A method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound cooperation

Abstract

The application discloses a kind of bitter almond oil and protein peptide synchronous extraction method based on enzyme-ultrasonic cooperation, and it relates to the field of agricultural product processing.The method carries out pretreatment, two-stage supercritical carbon dioxide extraction and enzyme-ultrasonic coupling processing to bitter almond raw material in turn.Oil component is extracted under high pressure and high temperature conditions in the first step of supercritical extraction, then enzyme-ultrasonic coupling processing is used to hydrolyze protein into active peptide substances, and residual oil is extracted under optimized conditions in the second step of supercritical extraction, and product separation is completed simultaneously.The application designs and implements the coupling process of segmented supercritical extraction and enzyme-ultrasonic coupling processing, effectively resolves the polarity contradiction between oil fat solubility and protein peptide water solubility, and synchronously obtains two kinds of products of bitter almond oil and protein peptide in a single process flow, the oil yield is more than 92%, the protein peptide content is more than 75%, and the comprehensive utilization efficiency of bitter almond resources is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product processing technology, and in particular relates to a method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy. Background Technology

[0002] Bitter almonds are rich in oils and high-quality plant protein resources. Achieving efficient simultaneous extraction of oils and protein peptides while ensuring product activity is a key technical challenge for enhancing the comprehensive utilization value of bitter almonds. The fat solubility of oils and the water solubility of protein peptides present a fundamental contradiction in terms of the polarity requirements of the extraction medium, making it difficult for traditional processes to simultaneously obtain high yields of both oils and protein peptides in a single process.

[0003] Common methods for extracting bitter almond oil primarily rely on supercritical carbon dioxide extraction (SCCO) technology to achieve low-temperature, high-efficiency separation of oil components. Protein peptide preparation, on the other hand, depends on enzymatic hydrolysis and ultrasonic disruption to break down cell walls and promote protein hydrolysis. However, SCCO extraction requires a non-polar, high-pressure environment to ensure oil solubility, while the enzymatic hydrolysis of protein peptides requires a polar aqueous environment to ensure enzyme activity and substrate contact efficiency. The two processes are mutually constrained by their requirements for key parameters such as temperature, pressure, and medium polarity. This forces existing technologies to balance separation and stepwise extraction, failing to achieve true simultaneous and efficient synergistic extraction of oil and protein peptides. This technological bottleneck severely restricts the high-value comprehensive utilization of bitter almond resources. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for the simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy, which can effectively solve the problems mentioned in the background technology.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy, the method comprising the following steps: The bitter almond raw material is pretreated to obtain pretreated bitter almond material; the pretreatment includes impurity removal, crushing and debittering treatment. The pretreated bitter almond material is placed in a supercritical carbon dioxide extraction device for the first extraction step to obtain the first extracted solid material and the first oil extract; the extraction pressure of the first extraction step is 25-35 MPa, the extraction temperature is 35-50℃, the carbon dioxide flow rate is 15-25 L / h, and the extraction time is 1-2 h. The first extracted solid phase material is mixed with an enzymatic hydrolysis buffer, and a compound enzyme preparation is added for enzymatic hydrolysis. At the same time, ultrasonic assistance is applied to obtain an enzymatic hydrolysis reaction system. The enzymatic hydrolysis temperature is 40-55℃, the pH value is 6.5-8.5, the enzymatic hydrolysis time is 2-4h, the ultrasonic power is 200-400W, and the ultrasonic frequency is 20-40kHz. The enzymatic hydrolysis system was subjected to a second step of supercritical carbon dioxide extraction to obtain a second oil extract and a protein peptide extract. The extraction pressure of the second step was 20-30 MPa, the extraction temperature was 30-45℃, the carbon dioxide flow rate was 10-20 L / h, and the extraction time was 1-2 h. The first and second oil extracts were combined to obtain bitter almond oil product; the protein peptide extract was separated and purified to obtain bitter almond protein peptide product.

[0006] Furthermore, the compound enzyme preparation includes at least two of neutral protease, alkaline protease, and flavor enzyme; the neutral protease has an activity of 80,000-120,000 u / g, the alkaline protease has an activity of 100,000-150,000 u / g, and the flavor enzyme has an activity of 50,000-80,000 u / g; the total amount of each enzyme preparation added is 0.5-2% of the mass of the first extraction solid material.

[0007] Furthermore, the enzymatic hydrolysis buffer is a phosphate buffer or a citrate-disodium hydrogen phosphate buffer; the concentration of the buffer is 0.01-0.05 mol / L; and the mass ratio of the added buffer to the first extraction solid material is 3-8:1 mL / g.

[0008] Furthermore, a pressure release step is included between the first extraction step and the second extraction step; the pressure release step adopts a graded pressure reduction method, gradually reducing the pressure in the extraction device from the extraction pressure of the first extraction step to atmospheric pressure, with each pressure reduction being 3-5 MPa, and the stabilization time after each pressure reduction being 5-15 minutes.

[0009] Furthermore, the separation and purification process includes ultrafiltration, ion exchange chromatography, and vacuum freeze-drying. The ultrafiltration membrane has a molecular weight cutoff of 3000-10000 Da and an operating pressure of 0.1-0.3 MPa. The ion exchange chromatography uses cation exchange resin or anion exchange resin with a column diameter-to-height ratio of 1:5-1:10 and a flow rate of 1-3 mL / min. The vacuum freeze-drying process has a cold trap temperature of -40 to -60°C, a vacuum degree of 10-50 Pa, and a drying time of 24-48 h.

[0010] Furthermore, the debittering treatment is performed using either boiling water soaking or ethanol extraction. In the boiling water soaking method, the crushed bitter almonds are soaked in boiling water for 5-15 minutes and then drained. In the ethanol extraction method, the crushed bitter almonds are soaked in an ethanol solution with a concentration of 60-80% for 30-60 minutes, and the mass ratio of the ethanol solution to the bitter almonds is 5-10:1 mL / g.

[0011] Preferably, during the enzymatic hydrolysis process, the ultrasound is operated in a pulsed mode; the pulse on-time of the pulsed mode is 2-5s, and the pulse off-time is 3-8s; the ultrasound-assisted effect can disrupt the cell wall structure of bitter almonds, improve the contact efficiency between enzyme molecules and protein substrates, and promote the release of peptide molecules generated by the enzymatic hydrolysis reaction from the cell solid phase into the liquid phase.

[0012] Preferably, in the second step of supercritical carbon dioxide extraction, a modifier is added to the extraction medium; the modifier is at least one of ethanol, acetone or ethyl acetate; the amount of the modifier added is 3-8% of the mass of carbon dioxide; the addition of the modifier can improve the solubility of residual oil components in the second step of extraction, while not affecting the activity of the generated protein peptide products.

[0013] Preferably, the direction of the ultrasonic assistance is arranged in the same direction or opposite to the flow direction of the enzymatic reaction system; when arranged in the same direction, the ultrasonic waves can enhance the mass transfer effect of the reaction system; when arranged in opposite direction, the ultrasonic waves can increase the turbulence in the reaction system and increase the collision frequency between the enzyme and the substrate.

[0014] Preferably, before the first extraction step, the supercritical carbon dioxide is further subjected to a preheating and prepressurization process; the preheating temperature is the same as the extraction temperature of the first extraction step, and the prepressurization pressure is the same as the extraction pressure of the first extraction step; this step can ensure that the carbon dioxide has reached a supercritical state when it enters the extraction vessel, avoiding fluctuations in extraction efficiency due to unstable state.

[0015] In this invention, the first extraction step and the second extraction step have substantial differences in process parameters: the first extraction step uses a relatively high extraction pressure (25-35MPa) and extraction temperature (35-50℃) to maximize the solubility of the oil components and achieve the main extraction of the oil; the second extraction step uses a relatively low extraction pressure (20-30MPa) and extraction temperature (30-45℃) to extract residual oil under low energy consumption conditions, while avoiding the adverse effects of high temperature and high pressure on the activity of protein peptide products.

[0016] Compared with existing technologies, this invention solves the fundamental contradiction between the lipid solubility of oils and the water solubility of protein peptides by designing a coupled process of segmented supercritical carbon dioxide extraction and enzyme-ultrasound synergistic treatment, achieving the goal of simultaneously extracting bitter almond oil and protein peptides in a single process flow. The first step, supercritical carbon dioxide extraction, efficiently extracts the oil components from bitter almonds under high pressure and temperature conditions while maintaining the relative integrity of the protein structure. Subsequently, the cell wall structure in the solid phase material after the first extraction is destroyed by the synergistic effect of enzymatic hydrolysis and ultrasound, allowing the protein to be fully hydrolyzed into active peptides. The second step, supercritical carbon dioxide extraction, further extracts the residual oil components under optimized lower pressure conditions, while simultaneously achieving complete separation of oil and protein peptide products. This invention overcomes the technical bottleneck of mutual constraints in parameter requirements between supercritical extraction and enzymatic hydrolysis processes in existing technologies, significantly improving the comprehensive utilization efficiency of bitter almond resources and providing an efficient and feasible technical solution for the simultaneous extraction of bitter almond oil and protein peptides. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the process for simultaneous extraction of bitter almond oil and protein peptides. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0025] Example 1 A method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy, the process is as follows: Figure 1 As shown, the specific steps include the following: S1. Crush bitter almonds into coarse powder of 20-40 mesh. Soak the crushed bitter almond material in a 70% ethanol solution for 45 minutes. The mass ratio of ethanol solution to bitter almonds is 8:1 mL / g. After soaking, filter and separate the almonds, collect the filter residue and dry it to obtain the debittered bitter almond material. Perform quality testing to ensure that the residual amygdalin content is controlled within the safe range.

[0026] S2. The pretreated bitter almond material is placed in a supercritical carbon dioxide extraction device for the first step of extraction. The supercritical carbon dioxide extraction device is an existing device, mainly composed of a carbon dioxide storage tank, a refrigeration system, a high-pressure pump, an extraction vessel, a separation vessel, and a circulation system. This embodiment only utilizes it for supercritical carbon dioxide extraction. The specific operation process is as follows: The debittering pretreatment bitter almond material is loaded into the extraction vessel. Liquid carbon dioxide in the carbon dioxide storage tank is cooled by the refrigeration system, pressurized to the set pressure by the high-pressure pump, and then heated to the set temperature by the preheater before entering the extraction vessel. The extraction pressure is set to 30 MPa, the extraction temperature is set to 45℃, the carbon dioxide flow rate is set to 20 L / h, and the extraction time is set to 90 min.

[0027] Supercritical carbon dioxide fluid selectively dissolves oil components as it passes through the material bed. The carbon dioxide fluid carrying the oil enters the separation vessel, where, due to pressure reduction and temperature changes, the oil components precipitate from the carbon dioxide fluid and are collected at the bottom of the vessel, yielding the first oil extract. The material that has lost its oil components remains in the extraction vessel, becoming the first extraction solid phase. During the first extraction process, carbon dioxide circulates within the extraction vessel, continuously extracting the oil components from the material. The amount of oil collected in the separation vessel is monitored in real time during the extraction process. When the collection rate significantly slows down and approaches equilibrium, the first extraction step is considered complete.

[0028] S3. Transfer the first extracted solid phase material to the enzymatic hydrolysis reactor, and add 32mM phosphate buffer (pH 7.5) at a solid-liquid ratio of 5:1 mL / g. Stir to fully disperse the solid phase material in the buffer solution. After uniform dispersion, add 2% (by weight) of the first extracted solid phase material's compound enzyme preparation to the reaction system. The compound enzyme preparation consists of a neutral protease and an alkaline protease in a 2:3 mass ratio, with the neutral protease activity at 100,000 U / g and the alkaline protease activity at 120,000 U / g. The enzymatic hydrolysis temperature is 48℃, and the hydrolysis time is 3 hours.

[0029] The ultrasound operates in a pulsed mode, with a pulse on-time of 3 seconds and a pulse off-time of 5 seconds. The ultrasonic power is 300W, and the ultrasonic frequency is 28kHz. After the enzymatic hydrolysis reaction is completed, a reaction system containing the enzymatic hydrolysis products is obtained, i.e., the enzymatic hydrolysis reaction system.

[0030] S4. The enzymatic hydrolysis reaction system is subjected to a second step of supercritical carbon dioxide extraction to obtain a second oil extract and a protein peptide extract. Specifically, this includes the following steps: Pressure was released using a staged depressurization method, with each depressurization step involving 4 MPa and a stabilization time of 10 minutes after each depressurization. After pressure release, the enzymatic hydrolysis reaction system was directly fed into a supercritical carbon dioxide extraction device for the second extraction step. The extraction pressure was set at 25 MPa, the extraction temperature at 38°C, the carbon dioxide flow rate at 15 L / h, and the extraction time at 75 minutes. During the extraction process, ethanol, a modifier, was added to the extraction medium at a concentration of 5.5% of the carbon dioxide mass.

[0031] After the second step of supercritical carbon dioxide extraction is completed, the second oil extract and protein peptide extract are collected separately.

[0032] S5. The first and second oil extracts are combined to obtain bitter almond oil product. The protein peptide extract is then separated and purified to obtain bitter almond protein peptide product.

[0033] The separation and purification of protein peptide extracts includes three stages: ultrafiltration, ion exchange chromatography, and vacuum freeze-drying.

[0034] The ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the operating pressure is set at 0.2 MPa.

[0035] Ion exchange chromatography was used to further purify the ultrafiltered protein peptide solution using cation exchange resin to remove ionic impurities. The resin column diameter-to-height ratio for ion exchange chromatography was 1:8, and the flow rate was 2 mL / min.

[0036] The purified protein peptide solution was pre-frozen to below -50°C and then dried under a cold trap temperature of -52°C and a vacuum degree of 30 Pa for 36 hours to obtain dried bitter almond protein peptide powder.

[0037] The quality verification analysis was performed on bitter almond oil products and bitter almond protein peptide products respectively.

[0038] (1) Quality verification of oil products includes acid value determination, peroxide value determination and fatty acid composition analysis.

[0039] ① The acid value was determined according to GB 5009.229 "National Food Safety Standard - Determination of Acid Value in Food". Accurately weigh an appropriate amount of bitter almond oil sample (approximately 5-10g), place it in a 250mL Erlenmeyer flask, add 50mL of a 1:1 mixture of ether and isopropanol to completely dissolve the sample, add 2-3 drops of phenolphthalein indicator, and titrate with 0.1mol / L potassium hydroxide standard titration solution until the solution turns a faint pink color that does not fade within 30 seconds. Record the volume of potassium hydroxide standard titration solution consumed. A blank test was also performed simultaneously. The acid value was calculated using the following formula: ; In the formula: X is the acid value (mg / g); The volume (mL) of potassium hydroxide standard titration solution consumed for the sample. The volume (mL) of potassium hydroxide standard titration solution consumed for blank control. c 56.1 represents the actual concentration (mol / L) of the potassium hydroxide standard titration solution; 56.1 represents the molar mass (g / mol) of potassium hydroxide. m The sample mass is in g. The acid value was measured to be 0.7 mg / g.

[0040] ② The peroxide value was determined according to GB 5009.227 "National Food Safety Standard - Determination of Peroxide Value in Food". Accurately weigh approximately 2-3 g of bitter almond oil sample and place it in a 250 mL iodine flask. Add 30 mL of a chloroform-glacial acetic acid mixture (2:3) to dissolve the sample. Add 1 mL of saturated potassium iodide solution, stopper tightly, and shake for 0.5 min. Place in the dark for 3 min. Remove the flask and add 100 mL of water, shake well, and immediately titrate with 0.002 mol / L sodium thiosulfate standard solution until a pale yellow color appears. Add 1 mL of starch indicator and continue titrating until the blue color disappears. A blank test was performed simultaneously. The peroxide value was calculated using the following formula: ; In the formula: Peroxide value (g / 100g); The volume (mL) of sodium thiosulfate standard titration solution consumed by the sample. The volume (mL) of sodium thiosulfate standard titration solution consumed for blank control. c 0.1269 represents the actual concentration (mol / L) of the sodium thiosulfate standard titration solution; 0.1269 represents the mass (g) of iodine equivalent to 1.00 mL of sodium thiosulfate standard titration solution (1.000 mol / L). m The sample mass is in g. The measured peroxide value is 0.12 g / 100 g.

[0041] ③ The fatty acid composition was determined according to GB 5009.168 "National Food Safety Standard - Determination of Fatty Acids in Food". Gas chromatography (GC) was used: the bitter almond oil sample was methylated and then analyzed by gas chromatography. The chromatographic conditions were as follows: the column was a polyethylene glycol capillary column (30m × 0.32mm × 0.25μm); the injection port temperature was 250℃; the detector (FID) temperature was 280℃; the temperature program was: initial temperature 100℃ held for 5 min, then increased to 240℃ at 4℃ / min and held for 15 min; the carrier gas was high-purity nitrogen, the flow rate was 1.0mL / min; the split ratio was 50:1; and the injection volume was 1μL. The fatty acid methyl ester standard was used for qualitative analysis, and the relative percentage content of each fatty acid component was calculated using the peak area normalization method. The detected content was 68% oleic acid, 25% linoleic acid, and 3% linolenic acid. Oleic acid, linoleic acid, and linolenic acid are all unsaturated fatty acids.

[0042] (2) Quality verification of protein peptide products includes peptide content determination, molecular weight distribution determination and amino acid composition analysis.

[0043] ① The peptide content was determined using the biuret method. An appropriate amount of bitter almond protein peptide powder sample was accurately weighed, dissolved in deionized water, and diluted to a suitable concentration. 1.0 mL of the sample solution was placed in a centrifuge tube, and 1.0 mL of 10% trichloroacetic acid (TCA) solution was added. After shaking well, the mixture was allowed to stand for 10 min, then centrifuged at 4000 r / min for 15 min. 1.0 mL of the supernatant was mixed with 4.0 mL of biuret reagent and reacted in a 50℃ water bath for 10 min. After cooling to room temperature, the absorbance was measured at 550 nm using a reagent blank as a reference. A standard curve was plotted using bovine serum albumin (0-10 mg / mL) as a standard, and the peptide content in the sample was calculated based on the standard curve. The peptide content was determined to be 78%.

[0044] ② The molecular weight distribution of the protein peptides was determined using high-performance gel filtration chromatography (HPGFC). The molecular weight distribution was found to be mainly concentrated in the range of 500-5000 Da.

[0045] ③ Amino acid composition analysis was performed using an amino acid analyzer. An appropriate amount of bitter almond protein peptide powder sample (approximately 50-100 mg) was accurately weighed and placed in an ampoule. 10 mL of 6 mol / L hydrochloric acid solution was added, and the ampoule was sealed with nitrogen and placed in a 110℃ oven for hydrolysis for 22-24 h. After hydrolysis, the hydrolysate was transferred to an evaporating dish and evaporated to dryness in a 60℃ water bath. The solution was then dissolved in 0.02 mol / L hydrochloric acid solution and diluted to a suitable volume. After filtration through a 0.22 μm filter membrane, the amino acid composition was determined using a fully automated amino acid analyzer. The results showed that essential amino acids accounted for 38% of the total amino acid content.

[0046] (3) Calculation of oil extraction rate: The total oil content in the raw material was determined by Soxhlet extraction according to GB 5009.6 "National Food Safety Standard - Determination of Fat in Food". Accurately weigh about 2-5g of bitter almond powder, wrap it in filter paper, place it in a Soxhlet extractor, use petroleum ether (boiling range 30-60℃) as solvent, heat in a water bath and reflux for 6-8h. After recovering the solvent, place the extraction bottle in a 105℃ oven to dry to constant weight, weigh and calculate the total oil content in the raw material.

[0047] Oil extraction rate = (mass of oil obtained from the first extraction step + mass of oil obtained from the second extraction step) / total oil mass in the raw material × 100%.

[0048] The measured oil extraction rate was 92.5%.

[0049] (4) Calculation of protein peptide extraction rate: The total protein content in the raw material was determined by the Kjeldahl method in GB 5009.5 "National Food Safety Standard - Determination of Protein in Food". Approximately 0.5-1g of bitter almond powder was accurately weighed and digested, distilled and titrated using an automatic Kjeldahl nitrogen analyzer. The total protein content in the raw material was calculated using a nitrogen-to-protein conversion factor of 6.25.

[0050] Protein peptide extraction rate = mass of peptides in extract / mass of total protein in raw material × 100%.

[0051] The protein peptide extraction rate was measured to be 85.3%.

[0052] Example 2 A method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy differs from Example 1 in that a ternary complex enzyme preparation is used. The complex enzyme preparation is a ternary combination of neutral protease, alkaline protease, and flavor enzyme in a mass ratio of 2:3:2. The neutral protease has an activity of 80,000 u / g, the alkaline protease has an activity of 100,000 u / g, and the flavor enzyme has an activity of 60,000 u / g. The enzymatic hydrolysis temperature is set at 45°C, and the hydrolysis time is 3.5 h.

[0053] Example 3 A method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy differs from Example 1 in that the ultrasound is conducted in an intermittent mode, with an ultrasound treatment time of 15 min and a pause time of 5 min, repeated cyclically. The ultrasound power is 250 W, and the ultrasound frequency is 25 kHz. The intermittent ultrasound treatment cycle includes two phases: an activation phase and a quiescent phase. The time allocation of each cycle is dynamically adjusted according to the progress of the enzymatic hydrolysis reaction. A shorter activation phase and a longer quiescent phase are used in the early stages of the reaction. As the substrate concentration decreases, the proportion of the activation phase is gradually increased to accelerate the completion of the hydrolysis reaction.

[0054] Test Example 1 The process stability of the method described in Example 1 was verified. Using the same batch of bitter almond raw materials, three independent replicate experiments were conducted according to the process parameters of Example 1. The results are shown in Table 1.

[0055] Table 1 Results of process repeatability verification

[0056] The oil extraction rates in the three experiments were 92.5%, 92.1%, and 93.0%, with an average of 92.53% and a standard deviation of 0.46%. The protein and peptide extraction rates in the three experiments were 85.3%, 84.9%, and 86.2%, with an average of 85.47% and a standard deviation of 0.68%.

[0057] The acid values ​​of the oil products in the three experiments were 0.7, 0.71, and 0.69 mg / g, respectively, with an average of 0.7 mg / g. The peptide contents of the protein peptide products in the three experiments were 78%, 77.5%, and 74.4%, respectively, with an average of 77.63%.

[0058] Test results show that the method of the present invention has good process stability, and the batch-to-batch differences of various indicators are within an acceptable range.

[0059] Test Example 2 The effect of the direction of ultrasound-assisted action on the enzymatic hydrolysis effect was verified.

[0060] Enzymatic hydrolysis was performed using both co-current and counter-current arrangement methods, with other process parameters remaining consistent. In the co-current arrangement, the direction of ultrasonic assistance was the same as the flow direction of the enzymatic hydrolysis system, enhancing axial mass transfer. In the counter-current arrangement, the direction of ultrasonic assistance was opposite to the flow direction, creating turbulence within the reactor and increasing the collision frequency between the enzyme and substrate. The protein peptide extraction rate was 84.2% with the co-current arrangement and 86.1% with the counter-current arrangement. The test results indicate that the enzymatic hydrolysis effect of the counter-current arrangement is superior to that of the co-current arrangement. This is because the counter-current arrangement increases the turbulence within the reaction system, raising the collision frequency between the enzyme and substrate, thereby promoting the enzymatic hydrolysis reaction.

[0061] Test Example 3 The enzymatic hydrolysis effects of the ternary complex enzyme preparation in Example 2 and the binary complex enzyme preparation in Example 1 were compared and verified. An evaluation team was formed according to GB / T 16291.1 "General Guidelines for the Selection, Training and Management of Sensory Analysis Evaluators," and a 5-point scale was used to score the flavor quality of the protein peptide products. The specific standards are shown in Table 2.

[0062] Table 2 Sensory Evaluation Scoring Criteria

[0063] The protein peptide product scoring results of Example 1 (binary complex enzyme) and Example 2 (ternary complex enzyme) are shown in Table 3.

[0064] Table 3. Scoring Results of Protein Peptide Products

[0065] Sensory evaluation results showed that the protein peptide products obtained by ternary enzymatic hydrolysis exhibited better flavor characteristics, with significantly reduced bitterness and a more mellow aroma. The protein peptide products obtained by binary enzymatic hydrolysis retained a certain degree of bitterness, affecting their edible quality. The test results indicate that the addition of flavor enzymes can effectively improve the flavor characteristics of protein peptide products, meeting the application requirements where the primary goal is to achieve optimal product flavor.

[0066] Test Example 4 The enzymatic hydrolysis effects of intermittent and continuous ultrasonic working modes were compared and verified.

[0067] The protein peptide extraction rate was 86.5% in intermittent working mode and 84.8% in continuous working mode. The test results indicate that the enzymatic hydrolysis effect is superior to that in continuous working mode. This is because the intermittent working mode can reduce excessive shearing of enzyme molecules by ultrasound while ensuring the effectiveness of ultrasound treatment, thus protecting enzyme activity and improving hydrolysis efficiency.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for simultaneous extraction of bitter almond oil and protein peptides based on enzyme-ultrasound synergy, characterized in that, Includes the following steps: S1. Pre-treat the bitter almond raw material to obtain pre-treated bitter almond material; S2. The pretreated bitter almond material is placed in a supercritical carbon dioxide extraction device for the first step of extraction to obtain the first extraction solid material and the first oil extract. S3. Mix the first extraction solid phase material with the enzymatic hydrolysis buffer, add the compound enzyme preparation for enzymatic hydrolysis, and apply ultrasonic assistance to obtain the enzymatic hydrolysis reaction system. S4. The enzymatic hydrolysis reaction system is subjected to a second step of supercritical carbon dioxide extraction to simultaneously complete the extraction of residual oil and the separation of protein peptide products, resulting in a second oil extract and a protein peptide extract. S5. Combine the first and second oil extracts to obtain bitter almond oil product; separate and purify the protein peptide extract to obtain bitter almond protein peptide product.

2. The method according to claim 1, characterized in that: In S1, the pretreatment includes impurity removal, crushing, and debittering. The debittering is performed by boiling water soaking or ethanol extraction. In the boiling water soaking method, the crushed bitter almonds are soaked in boiling water for 5-15 minutes. In the ethanol extraction method, the crushed bitter almonds are soaked in an ethanol solution with a concentration of 60%-80% for 30-60 minutes, and the mass ratio of ethanol solution to bitter almonds is 5-10:

1.

3. The method according to claim 2, characterized in that: In S2, the extraction pressure of the first extraction step is 25-35 MPa, the extraction temperature is 35-50℃, the carbon dioxide flow rate is 15-25 L / h, and the extraction time is 1-2 h.

4. The method according to claim 1, characterized in that: In step S3, the compound enzyme preparation includes at least two of the following: neutral protease, alkaline protease, and flavor enzyme; the activity of the neutral protease is 80,000-120,000 u / g, the activity of the alkaline protease is 100,000-150,000 u / g, and the activity of the flavor enzyme is 50,000-80,000 u / g; the amount of the compound enzyme preparation added is 0.5%-2% of the mass of the first extraction solid phase material.

5. The method according to claim 1, characterized in that: In S3, the enzymatic hydrolysis buffer is a phosphate buffer or a citrate-disodium hydrogen phosphate buffer; the concentration of the enzymatic hydrolysis buffer is 0.01-0.05 mol / L; and the mass ratio of the amount of enzymatic hydrolysis buffer added to the first extraction solid phase material is 3-8:1 mL / g.

6. The method according to claim 1, characterized in that: In step S5, the separation and purification process includes ultrafiltration, ion exchange chromatography, and vacuum freeze-drying. The ultrafiltration membrane has a molecular weight cutoff of 3000-10000 Da and an operating pressure of 0.1-0.3 MPa. The ion exchange chromatography uses cation exchange resin or anion exchange resin with a column diameter-to-height ratio of 1:5-10 and a flow rate of 1-3 mL / min. The vacuum freeze-drying process has a cold trap temperature of -40 to -60°C, a vacuum degree of 10-50 Pa, and a drying time of 24-48 h.

7. The method according to claim 1, characterized in that: In the second step of supercritical carbon dioxide extraction, a modifier is added to the extraction medium; the modifier is at least one of ethanol, acetone or ethyl acetate; the amount of the modifier added is 3-8% of the mass of carbon dioxide.

8. The method according to claim 1, characterized in that: The direction of the ultrasonic-assisted action is either in the same direction or in the opposite direction to the flow direction of the enzymatic reaction system. When the same direction is used, the ultrasonic waves enhance the mass transfer effect of the reaction system. When the opposite direction is used, the ultrasonic waves increase the turbulence in the reaction system and increase the collision frequency between the enzyme and the substrate.

9. The method according to claim 1, characterized in that: Before the first extraction step, the process includes preheating and prepressurizing the supercritical carbon dioxide; the preheating temperature is the same as the extraction temperature of the first extraction step, and the prepressurization pressure is the same as the extraction pressure of the first extraction step.

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