicity. It can be detoxified by boiling and soaking. Selected almonds are boiled in boiling water for 3 minutes, then cooled in cold water, peeled, and soaked in 0.2% citric acid solution at 70℃ for 48 hours. The volume of the solution is 3 times that of the almonds. The water is changed every 12 hours for detoxification treatment. After soaking, the hydrocyanic acid content in the soaking solution is 1.15 mg / L.

[0156] (2) Drying: Place the detoxified almonds in a 45℃ vacuum constant temperature drying oven and dry them until the moisture content is 5%, then grind them to 80 mesh in a high-speed multi-functional pulverizer;

[0157] (3) Oil extraction: Almond oil was extracted using supercritical carbon dioxide at an extraction temperature of 55°C, an extraction pressure of 20 MPa, and an extraction time of 60 min. Separation pressure was 9 MPa and separation temperature was 50°C. Finally, almond oil and almond protein meal were obtained, with an almond oil yield of 87.2%.

[0158] Comparative Example 2

[0159] 1. Functional Oil Preparation Process: Referring to Example 2, in the preparation of almond oil diglycerides, instead of using Lipase G50 for pH-controlled conformation esterification, molecular distillation is used to directly remove fatty acids. The specific differences are as follows:

[0160] 2. Preparation of almond oil diglyceride

[0161] Almond oil was added to a batch reactor and purged with nitrogen for protection. Simultaneously, imprinted lipase LipaseAY-30SD was added at a rate of 12 wt% of the weight of the almond oil. The temperature was raised to 60°C and stirring was started at a speed of 600 rpm. At the same time, food-grade glycerol was pumped into the reactor at a molar ratio of 3:1 between the amount of glycerol pumped and the amount of almond oil.

[0162] The glycerol was pumped in at three stages: the first stage involved a uniform pumping of 20% glycerol over 1 hour; the second stage involved pumping in 40% glycerol over 1 hour; and the third stage involved pumping in 40% glycerol over 0.5 hours. After the glycerol pumping was complete, the reaction continued for 6 hours. Lipase was then filtered out to obtain a mixture of glycerides. The resulting glyceride mixture was heated to 85°C and slowly stirred at 50 rpm for 0.5 hours to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for a new glycerolysis reaction.

[0163] The product contained 53.6% monoglycerides, 40.5% diglycerides, and 5.8% triglycerides.

[0164] Fatty acids were removed by molecular distillation under the following conditions: distillation temperature 160℃, pressure 3Pa, and condenser temperature 30℃. The resulting product contained 41.0% diglycerides, 53.1% monoglycerides, and 5.9% triglycerides, with a diglyceride yield of 41.0%. The glycidyl ester content was 2.16 mg / kg, and the chloropropanol ester content was 0.42 mg / kg. Monoglycerides were removed by two-stage molecular distillation under the following conditions: distillation temperature 200℃, pressure 2Pa, and condenser temperature 25℃. The resulting product contained 86.5% diglycerides, 0.8% monoglycerides, 12.7% triglycerides, 5.78 mg / kg glycidyl esters, and 1.25 mg / kg chloropropanol esters.

[0165] Comparative Example 3

[0166] 1. Referring to Example 2, in the preparation of almond oil diglycerides, instead of using the method of adding glycerol in stages and homogenizing, glycerol is added directly for reaction. The difference is:

[0167] 2. Preparation of almond oil diglycerides

[0168] Almond oil was added to a batch reactor under nitrogen protection, and imprinted lipase LipaseAY-30SD was added simultaneously at a rate of 12 wt% of the almond oil weight. The temperature was raised to 60°C, the molar ratio of glycerol to almond oil was 3:1, the stirring speed was 600 rpm, and the reaction time was 8.5 h. After the reaction, the lipase was filtered to obtain a mixture of glycerides. The obtained glyceride mixture was heated to 85°C and slowly stirred at 50 rpm for 0.5 h to accelerate the separation of glycerol and glycerides. The glycerol was then separated from the glycerides, and the recovered glycerol was used as a raw material for a new glycerolysis reaction.

[0169] The product contained 44.6% monoglycerides, 36.8% diglycerides, and 18.6% triglycerides. This demonstrates that the reaction efficiency of the system without homogeneous mixing was significantly reduced.

[0170] After esterification and molecular distillation, the product contains 0.6% monoglycerides, 80.6% diglycerides, and 18.8% triglycerides.

[0171] Comparative Example 4

[0172] Under the conditions of Example 1, almond oil diglycerides were not prepared; instead, almond oil was directly substituted. The almond milk beverage was prepared in the same manner as in Example 1. The stability of the almond milk beverage was determined, and the results of the stability were obtained by referring to the method for determining the centrifugal sedimentation rate.

[0173] The measurement results are shown in Table 17.

[0174] Table 17

[0175] Centrifugal sedimentation rate (%) Example 1 0.6 Comparative Example 4 1.1

[0176] It can be seen that when almond oil diglycerides are not prepared and are directly replaced with almond oil, the resulting dairy product has poor stability. Since almond oil is composed of triglycerides and does not possess emulsifying properties, it cannot help stabilize the almond milk beverage emulsion system.

[0177] Comparative Example 5

[0178] Under the conditions of Example 1, only α-amylase, cellulase and pectinase were used to prepare enzymatically hydrolyzed almond protein paste, without using protease for enzymatic hydrolysis. Other processes were the same as in Example 2. Milk beverage was prepared, and the stability of the almond milk beverage was determined. The stability results were obtained by referring to the method for determining the centrifugal sedimentation rate.

[0179] The measurement results are shown in Table 18.

[0180] Table 18

[0181]

[0182]

[0183] It can be seen that without the addition of protease to generate peptides, the final beverage exhibits poor stability. Because the generated peptides possess emulsifying properties and better solubility, they work synergistically with other substances to improve the stability of the almond milk beverage emulsion system. Simultaneously, the enzymatic hydrolysis processes of α-amylase, cellulase, and pectinase release a certain amount of polyphenols, which bind to proteins, reducing protein solubility to some extent.

[0184] Comparative Example 6

[0185] Under the conditions of Example 1, almond meal was prepared into almond protein slurry. Without adding α-amylase, cellulase and pectinase for hydrolysis, protease was directly added to generate peptides. Other processes were the same as in Example 2. Milk beverage was prepared and the stability of almond milk beverage was determined. The stability results were obtained by referring to the method for determining centrifugal sedimentation rate.

[0186] The measurement results are shown in Table 19.

[0187] Table 19

[0188] Centrifugal sedimentation rate (%) Example 2 0.6 Comparative Example 6 1.4

[0189] It can be seen that adding only protease to generate peptides, without adding other enzymes, has a certain negative impact on the stability of the final beverage. Since almond meal contains a certain amount of insoluble starch, cellulose, and other substances, if not hydrolyzed, it will adversely affect the stability of the beverage system.

[0190] Comparative Example 7

[0191] Under the conditions of Example 1, almond meal was prepared into almond protein slurry without the addition of α-amylase, cellulase and pectinase for hydrolysis, and without the addition of protease to generate peptides. Other processes were the same as in Example 2. Milk beverage was prepared and the stability of the almond milk beverage was determined. The stability results were obtained by referring to the method for determining the centrifugal sedimentation rate.

[0192] The measurement results are shown in Table 20.

[0193] Table 20

[0194] Centrifugal sedimentation rate (%) Example 2 0.6 Comparative Example 7 2.1

[0195] It can be seen that when almond meal is formulated into almond protein slurry, without the addition of α-amylase, cellulase and pectinase for hydrolysis, and without the addition of protease to generate peptides, the resulting beverage has poor stability.

[0196] Comparative Example 8

[0197] Referring to Example 3, different batches of reactions were carried out using double-imprinted lipase Lipase G50 and non-imprinted Lipase G50, respectively, and the activity changes of different lipases in different batches were compared to compare their catalytic stability.

[0198] The activity and enzyme inactivation rate of double-blotted and non-blotted Lipase G50 in different reaction batches are shown in Table 21.

[0199] Table 21

[0200] batch Double Imprint No trace 1 32.4％ 15.7％ 2 31.5％ 13.5％ 3 30.6％ 12.7％ 4 28.3％ 11.4％ 5 27.4％ 9.2％ 6 25.5％ 6.1％ Enzyme inactivation rate 21.3％ 61.1％

[0201] First, the active catalytic structure of Lipase G50 is fixed by pH. Then, a nonionic surfactant interacts with the lipase through hydrophobic interactions, allowing the polar head of the nonionic surfactant to bind to the hydrophilic group of the lipase, and the nonpolar head to bind to its hydrophobic group. This alters the hydrophilicity and hydrophobicity of the lipase surface, preventing excessive contact between the lipase and hydrophilic substances in the system, thus preventing enzyme dehydration and ensuring the stability of the lipase catalytic structure. Simultaneously, the interaction between the surfactant and the active site of the lipase opens the cap of the active site, allowing water to be removed and maintaining the catalytic conformation of the active site, thereby further enhancing the catalytic activity of the lipase.

[0202] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing functional almond milk beverages using a bio-enzymatic method, characterized in that: include, Boil the almonds in boiling water for 5-12 minutes and remove the skin; soak them in a 0.2-0.6% citric acid solution at 60-80℃ for 48-72 hours, using 3-5 times the volume of the almonds, changing the water every 12-24 hours, and ensuring that the hydrocyanic acid in the soaking solution is less than 1 mg / L. Almond oil and almond meal were obtained by low-temperature drying and supercritical carbon dioxide extraction. Almond meal was prepared into almond protein slurry, and α-amylase, cellulase hydrolysis, and pectinase were added. Neutral protease and alkaline protease were then added to obtain enzymatically hydrolyzed almond protein slurry. Specifically, the amount of α-amylase added was 1-2%, the amount of cellulase added was 0.3-0.5%, and the amount of pectinase added was 0.1-0.3%. The reaction was carried out at pH 6, temperature 50℃, and time 0.5-1.5 h. The amount of neutral protease added was 0.01-0.05%, the reaction was carried out at pH 7, temperature 50℃, and time 0.5-1.5 h. The amount of alkaline protease added was 0.03-0.08%, the temperature was 60℃, and time 0.5-1.5 h. A glycerol ester intermediate was prepared by lipase-catalyzed hydrolysis of almond oil. The addition of glycerol was carried out in three stages, each stage involving thorough mixing using high-pressure homogenization. The lipase used was an imprinted lipase. The imprinting process included dissolving a nonionic surfactant in isopropanol at a concentration of 10-80 mg / L, adding 5-30% lipase, stirring the mixture at 50-100 rpm for 30-60 min at 25°C, and filtering to obtain the lipase. Excess imprint template was eluted with a nonpolar solvent, and the mixture was dried in a vacuum dryer at room temperature for 12-36 h to remove organic solvent, yielding the imprinted lipase. The nonionic surfactant was Tween 20, Tween 40, Tween 60, or Tween 80; the lipase was Lipase R, Lipase AY-30SD, or Lipase DF-15; and the nonpolar solvent was n-hexane or octane. The glycerol hydrolysis reaction process involves three stages of glycerol injection: the first stage involves a uniform injection of 10-20% glycerol over 1 hour; the second stage involves an injection of 20-40% glycerol over 1 hour; and the third stage involves an injection of 40-65% glycerol over 0.5 hours. The molar ratio of glycerol to almond oil is 1.5-4:

1. After the glycerol injection is complete, the reaction continues for 4-7.5 hours. The amount of imprinted lipase added is 8-15 wt% of the weight of almond oil. The temperature is 50-70℃, and the stirring speed is 600-800 rpm. After glycerol hydrolysis, a glycerol lipase (Lipase G50) with a double-imprinted conformation immobilized by pH and a nonionic surfactant is used to catalyze the esterification reaction of the glycerol hydrolysis product with fatty acids to obtain amygdalase diglyceride. The double-imprinting process of the glycerol lipase (Lipase G50) includes: preparing a buffer solution with a pH of 5-6; adding a nonionic surfactant at a concentration of 20-80 mg / mL to the buffer solution; adding 10-30% (w / w) of Lipase G50; stirring at 25°C for 30-60 min; freeze-drying for 12-36 h; eluting the imprinted template with a nonpolar solvent; and removing the solvent by vacuum drying to obtain the imprinted lipase. In the esterification process, the molar ratio of free fatty acids in almond oil to the glycerol backbone of glycerol ester is 1.5-5:1, the reaction temperature is 30-50℃, the reaction stirring speed is 600-800 rpm, the amount of imprinted lipase G50 added is 4-8%, the vacuum degree is 10-30 mbar, and the reaction time is 8-12 h; the content of diglyceride is above 90%, and the yield is above 90%. A functional almond milk beverage is obtained by mixing enzymatically hydrolyzed almond protein slurry, deionized water, almond diglycerides, monoglycerides, sucrose esters, sodium caseinate, CMC, sodium alginate, xanthan gum, pectin, and sucrose, adjusting the pH to 6-8, homogenizing under high pressure, and sterilizing. The beverage comprises, by weight percentage of raw materials: 20-30% enzymatically hydrolyzed almond protein slurry, 48-64% deionized water, 8-16% almond diglycerides, 0.04-0.08% monoglycerides, 0.01-0.03% sucrose esters, 0.03-0.05% sodium caseinate, 0.04-0.08% CMC, 0.02-0.06% sodium alginate, 0.03-0.05% xanthan gum, 0.03-0.07% pectin, and 7-10% sucrose.

2. The preparation method according to claim 1, characterized in that: The low-temperature drying is carried out in a vacuum constant temperature drying oven at 45-60℃ until the moisture content is 3-5%, and then pulverized to 100-150 mesh; the supercritical carbon dioxide extraction temperature is 50-60℃; the extraction pressure is 20-30MPa; the extraction time is 45-75min; the separation pressure is 6-12MPa; the separation temperature is 30-50℃; and the almond oil yield is over 90%.

3. The functional almond milk beverage prepared by the preparation method according to claim 1 or 2.

Images