Rice bran degreasing and nutrition retention edible process synergistic treatment method

Abstract

The invention relates to the technical field of rice bran processing, and discloses a rice bran degreasing and nutrition retention edible process synergistic treatment method which comprises the following steps: (a) in an NADES-aqueous solution medium, adding an immobilized catalyst A, and hydrolyzing ferulate bonds and phospholipids in rice bran at low temperature; (b) carrying out intermediate dehydration on the slurry obtained in the step (a) to reduce the water activity; (c) adding an immobilized catalyst B into the dehydrated slurry in the step (b), carrying out esterification reaction, and grafting free ferulic acid generated by hydrolysis to glyceride in situ; and (d) carrying out solid-liquid separation on the slurry obtained in the step (c) to obtain finished oil and finished powder. According to the method, an NADES medium is adopted to replace an organic solvent, the whole process is carried out at low temperature, degradation of heat-sensitive nutrient substances is avoided, and resource utilization of ferulic acid is achieved. The prepared functional rice bran oil is rich in feruloyl glyceride, total tocopherol and oryzanol, and is low in acid value and peroxide value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of rice bran processing, in particular to a kind of edible process synergistic treatment method of rice bran degreasing and nutrition retention. BACKGROUND

[0002] Rice bran is the main by-product of rice processing, rich in oil, protein and various functional nutrients such as oryzanol and tocopherol, and has high comprehensive utilization value. However, fresh rice bran contains high-activity endogenous lipase, which is activated after the rice bran is in contact with air, rapidly catalyzing the hydrolysis of oil in the rice bran, resulting in a sharp increase in free fatty acid content and the occurrence of rice bran rancidity. The rancidity of rice bran not only makes it lose its edible value, but also severely limits the subsequent high-value deep processing utilization.

[0003] Currently, in order to inhibit the activity of rice bran lipase in industry, heat treatment (such as high-temperature steaming and frying, extrusion puffing) is mainly used for stabilization treatment. Such methods rely on high temperature (usually higher than 110℃) to denature and inactivate enzyme proteins. Although high-temperature treatment achieves the purpose of enzyme inactivation, it also causes significant degradation and loss of heat-sensitive nutrients (such as oryzanol and tocopherol) in rice bran.

[0004] In the rice bran oil production process, traditional processes mostly use organic solvent extraction (such as using n-hexane). The solvent extraction process also requires high temperature in the subsequent desolventization step, which further exacerbates the loss of heat-sensitive nutrients. More importantly, the use of organic solvents poses a risk of solvent residue in the finished product and environmental safety problems in the production process, making it difficult to meet the production requirements of organic edible products or green food.

[0005] In addition, rice bran also contains ferulic acid bound to cell wall polysaccharides in the form of ferulic acid ester bonds. Existing heat treatment or solvent extraction processes do not involve the effective utilization of this component, resulting in the loss of ferulic acid resources with rice bran, reducing the overall economic value of rice bran.

[0006] Therefore, the present application proposes an edible process synergistic treatment method of rice bran degreasing and nutrition retention to solve the problems of the prior art. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides an edible process synergistic treatment method of rice bran degreasing and nutrition retention, which solves the problems of degradation of heat-sensitive nutrients (such as oryzanol and tocopherol) caused by high temperature in the existing process, and the inability to effectively utilize ferulic acid resources in rice bran.

[0008] To solve the above problems, the present application provides the following technical solutions: In a first aspect, the present application provides a method for the simultaneous defatting and nutrient retention of rice bran, which comprises the following technical solutions: A method for the simultaneous defatting and nutrient retention of rice bran, comprising the following steps: (a) First-stage hydrolysis: mixing rice bran raw material, NADES-aqueous solution, and immobilized catalyst A, and reacting to hydrolyze ferulic acid ester bonds and phospholipids in the rice bran raw material; (b) Intermediate dehydration: dehydrating the slurry obtained in step (a) to reduce the water activity of the slurry to a preset esterification synthesis threshold; (c) Second-stage esterification: adding immobilized catalyst B to the dehydrated slurry of step (b) and reacting to graft free ferulic acid produced in step (a) to glycerides to form feruloylated glycerides; (d) Product separation: performing solid-liquid separation on the slurry obtained in step (c) to obtain finished oil and finished powder.

[0009] By using the above technical solutions, the present application uses NADES-aqueous solution as the reaction medium, so that the entire process (hydrolysis and esterification) can be carried out at low temperature (e.g., 45-55°C). This low-temperature condition avoids the destruction of heat-sensitive nutrients caused by traditional high-temperature pressing, thereby retaining the original content of oryzanol and total tocopherols in rice bran. At the same time, the low-temperature environment combined with the pre-hydrolysis of phospholipids in step (a) effectively inhibits the hydrolysis and oxidation of oil, ensuring the low acid value and low peroxide value of the finished oil.

[0010] The core innovation of the technical solutions of the present application lies in the design of a two-stage (hydrolysis-esterification) enzymatic reaction, the mechanism of which is as follows: In the first-stage hydrolysis of step (a), the immobilized catalyst A (containing ferulic acid esterase) acts on the solid phase of rice bran, specifically cutting the ester bonds between ferulic acid and cell wall polysaccharides, releasing ferulic acid as free ferulic acid and dissolving it in the NADES liquid phase; at the same time, the immobilized catalyst A (containing phospholipase A2) hydrolyzes phospholipids.

[0011] In the intermediate dehydration of step (b), by reducing the water activity of the system to the esterification synthesis threshold (e.g., aw≤0.15), the thermodynamic equilibrium of the enzymatic reaction is shifted from hydrolysis (high aw) to synthesis (low aw).

[0012] In the second-stage esterification of step (c), under low aw conditions, the immobilized catalyst B (non-specific lipase) uses the free ferulic acid released in step (a) as a substrate to catalyze the esterification or transesterification of free ferulic acid with glycerides in the system, grafting ferulic acid to the glyceride backbone in situ to form feruloylated glycerides (FAGs).

[0013] By the technical scheme, the ferulic acid resource lost with the rice bran meal is converted into the high-value functional ingredient (feruloylated glycerides) in the oil phase, and meanwhile, the natural nutrients are retained, and the finished oil with low acid value, low peroxide value and high oxidation stability is obtained.

[0014] Preferably, the NADES-water solution is prepared by mixing choline chloride with an acidic hydrogen bond donor selected from L-malic acid or citric acid.

[0015] Preferably, the immobilized catalyst A is prepared by immobilizing ferulic acid esterase and phospholipase A2 on the same carrier.

[0016] Further preferably, the activity ratio of the ferulic acid esterase to the phospholipase A2 is 1:2 to 2:1 U / U.

[0017] Preferably, the immobilized catalyst B is prepared by immobilizing a non-specific lipase on a carrier.

[0018] Preferably, in the step (b), the preset esterification synthesis threshold value is a slurry water activity ≤0.15; the reaction temperature of the step (a) is 45-55℃; and the reaction temperature of the step (c) is 45-55℃.

[0019] Preferably, the method is carried out under nitrogen protection. By adopting nitrogen protection, the oxidation reaction in the whole process can be inhibited, and the finished oil with low peroxide value is ensured.

[0020] Preferably, the finished powder obtained in the step (d) has a free ferulic acid content of 50-70 mg / kg on a dry basis. This feature confirms that the hydrolysis of the ferulic acid ester bond in the step (a) indeed occurs, and part of the free ferulic acid not involved in esterification remains in the finished powder.

[0021] In a second aspect, the application provides a functional rice bran oil composition, which adopts the following technical scheme: A functional rice bran oil composition, which is prepared by the rice bran degreasing and nutrient-retaining edible process synergistic treatment method according to the first aspect of the application, and comprises: feruloylated glycerides, content greater than 1000 mg / kg; total tocopherols, content greater than 700 mg / kg; and oryzanol, content greater than 8000 mg / kg; the acid value of the functional rice bran oil composition is ≤1.8 mg KOH / g; and the peroxide value of the functional rice bran oil composition is ≤2.1 mmol / kg.

[0022] By adopting the technical scheme, the functional rice bran oil composition is prepared by the low-temperature two-stage enzymatic process. The process (a) is operated at low temperature (for example, 45-55 DEG C), which avoids degradation of heat-sensitive total tocopherols and oryzanol, so that high content of the oryzanol is retained; (b) the in-situ esterification reaction of the process step (c) converts the original ferulic acid in the rice bran into feruloylated glycerides, which improves the antioxidant capacity and functional value of the oil product; (c) the low-temperature condition of the process combined with the phospholipid hydrolysis of step (a) inhibits the degradation of oil and fat, so that the final product has low acid value and low peroxide value.

[0023] Preferably, the feruloylated glyceride content is 1100-1200 mg / kg; the total tocopherol content is 750-810 mg / kg; and the oryzanol content is 8800-9150 mg / kg.

[0024] The application provides an edible process synergistic treatment method for rice bran degreasing and nutrient retention. The method is operated at low temperature, which avoids damage to heat-sensitive substances in the rice bran caused by traditional high-temperature pressing; therefore, high content of natural nutrients, such as oryzanol and total tocopherols, is retained in the finished oil. The application uses immobilized catalyst A (ferulic acid esterase) to hydrolyze and release ferulic acid in the cell wall of rice bran in step (a), and uses immobilized catalyst B (lipase) to esterify the free ferulic acid into feruloylated glycerides in step (c); this method converts the original ferulic acid resources in the rice bran into functional ingredients in the oil phase, which improves the antioxidant capacity and functional value of the finished oil. In step (a), the application uses immobilized catalyst A (phospholipase A2) to hydrolyze phospholipids, which, combined with low-temperature operation and nitrogen protection, effectively inhibits oil hydrolysis and oxidation side reactions in the process. Therefore, the finished oil has low acid value and peroxide value, which improves the quality and storage stability of the oil product. The application uses NADES-aqueous solution as a reaction medium, which replaces traditional organic solvents; NADES is composed of components of natural origin, has the characteristics of biodegradability and low toxicity, and, combined with the use of biological catalysts (enzymes), makes the entire process environmentally friendly, and there is no harmful solvent residue in the finished oil. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0026] The main raw materials and reagents used in the following examples and comparative examples are as follows, and the reagents not specifically described are commercially available analytical pure or higher grade products.

[0027] Rice bran: Fresh rice bran was purchased from the market, which was derived from japonica rice, and was used within 6 hours after milling. Before use, it was sieved through a 40-mesh sieve.

[0028] Choline chloride: The chemical name is 2-hydroxyethyltrimethylammonium chloride, and the CAS number is 67-48-1. The food grade used in the present application has a purity of ≥99.0%.

[0029] L-malic acid: The chemical name is (S)-2-hydroxybutanedioic acid, and the CAS number is 97-67-6. The food grade used in the present application has a purity of ≥99.5%.

[0030] Citric acid: The chemical name is 2-hydroxypropane-1, 2, 3-tricarboxylic acid, and the CAS number is 77-92-9 (anhydrous). The food grade used in the present application has a purity of ≥99.5%.

[0031] Ferulic acid esterase: A hydrolytic enzyme. The commercially available food grade enzyme preparation derived from the fermentation product of Aspergillus niger was used.

[0032] Phospholipase A2: A hydrolytic enzyme. The commercially available food grade enzyme preparation derived from the fermentation product of Aspergillus niger was used.

[0033] Lipase: A non-specific hydrolytic enzyme. The commercially available food grade enzyme preparation derived from the fermentation product of Rhizomucor miehei was used.

[0034] Immobilized carrier: Commercially available food grade macroporous acrylate adsorption resin. The skeleton is acrylate copolymer. The appearance is white spherical particles. The particle size range is 1.5 mm-2.0 mm. The specific surface area is 100-150 m 2 / g. The average pore size is 100-150 Å.

[0035] Glutaraldehyde: The chemical name is glutaraldehyde, and the CAS number is 111-30-8. The commercially available 25% (w / w) aqueous solution was used as a crosslinking agent.

[0036] Glycine: The chemical name is aminoacetic acid, and the CAS number is 56-40-6. The commercially available analytical pure was used as a blocking agent.

[0037] Rice bran shell ultrafine powder: commercially available organic rice bran shell powder with a particle size <100 μm, used as a pressing aid.

[0038] Preparation Example 1: This preparation example provides a method for preparing NADES medium-1 (ChCl-malic acid), comprising the following steps: Choline chloride and L-malic acid were placed in a jacketed reactor in a molar ratio of 1:1.2; 25.0% (w / w) of purified water was added based on the total mass; mechanical stirring (150-250 rpm) was started, and the reaction was continued for 2-4 hours under the condition of heating in a 60°C water bath until a homogeneous, clear, transparent liquid NADES-aqueous solution was formed; cooled to room temperature for standby use.

[0039] Preparation Example 2: This preparation example provides a method for preparing NADES medium-2 (ChCl-citric acid), comprising the following steps: Choline chloride and citric acid were placed in a jacketed reactor in a molar ratio of 1:1; 30.0% (w / w) of purified water was added based on the total mass; mechanical stirring (150-250 rpm) was started, and the reaction was continued for 2-4 hours under the condition of heating in a 60°C water bath until a homogeneous, clear, transparent liquid NADES-aqueous solution was formed; cooled to room temperature for standby use.

[0040] Preparation Example 3: This preparation example provides a method for preparing immobilized catalyst A (FAE+PLA2) condition 1, comprising the following steps: (1) Carrier activation: 100 g of macroporous acrylate adsorption resin (particle size 1.5-2.0 mm) was washed with ethanol and purified water, and then placed in 1000 mL of 2.5% (v / v) glutaraldehyde aqueous solution (prepared using 0.1M, pH 7.5 PBS); activated in a 25°C, 100 rpm shaker for 2 hours; washed with purified water until neutral, and then suction filtered for standby use.

[0041] (2) Enzyme solution preparation: According to the original specific activity (U / mg) of the enzyme preparation used, ferulic acid esterase freeze-dried powder and phospholipase A2 freeze-dried powder were dissolved in 0.1M, pH 7.0 PBS in an activity ratio (U / U) of 1:2 to prepare a mixed enzyme solution. The total amount of the two enzymes added in the enzyme solution was calculated to make the theoretical immobilization load of ferulic acid esterase 1000 U / g carrier and phospholipase A2 2000 U / g carrier.

[0042] (3) Immobilization reaction: The activated carrier of step (1) was added to the mixed enzyme solution of step (2) (solid-liquid ratio 1:10, w / v); reacted on a low-speed shaker at 4°C, 60 rpm for 24 hours.

[0043] (4) Washing and blocking: The carrier was recovered by filtration and washed with 0.1 M, pH 7.0 PBS for 3 times; then, 1000 mL of 0.5 M glycine solution (pH 7.0) was added, and the unreacted aldehyde groups were blocked at 25 °C for 2 hours.

[0044] (5) Finished product: The carrier was washed to neutral with purified water, and after suction filtration, it was stored in 0.1 M PBS (pH 7.0) buffer containing 20% (v / v) glycerol at 4 °C for standby.

[0045] Preparation Example 4: The preparation method of the immobilized catalyst A (FAE + PLA2) condition 2 provided by the present preparation example includes the following steps: (1) Carrier activation: 100 g of macroporous acrylate adsorption resin (particle size 1.5-2.0 mm) was washed with ethanol and purified water, and then placed in 1000 mL of 4.0% (v / v) glutaraldehyde aqueous solution (prepared using 0.1 M, pH 7.5 PBS); activated in a 25 °C, 100 rpm shaker for 4 hours; washed with purified water to neutral, and suction filtered for standby.

[0046] (2) Enzyme solution preparation: According to the original specific activity (U / mg) of the enzyme preparation used, the ferulic acid esterase freeze-dried powder and the phospholipase A2 freeze-dried powder were dissolved in 0.1 M, pH 7.0 PBS according to the activity ratio (U / U) of 2:1 to prepare a mixed enzyme solution. The total amount of the two enzymes in the enzyme solution was calculated to make the theoretical immobilization load of ferulic acid esterase 2000 U / g carrier and phospholipase A2 10000 U / g carrier.

[0047] (3) Immobilization reaction: The same as step (3) of preparation example 3.

[0048] (4) Washing and blocking: The same as step (4) of preparation example 3.

[0049] (5) Finished product: The same as step (5) of preparation example 3.

[0050] Preparation Example 5: The preparation method of the immobilized catalyst B condition 1 provided by the present preparation example includes the following steps: (1) Carrier activation: 100 g of macroporous acrylate adsorption resin (particle size 1.5-2.0 mm) was washed with ethanol and purified water, and then placed in 1000 mL of 3.0% (v / v) glutaraldehyde aqueous solution (prepared using 0.1 M, pH 7.5 PBS); activated in a 25 °C, 100 rpm shaker for 3 hours; washed with purified water to neutral, and suction filtered for standby.

[0051] (2) Enzyme solution preparation: According to the original specific activity (U / mg) of the enzyme preparation used, the non-specific lipase freeze-dried powder was dissolved in 0.1 M, pH 7.5 PBS to prepare an enzyme solution; the total amount of enzyme added in the enzyme solution was calculated to make the theoretical immobilized load reach 5000 U / g carrier.

[0052] (3) Immobilization reaction: The activated carrier of step (1) was added to the enzyme solution of step (2) (solid-liquid ratio 1:10, w / v); the reaction was carried out on a low-speed shaker at 4°C and 60 rpm for 24 hours.

[0053] (4) Washing and blocking: Same as step (4) of Preparation Example 3.

[0054] (5) Finished product: Same as step (5) of Preparation Example 3.

[0055] Preparation Example 6: This preparation example provides a preparation method of immobilized catalyst B condition 2, including the following steps: (1) Carrier activation: Same as step (1) of Preparation Example 5.

[0056] (2) Enzyme solution preparation: Same as step (2) of Preparation Example 5.

[0057] (3) Immobilization reaction: The activated carrier of step (1) was added to the enzyme solution of step (2) (solid-liquid ratio 1:10, w / v); the reaction was carried out on a shaker at 25°C and 100 rpm for 4 hours.

[0058] (4) Washing and blocking: Same as step (4) of Preparation Example 3.

[0059] (5) Finished product: Same as step (5) of Preparation Example 3.

[0060] Example 1: This example provides a rice bran synergistic treatment method based on NADES-1 (ChCl-malic acid) and benchmark process parameters, including the following steps: (1) Material mixing and first stage reaction: 100 kg of fresh rice bran (passed through a 40 mesh sieve) was pumped into a first reaction kettle (R-01) with 350 kg (solid-liquid ratio 1:3.5, w / w) of NADES medium-1 prepared in Preparation Example 1; the reaction kettle was sealed, food-grade nitrogen was introduced to replace the air in the kettle, until the headspace oxygen content was less than 1.0%, and a micro-positive pressure of 0.01 MPa was maintained; "Catalyst A condition 1" prepared in Preparation Example 3 (equilibrated with NADES-1 before use) was added, the addition amount was 1.0% (w / w) of the dry weight of rice bran; the stirring was started (the stirring power and system viscosity were recorded during the process), the jacket temperature was controlled at 45°C, and the reaction was carried out for 60 minutes.

[0061] (2) Intermediate separation A: After the reaction is completed, the slurry in R-01 is pumped out to an intermediate dewatering unit through an online basket filter with a pore size of 0.5 mm; "Catalyst A" is trapped in R-01 and is cleaned according to the established SOP before being recovered.

[0062] (3) Intermediate dewatering: The slurry obtained in step (2) without catalyst A is pumped into a low-temperature vacuum flash kneader; the jacket temperature is set to 50°C, the vacuum degree is maintained at -0.08 MPa, and kneading stirring is performed for 30 minutes; sampling detection is performed to confirm that the water activity (aw) of the material is ≤0.10.

[0063] (4) Second-stage reaction: The dewatered slurry of step (3) is pumped into a second reaction kettle (R-02) (which has been replaced with nitrogen); "Catalyst B Condition 1" prepared in Preparation Example 5 (equilibrated with NADES-1 before use) is added, and the addition amount is 3.0% (w / w) of the dry weight of rice bran; stirring is maintained, and the jacket temperature is controlled at 45°C, and the reaction is performed for 60 minutes.

[0064] (5) Separation B and pressing: After the reaction is completed, the slurry in R-02 is pumped out to a conditioning unit through a 0.5 mm online filter; "Catalyst B" is trapped in R-02 and is cleaned according to the SOP before being recovered; 3.0% (w / w) of the dry weight of rice bran of rice husk ultrafine powder is added to the slurry, and after being uniformly mixed, it is sent to a low-temperature screw press (pressing temperature <60°C, nitrogen protection) for pressing to separate crude oil and wet meal.

[0065] (6) Refining and recovery: The crude oil is subjected to high-speed centrifugation to remove impurities, vacuum dewatering, and then is packaged under nitrogen to obtain finished oil; the wet meal is subjected to multi-stage countercurrent washing and vacuum drying to obtain finished powder; the NADES recovery liquid (washing liquid) is first subjected to centrifugal separation to remove residual oil, and then is subjected to nanofiltration concentration and thin film evaporation to recover the NADES solvent.

[0066] Example 2: This example provides a rice bran synergistic treatment method based on NADES-2 (ChCl-citric acid) and high-temperature process parameters, including the following steps: (1) Material mixing and first-stage reaction: 100 kg of fresh rice bran and 400 kg (solid-liquid ratio 1:4, w / w) of NADES medium-2 prepared in Preparation Example 2 are pumped into R-01; nitrogen protection is the same as in Example 1; "Catalyst A Condition 2" prepared in Preparation Example 4 is added, and the addition amount is 1.0% (w / w) of the dry weight of rice bran. The jacket temperature is controlled at 55°C, and the reaction is performed for 45 minutes.

[0067] (2) Intermediate separation A: The same as step (2) of Example 1.

[0068] (3) Intermediate dewatering: The same as step (3) of Example 1, but the dewatering target is controlled to be a material water activity (aw) ≤0.15 detected.

[0069] (4) Second stage reaction: Pump the dehydrated slurry of step (3) into R-02; add "Catalyst B Condition 2" prepared in Preparation Example 6 at an addition amount of 3.0% (w / w) of the dry weight of the rice bran; control the jacket temperature at 55°C, and react for 45 minutes.

[0070] (5) Separation B and pressing: Same as step (5) of Example 1.

[0071] (6) Finishing and recovery: Same as step (6) of Example 1.

[0072] Example 3: This example provides a rice synergistic treatment method based on NADES-1 and high solid-liquid ratio process parameters, including the following steps: (1) Material mixing and first stage reaction: Pump 100 kg of fresh rice bran and 300 kg (solid-liquid ratio 1:3, w / w) of NADES medium-1 prepared in Preparation Example 1 into R-01; nitrogen protection is the same as in Example 1; add "Catalyst A Condition 2" prepared in Preparation Example 4 at an addition amount of 1.5% (w / w) of the dry weight of the rice bran. Control the jacket temperature at 50°C, and react for 75 minutes.

[0073] (2) Intermediate separation A: Same as step (2) of Example 1.

[0074] (3) Intermediate dehydration: Same as step (3) of Example 1, and the dehydration target is controlled to be a material water activity (aw) of ≤0.10 detected.

[0075] (4) Second stage reaction: Pump the dehydrated slurry of step (3) into R-02; add "Catalyst B Condition 2" prepared in Preparation Example 6 at an addition amount of 3.5% (w / w) of the dry weight of the rice bran; control the jacket temperature at 50°C, and react for 75 minutes.

[0076] (5) Separation B and pressing: Same as step (5) of Example 1.

[0077] (6) Finishing and recovery: Same as step (6) of Example 1.

[0078] Comparative Example 1: This comparative example describes a traditional hexane extraction method; 100 kg of rice bran is subjected to enzyme inactivation at high temperature (120°C for 20 minutes), and then Soxhlet extracted with n-hexane at 55°C for 6 hours.

[0079] Comparative Example 2: This comparative example describes a traditional hot pressing method; 100 kg of rice bran is subjected to enzyme inactivation at high temperature (120°C for 20 minutes), and then high-temperature spiral pressing is performed at 110°C.

[0080] Comparative Example 3: The difference compared with Example 1 is that no "catalyst A" is added in step (1), and no "catalyst B" is added in step (4), and the rest are the same.

[0081] Comparative Example 4: The difference compared with Example 1 is that after the intermediate dehydration in step (3) is performed, the second-stage enzymatic reaction in step (4) is skipped (i.e., catalyst B is not added), and step (5) of separating B and pressing is directly entered, and the rest are the same.

[0082] Comparative Example 5: The difference compared with Example 1 is that all the reaction kettles (R-01, R-02) and the pressing process are carried out in an open air environment, without the protection of food-grade nitrogen, and the rest are the same.

[0083] Comparative Example 6: The difference compared with Example 1 is that catalyst A of Preparation Example 3 and catalyst B of Preparation Example 5 are simultaneously added to R-01 in step (1), and the mixed reaction is carried out for 120 minutes (i.e., a "one-pot method" is adopted), and steps (2), (3), and (4) are skipped, and the rest are the same.

[0084] Comparative Example 7: The difference compared with Example 1 is that after the intermediate separation A in step (2) is performed, the intermediate dehydration step in step (3) is skipped, and the slurry (not dehydrated, aw>0.9) is directly pumped into R-02 for the second-stage reaction in step (4), and the rest are the same.

[0085] Test Example 1: Catalyst A Function Verification This test example is used to verify whether the immobilized catalyst A (co-immobilized enzyme of ferulic acid esterase FAE and phospholipase A2 PLA2) has the preset catalytic activity in the NADES-water solution system, i.e., the hydrolysis (unlocking) function of ferulic acid ester bond and the hydrolysis (degumming) function of phospholipid.

[0086] The experimental steps are as follows: Take the slurry of Example 1 after completing step (1) (first-stage enzymatic reaction) and before entering step (3) (intermediate dehydration), and mark it as sample A; take the slurry of Comparative Example 3 (NADES treatment without any enzyme addition) after the same reaction time (60 minutes) is ended, and mark it as sample B.

[0087] Place sample A and sample B in centrifuge tubes, respectively, and centrifuge at 4°C and 10,000xg for 15 minutes, and take the supernatant; filter the supernatant through a 0.45 μm nylon filter membrane, and collect the filtrate for analysis.

[0088] Free ferulic acid content was determined by HPLC; column: C18 reversed-phase column (4.6 x 250 mm, 5 pm); mobile phase A: 0.1% (v / v) formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution; detection wavelength: 320 nm. Quantification was performed using external standard (ferulic acid standard) curve.

[0089] Lysophospholipid content was determined by HPLC-ELSD; column: C18 column; mobile phase A: acetonitrile-methanol (80:20, v / v); mobile phase B: isopropanol containing 0.5% triethylamine; gradient elution; ELSD detector parameters: drift tube temperature 60 °C, nebulizing gas (nitrogen) pressure 3.5 Bar. Quantification was performed using external standard method.

[0090] All samples were tested in triplicate, and the results are expressed as mean ± standard deviation.

[0091] The experimental data are shown in Table 1. Table 1: Catalyst A functional verification data (n = 3, mean ± SD) Conclusion: The data of Test Example 1 (Table 1) show that, when sample B (Comparative Example 3) is treated with NADES medium only, the content of free ferulic acid and lysophospholipid in the liquid phase is at a low level (11.3 ± 0.8 mg / L and 1.2 ± 0.3 mg / L, respectively); this result shows that, in the absence of exogenous enzyme catalysis, the physicochemical properties (such as acidity or swelling effect) of the NADES medium are not enough to hydrolyze the covalently linked ferulic acid ester bond on the cell wall of rice bran, nor can it achieve the hydrolysis of phospholipids (glue) in rice bran.

[0092] In contrast, after adding immobilized catalyst A to the NADES medium for reaction, sample A (Example 1), the content of free ferulic acid in the liquid phase is increased to 146.6 ± 4.2 mg / L, and lysophospholipid (78.8 ± 3.1 mg / L) is also detected.

[0093] This comparative data proves that the immobilized catalyst A (FAE-PLA2 co-immobilization) prepared in the preparation example maintains its dual catalytic activity in the NADES-aqueous solution: The ferulic acid esterase (FAE) component in catalyst A catalyzes the hydrolysis of the ferulic acid ester bond on the polysaccharide-lignin complex of the cell wall of rice bran, releasing ferulic acid molecules into the NADES liquid phase.

[0094] The phospholipase A2 (PLA2) component in catalyst A simultaneously hydrolyzes the phospholipids in rice bran, converting them into lysophospholipids that are easily soluble in the NADES liquid phase, achieving in-situ degumming.

[0095] This test example confirmed the feasibility of the first stage reaction (step one) in the process flow; this step achieved the degumming of the rice bran and provided the reaction substrate (free ferulic acid) for the subsequent second stage reaction (step four, in situ esterification grafting).

[0096] Test Example 2: Necessity verification of intermediate dehydration and catalyst B function This test example aimed to verify the necessity of two key elements in the process flow of the present application: intermediate dehydration (control of water activity aw) in step (3) and addition of catalyst B (lipase) in step (4), for achieving the second stage feruloylated glycerides (FAGs) in situ esterification grafting.

[0097] The experimental procedure was as follows: (1) Obtain the final product oil of Example 1 (complete process: dehydration + catalyst B), Comparative Example 4 (dehydrated, but without catalyst B), and Comparative Example 7 (with catalyst B, but without dehydration).

[0098] (2) Accurately weigh 0.1 g of the above three kinds of product oil samples, and use isopropyl alcohol-acetonitrile (1:1, v / v) mixed solvent to make up to 10 mL; after vortex mixing, filter through a 0.45 μm organic phase filter membrane, and take the filtrate for analysis.

[0099] (3) Use liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the content of feruloylated glycerides (FAGs); chromatographic column: C18 column (2.1x100mm, 1.8 μm); gradient elution is used; mass spectrometry uses electrospray ion source (ESI), and is detected in multiple reaction monitoring (MRM) mode, and is quantified by monitoring the characteristic ion pair of feruloylated glycerides; the external standard method is used to make a standard curve for quantification.

[0100] (4) Determine the acid value (AV) of the product oil according to the indicator titration method in GB-5009.229-2016. Weigh the sample, dissolve it in a mixture of diethyl ether-ethanol, and use a standard solution of potassium hydroxide to titrate to the end point.

[0101] All samples were subjected to three independent repeated experiments, and the results were expressed as mean ± standard deviation.

[0102] The experimental data is shown in Table 2: Table 2: Verification data of intermediate dehydration and catalyst B function (n=3, mean ± SD) Conclusion: The data in Table 2 verified the necessity of the intermediate dehydration step and catalyst B (lipase) for the second stage reaction.

[0103] The feruloylated glycerides (FAGs) content (15.2 ± 1.9 mg / kg) of Comparative Example 4 (dehydration, no catalyst B) was at the baseline level; this data indicates that, under low water activity (aw≤ 0.10) and in the presence of free ferulic acid, the esterification reaction (grafting) cannot proceed without the addition of catalyst B (lipase), which is necessary to catalyze this esterification reaction.

[0104] The FAGs content (18.5 ± 2.1 mg / kg) of Comparative Example 7 (not dehydrated, with catalyst B) was also at the baseline level; this data indicates that, under high water activity (aw> 0.9), the reaction equilibrium is not favorable for esterification synthesis even with the addition of catalyst B. At the same time, the acid value of Comparative Example 7 (12.7 ± 0.8 mg KOH / g) was significantly higher than that of Example 1 (1.6 ± 0.2) and Comparative Example 4 (1.4 ± 0.1). This result indicates that, under high aw conditions, catalyst B (lipase) preferentially catalyzes the hydrolysis reaction of triglycerides, resulting in an increase in free fatty acids and an increase in the acid value of the oil.

[0105] Example 1 (FAGs content 1184.5 ± 21.7 mg / kg, acid value 1.6 ± 0.2 mg KOH / g) was significantly different from the results of Comparative Example 4 and Comparative Example 7.

[0106] In summary, the in situ esterification grafting reaction in the second stage must meet two conditions simultaneously: (1) The reaction system is converted to a low water activity (aw≤ 0.10) environment by intermediate dehydration to support the esterification reaction thermodynamically; (2) Catalyst B is added to provide the catalytic activity required for esterification. Omitting the dehydration step (Comparative Example 7) will result in lipase-catalyzed hydrolysis, causing the acid value to rise; omitting catalyst B (Comparative Example 4) will not initiate the esterification reaction.

[0107] Test Example 3: Catalyst recovery and stability test This test example aims to evaluate the reusability (operational stability) and physical stability (leakage rate) of the immobilized catalysts A and B prepared in the present invention when performing sequential reactions in the NADES system.

[0108] The experimental steps are as follows: (1) Take the recovered catalyst A (Preparation Example 3) and catalyst B (Preparation Example 5) produced at the end of Example 1 (first batch); use 0.1 M, pH 7.0 PBS buffer to rinse the recovered catalyst 3 times, and then use NADES medium-1 (Preparation Example 1) for equilibration.

[0109] (2) Put the catalysts A and B treated in step (1) into the next batch of fresh rice bran (100 kg) and NADES-1 (350 kg), and completely repeat all the process steps of Example 1.

[0110] (3) Repeat steps (1) and (2) until 5 batches of production are completed; sample at the end of the 1st batch, 3rd batch and 5th batch production, respectively.

[0111] (4) Sampling analysis: a. Obtain the slurry filtrate before intermediate dehydration (before step 2.5), determine the free ferulic acid content according to the method of Test Example 1, which is used to characterize the activity of catalyst A; b. Obtain the final product oil, determine the feruloylated glyceride content according to the method of Test Example 2, which is used to characterize the activity of catalyst B. c. Take the content of the product of the 1st batch as 100%, calculate the relative activity of the 3rd and 5th batches.

[0112] (5) Catalyst leakage rate test: collect the NADES slurry filtrate discharged in step (2) and step (5) of Example 1 (first batch); use Bradford method to determine the protein concentration in the filtrate, use bovine serum albumin (BSA) to make a standard curve, measure the absorbance at 595 nm, and calculate the leaked protein content (mg / L).

[0113] All samples were tested in triplicate, and the results were expressed as mean ± standard deviation.

[0114] The experimental data are shown in Tables 3 and 4: Table 3: Activity test of repeated use of catalysts A and B (n = 3, mean ± SD) Table 4: Catalyst leakage rate test (n = 3, mean ± SD) Conclusion: The data of this test example (Tables 3 and 4) collectively verify the stability of the immobilized catalyst system used in the present application.

[0115] Table 3 data shows that after 5 cycles of use, the catalytic activity of the immobilized catalysts A and B (characterized by the yield of free ferulic acid and feruloylated glyceride, respectively) in the high-viscosity, high-solid-content NADES system remains above 88% of the initial activity (89.7% and 88.2%, respectively). This result indicates that the immobilized enzyme has good operational stability; this stability can be attributed to the covalent cross-linking immobilization strategy used in the preparation example, in which glutaraldehyde fixes the enzyme protein on the carrier, enabling it to withstand the physical shear of the rice bran slurry and the chemical environment of the NADES medium.

[0116] Table 4 data complements the evidence of stability; after separation of catalysts A and B, the protein concentration in their filtrates was at low levels (4.5 ± 0.4 mg / L and 3.9 ± 0.6 mg / L, respectively); this confirms that physical leakage of enzyme protein was effectively controlled.

[0117] In summary, Test Example 3 demonstrates that the immobilized catalysts in the present application are reusable. This feature reduces the cost of enzyme preparation consumption in the process, providing data support for the economic feasibility of the process.

[0118] Test Example 4: Base quality and oxidative stability of finished oil This test example aims to compare the base quality of finished oil obtained from the inventive examples and different comparative examples (including traditional process and defective process); the quality evaluation is based on two core indicators: acid value (AV) and peroxide value (POV).

[0119] The experimental procedure is as follows: (1) Obtain the final finished oil of Example 1, Example 2, Example 3, Comparative Example 1 (hexane extraction), Comparative Example 2 (hot pressing), Comparative Example 5 (without nitrogen protection), and Comparative Example 7 (without intermediate dehydration).

[0120] (2) Determine the acid value (AV) according to the national standard GB-5009.229-2016 "Determination of Acid Value in Food"; accurately weigh about 3.0 g of sample and dissolve in 80 mL of mixed solvent of diethyl ether-ethanol (2:1, v / v); add phenolphthalein indicator, and titrate with 0.05 M potassium hydroxide standard solution until the solution appears reddish and does not fade for 30 seconds. Record the volume consumed and calculate the acid value (mg KOH / g).

[0121] (3) Determine the peroxide value (POV) according to the national standard GB-5009.227-2016 "Determination of Peroxide Value in Food" (first method, titration method); accurately weigh about 2.0 g of sample and dissolve in 25 mL of mixed solvent of chloroform-glacial acetic acid (2:3, v / v); add 1.0 mL of saturated potassium iodide solution, tightly cap, and react in the dark for 3 minutes; add 30 mL of purified water, titrate with 0.002 M sodium thiosulfate standard solution until the solution turns light yellow, add 1.0 mL of starch indicator, and continue titration until the blue color disappears; a blank test is also performed. Calculate the peroxide value (mmol / kg).

[0122] All samples were tested in triplicate, and the results are expressed as mean ± standard deviation.

[0123] The experimental data is shown in Table 5: Table 5: Base quality indicators of finished oil from different processes (n = 3, mean ± SD) Conclusion: The data of Test Example 4 (Table 5) showed the effect of different processes on the quality of rice bran oil.

[0124] The finished oils of Examples 1, 2, and 3 all had low acid values (1.6-1.8 mg KOH / g) and low peroxide values (1.8-2.1 mmol / kg); this result indicated that the process of the present application (sequential NADES enzymatic method) controlled the hydrolysis and oxidation of oil and fat, mainly due to low-temperature operation (<60°C) and nitrogen protection throughout the process.

[0125] The data of Comparative Example 2 (traditional hot-pressing method) (AV 5.5, POV 10.1) showed that high-temperature (110°C) treatment accelerated the thermal oxidation and hydrolysis of oil and fat, resulting in the deterioration of oil quality.

[0126] The data of Comparative Example 5 (without nitrogen protection) (AV 4.8, POV 15.2) illustrated the role of nitrogen protection; during the mixing and reaction of the NADES slurry, the material was in contact with air (oxygen), resulting in the peroxide value (POV) rising to 15.2 mmol / kg, which was higher than that of Example 1 (1.8 mmol / kg). This comparison showed that the process design of the present application inhibited the autoxidation pathway of oil and fat.

[0127] The data of Comparative Example 7 (without intermediate dehydration) (AV 12.7, POV 8.5) showed that its acid value (12.7 mg KOH / g) was relatively high. This result was consistent with the conclusion of Test Example 2, i.e., under high water activity, the function of catalyst B was changed to hydrolysis, resulting in the accumulation of free fatty acids.

[0128] The process of the present application avoided the deterioration of oil quality caused by the traditional hot-pressing method (thermal oxidation), conventional NADES treatment (autoxidation), and defective NADES enzymatic method (enzymatic hydrolysis) through low-temperature operation, nitrogen protection, and intermediate dehydration steps.

[0129] Test Example 5: Retention rate of heat-sensitive nutrients This test example aimed to quantitatively compare the retention contents of two key heat-sensitive nutrients, tocopherols (total vitamin E) and oryzanol, in the finished oils obtained from the inventive examples (low-temperature NADES process) and comparative examples (traditional high-temperature / solvent process).

[0130] The experimental steps were as follows: (1) Obtain the final finished oils of Example 1, Example 2, Example 3, Comparative Example 1 (hexane extraction), and Comparative Example 2 (hot-pressing).

[0131] (2) Total tocopherol content determination: Refer to national standard GB-5009.82-2016 (second method). Accurately weigh 0.5 g of sample oil, and make up to 10 mL with n-hexane; use high performance liquid chromatography-fluorescence detector (HPLC-FLD), normal phase silica gel column (4.6x250mm, 5μm), mobile phase is n-hexane-isopropyl alcohol (99.5:0.5, v / v), fluorescence detector excitation wavelength 295 nm, emission wavelength 330 nm. Use external standard method to quantitatively determine α, β, γ, δ-tocopherol, and calculate the total tocopherol content (mg / kg).

[0132] (3) Gossypol content determination: Refer to national standard GB-5009.243-2017; accurately weigh 0.1 g of sample oil, and make up to 10 mL with isopropyl alcohol; use high performance liquid chromatography-ultraviolet detector (HPLC-UV), C18 reversed phase column (4.6x250mm, 5μm), mobile phase is methanol-acetonitrile-isopropyl alcohol (50:45:5, v / v / v), ultraviolet detector detection wavelength 315 nm; use gossypol standard, and use external standard method for quantitative determination (mg / kg).

[0133] All samples were subjected to three independent repeated experiments, and the results were expressed as mean ± standard deviation.

[0134] The experimental data are shown in Table 6. Table 6: Heat-sensitive nutrient content of product oils of different processes (n=3, mean ± SD) Conclusion: The data of Test Example 5 (Table 6) show the effect of different processes on the retention of nutrients. Comparative Example 2 (traditional hot pressing method) has the lowest total tocopherol and gossypol content after high-temperature pressing at 110°C. This result shows that high-temperature treatment is the cause of the loss of these two heat-sensitive substances. Comparative Example 1 (hexane extraction method) also has a low level of nutrients. This process requires the rice bran to be treated at a high temperature (such as 120°C) for enzyme inactivation (stabilization) before solvent extraction, and this high-temperature pretreatment step causes the loss of heat-sensitive nutrients.

[0135] The data of Examples 1, 2 and 3 are all higher than those of the two comparative examples, and Example 1 (45°C) has the best retention effect; this result shows that the feature of the present application (NADES sequential enzyme method) is low-temperature processing. The present application process is carried out at a low temperature (<60°C) and under nitrogen protection throughout, avoiding thermal degradation and oxidative degradation. The inactivation of endogenous enzymes in rice bran is completed in the NADES medium environment and subsequent pressing separation, rather than relying on traditional high-temperature inactivation. Therefore, the present application process retains the original heat-sensitive nutrients in rice bran while extracting oil.

[0136] Test Example 6: Comparison between "one-pot method" and "sequential method" This test example aims to compare the process effect of the "sequential reaction" (i.e. step one hydrolysis, dehydration, step two esterification) employed in the present invention (Example 1) with the "one-pot" method (Comparative Example 6, i.e. hydrolysis enzyme and esterification enzyme added simultaneously at initial high water activity).

[0137] The experimental procedure is as follows: (1) Obtain the final product oil of "Example 1" (sequential method) and "Comparative Example 6" (one-pot method).

[0138] (2) Accurately weigh 0.1 g of the above two product oil samples, and use isopropanol-acetonitrile (1:1, v / v) mixed solvent to make up to 10 mL. After vortex mixing, filter through a 0.45 μm organic phase filter membrane, and take the filtrate.

[0139] (3) Free ferulic acid (FA) content determination: high performance liquid chromatography (HPLC-UV) method; chromatographic column: C18 reversed phase column (4.6x250 mm, 5 μm); mobile phase A: 0.1% (v / v) formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution; detection wavelength: 320 nm. Quantified by external standard (ferulic acid standard) curve.

[0140] (4) Feruloylated glycerides (FAGs) content determination: liquid chromatography-tandem mass spectrometry (LC-MS / MS) method; chromatographic column: C18 column (2.1x100 mm, 1.8 μm); gradient elution; mass spectrometry uses electrospray ion source (ESI), detects in multiple reaction monitoring (MRM) mode; quantified by external standard method.

[0141] All samples were independently repeated three times, and the results were expressed as mean ± standard deviation.

[0142] The experimental data is shown in Table 7: Table 7: Comparison of key components in product oil of "sequential method" and "one-pot method" (n=3, mean ± SD) Conclusion: The data (Table 7) of this test example compares the product differences between sequential method and one-pot method.

[0143] The product oil of Comparative Example 6 (one-pot method) contains a high concentration of free ferulic acid (151.3 ± 6.4 mg / kg), while the content of feruloylated glycerides (FAGs) (16.9 ± 1.1 mg / kg) is at the baseline level; this result shows that in the high water activity (aw>0.9) system of Comparative Example 6, catalyst A (FAE) has hydrolysis function and releases ferulic acid; but the catalyst B (lipase) added simultaneously fails to catalyze the esterification (grafting) reaction.

[0144] The mechanism of this phenomenon is that the two catalysts (catalyst A and B) have opposite water activity (aw) requirements. Catalyst A (hydrolytic enzyme) requires high aw (e.g. step one of Example 1) to perform hydrolysis; catalyst B (lipase) requires low aw (e.g. step three of Example 1) to push the reaction equilibrium towards ester synthesis.

[0145] As shown in the data of Test Example 2, at high aw, the catalytic behavior of catalyst B is thermodynamically controlled, its function is inhibited (unable to synthesize FAGs), or reversed to hydrolysis (resulting in high acid value). Comparative Example 6 (one-pot method) places both enzymes in a high aw environment, resulting in failure of the esterification reaction.

[0146] The data of Example 1 (low free ferulic acid 21.5 ± 1.8 mg / kg; high FAGs 1184.5 ± 21.7 mg / kg) confirms the operating mechanism of the sequential process; low free ferulic acid content indicates that the ferulic acid produced in step one (high aw) is consumed and synthesized into FAGs by catalyst B in step four (low aw) after intermediate dehydration (aw regulation).

[0147] This test example confirms that the "sequential reaction" design and the "intermediate dehydration" (aw regulation) step are technical means to achieve the compatibility of the two functions (unlocking and grafting) in the NADES system.

[0148] Test Example 7: Functional specificity evaluation of finished oil This test example aims to specifically evaluate the decisive role of step (4) (addition of catalyst B) in the process of the present application in achieving in situ grafting of feruloylated glycerides (FAGs).

[0149] The experimental steps are as follows: (1) Obtain the final finished oil of Example 1, Example 2, Example 3, and the final finished oil of Comparative Example 4; Comparative Example 4 is the same as Example 1 except that step (4) of adding catalyst B is omitted.

[0150] (2) Accurately weigh 0.1 g of the above four kinds of finished oil samples, and use isopropyl alcohol-acetonitrile (1:1, v / v) mixed solvent to make up to 10 mL; after vortex mixing, filter through a 0.45 μm organic phase filter membrane, and take the filtrate.

[0151] (3) Use liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the content of feruloylated glycerides (FAGs); chromatographic column: C18 column (2.1x100mm, 1.8μm); gradient elution is used; mass spectrometry uses electrospray ion source (ESI), and is detected in multiple reaction monitoring (MRM) mode, and is quantified by monitoring the characteristic ion pair of feruloylated glycerides; the external standard method is used to make a standard curve for quantification.

[0152] All samples were performed in triplicate and the results are expressed as mean ± standard deviation.

[0153] The experimental data is shown in Table 8: Table 8: Specific comparison of feruloylated glycerides (FAGs) content in finished oil (n = 3, mean ± SD) Conclusion: The feruloylated glycerides (FAGs) content of Comparative Example 4 (15.2 ± 1.9 mg / kg) without the addition of catalyst B is at baseline level; Comparative Example 4 has the same first stage reaction and intermediate dehydration step as Example 1; this result indicates that even with the presence of both the reaction substrate (free ferulic acid) and a thermodynamically favorable low water activity environment in the NADES system, the esterification (grafting) reaction does not proceed spontaneously.

[0154] The FAGs content of Examples 1, 2, 3 (1055.2-1184.5 mg / kg) with the addition of catalyst B is much higher than Comparative Example 4.

[0155] This data comparison rules out the possibility that the production of FAGs is a non-specific result of the NADES medium or the low-temperature physical process. The high FAGs content in the finished oil is confirmed to be produced by the enzymatic esterification reaction catalyzed by catalyst B under low water activity. This test example confirms the effectiveness and specificity of the second stage (step 4) in the process as a "functional grafting" step.

[0156] Test Example 8: Functional specificity evaluation of finished powder This test example aims to specifically evaluate the impact of step (1) (addition of catalyst A) in the process of the present invention on the chemical composition of the final finished powder (meal), i.e. to verify whether ferulic acid (FA) is effectively "unlocked" (hydrolyzed) from the rice bran solid matrix.

[0157] The experimental procedure is as follows: (1) Obtain the final finished powder of Example 1, Example 2, Example 3, and the final finished powder of Comparative Example 3 (Comparative Example 3 omits the addition of catalyst A and catalyst B compared to Example 1).

[0158] (1) Dry the finished powder samples at 40°C under vacuum to constant weight, grind using a high-speed pulverizer and pass through an 80-mesh sieve.

[0159] (1) Weigh 1.0 g of the powder sample into a 50 mL centrifuge tube with a plug, add 20 mL of 80% (v / v) methanol aqueous solution; extract in a 50°C water bath (40 kHz, 300 W) for 30 minutes.

[0160] (1) The extract was centrifuged at 8000 x g for 10 min at 4 °C. The supernatant was collected and filtered through a 0.45 pm organic phase microfiltration membrane. The filtrate was collected.

[0161] (1) The content of free ferulic acid in the filtrate was determined by high performance liquid chromatography (HPLC-UV). The column was a C18 reversed-phase column (4.6 x 250 mm, 5 pm). The mobile phase A was 0.1% (v / v) formic acid in water, and the mobile phase B was acetonitrile. Gradient elution was used. The column temperature was 30 °C, and the detection wavelength was 320 nm. The external standard curve was used for quantification.

[0162] The experiment was repeated three times independently, and the results were expressed as the mean ± standard deviation, with the unit converted to mg / kg (dry basis).

[0163] The experimental data are shown in Table 9. Table 9: Specificity comparison of free ferulic acid content in finished powder (n = 3, mean ± SD) Conclusion: The data in Table 9 show that the free ferulic acid content in the finished powder of Comparative Example 3 (NADES without enzyme treatment) is at a low baseline level (8.1 ± 1.1 mg / kg). This result indicates that ferulic acid in the rice bran raw material is mainly connected to the cell wall polysaccharides in the form of an ester bond, and the content of natural free state can be ignored. At the same time, it is confirmed that only NADES medium treatment (such as ChCl-malic acid) is not enough to break these covalent ester bonds.

[0164] The free ferulic acid content in the finished powder of Examples 1, 2, and 3 (58.9-65.4 mg / kg) is higher than that of Comparative Example 3 (7 to 8 times).

[0165] Combining the data of Test Example 1 (liquid phase free FA) and Test Example 7 (oil phase FAGs), the transfer path of ferulic acid can be determined: in step (1), the catalyst A (FAE) hydrolyzes the ferulic acid ester bond of the cell wall of the solid phase (rice bran powder), making ferulic acid free. The free ferulic acid partially dissolves into the NADES liquid phase (Test Example 1 data). In step (4), the ferulic acid in the NADES phase is consumed by catalyst B and grafted to glycerides and enters the oil phase (Test Example 7 data). The free ferulic acid measured in this test example (Table 9) is the part that is not consumed in step (4) and finally remains in the solid phase (finished powder) after being produced by step (1) hydrolysis.

[0166] The data of this test example confirm that catalyst A (FAE) is the cause of the change in the chemical composition (free ferulic acid) of the finished powder. This process simultaneously changes the oil phase (grafted FAGs) and the solid phase (unlocked FA), providing a chemical basis for the application of the finished powder (meal).

[0167] Test Example 9: Accelerated oxidative stability of finished oil This test example aims to quantitatively evaluate the difference in oxidative stability of the grafted oil obtained from the present invention (Example 1) compared to the traditional hot-pressed oil (Comparative Example 2) and the ungrafted NADES process oil (Comparative Example 4) through accelerated oxidation testing (Rancimat method).

[0168] The experimental procedure is as follows: (1) Obtain the final product oil of Example 1 (NADES sequential enzymatic method, grafted oil), Comparative Example 2 (traditional hot-pressed method) and Comparative Example 4 (NADES low-temperature process, ungrafted oil).

[0169] (2) Determine the oxidation induction period using a Rancimat analyzer (Switzerland Metrohm 892); accurately weigh 3.0 g ± 0.1 g of oil sample into the reaction tube; set the test conditions: test temperature 110°C, air flow rate 20 L / h; the collection pool is filled with 60 mL of purified water; the instrument automatically records the oxidation induction period (OSI) according to the change in conductivity.

[0170] The samples were subjected to three independent repeated experiments, and the results were expressed as mean ± standard deviation.

[0171] The experimental data is shown in Table 10: Table 10: Rancimat oxidation induction period (110°C) of product oils from different processes (n = 3, mean ± SD) Conclusion: The induction period of Comparative Example 2 (hot-pressed oil) (4.1 ± 0.3 h) is the shortest; traditional high-temperature pressing (> 110°C) leads to the degradation of natural antioxidants (such as tocopherols, oryzanol) in rice bran, reducing the antioxidant capacity of the finished oil.

[0172] The induction period of Comparative Example 4 (ungrafted oil) (7.2 ± 0.4 h) is higher than that of Comparative Example 2; both Comparative Example 4 and Example 1 are treated at low temperature (< 60°C), which retains the natural antioxidants; the 7.2 h induction period can be considered as the level of natural antioxidant retention by the low-temperature process.

[0173] The induction period of Example 1 (grafted oil) (10.3 ± 0.5 h) is higher than that of Comparative Example 4; the difference between Example 1 and Comparative Example 4 is that Example 1 performs step (4), grafting ferulic acid onto glycerides through catalyst B.

[0174] The data comparison shows that the in-situ grafting of ferulic acid (phenolic antioxidant) is the reason for the improvement of oxidative stability from 7.2 h to 10.3 h. Ferulic acid is covalently connected to the triglyceride backbone in the form of feruloylated glycerides (FAGs). This covalent binding makes the antioxidant group (feruloyl group) part of the oil molecule, which can avoid the decline in effectiveness caused by the volatilization of free antioxidants at high temperature tests or low solubility. The process of the present application retains natural antioxidants through low-temperature treatment and introduces covalent antioxidants by in-situ grafting of ferulic acid (FAGs) through catalyst B. These two mechanisms together improve the oxidative stability of the finished oil.

Claims

1. A method for synergistic treatment of rice bran degreasing and nutrient retention in an edible process, characterized by, The method comprises the following steps: (a) first stage hydrolysis: mixing the rice bran raw material, the NADES-aqueous solution, and the immobilized catalyst A, and reacting to hydrolyze ferulic acid ester bonds and phospholipids in the rice bran raw material; (b) intermediate dehydration: dehydrating the slurry obtained in step (a) to reduce the water activity of the slurry to a preset esterification synthesis threshold value; (c) second stage esterification: adding the immobilized catalyst B to the dehydrated slurry of step (b) to react to graft free ferulic acid produced in step (a) to glycerides to form feruloylated glycerides; (d) product separation: solid-liquid separation of the slurry obtained in step (c) to obtain finished oil and finished powder.

2. The method according to claim 1, wherein the method is characterized by, The NADES-aqueous solution is prepared by mixing choline chloride and an acidic hydrogen bond donor selected from L-malic acid or citric acid.

3. The method according to claim 1, wherein the method is characterized by, The immobilized catalyst A is prepared by co-immobilizing ferulic acid esterase and phospholipase A2 on the same carrier.

4. The method according to claim 3, wherein the method is characterized by, The activity ratio of the ferulic acid esterase to the phospholipase A2 is 1:2-2:1 U / U.

5. The method according to claim 1, wherein the method is characterized by, The immobilized catalyst B is prepared by immobilizing a non-specific lipase on a carrier.

6. The method according to claim 1, wherein the method is characterized by, The preset esterification synthesis threshold value in step (b) is a water activity of the slurry ≤0.15; the reaction temperature of step (a) is 45-55°C; and the reaction temperature of step (c) is 45-55°C.

7. The method according to claim 1, wherein the method is characterized by, The method is carried out under nitrogen protection.

8. The method according to claim 1, wherein the method is characterized by, The finished powder obtained in step (d) has a free ferulic acid content of 50-70 mg / kg on a dry basis.

9. A functional rice bran oil composition prepared by the method of synergistic processing of defatted rice bran with a nutrient-retaining edible process according to any one of claims 1 to 8, characterized in that, The functional rice bran oil composition comprises: feruloylated glycerides, content greater than 1000 mg / kg; total tocopherols, content greater than 700 mg / kg; The acid value of the functional rice bran oil composition is ≤1.8 mg KOH / g. The peroxide value of the functional rice bran oil composition is ≤2.1 mmol / kg. The feruloylated glyceride content is 1100-1200 mg / kg; the total tocopherol content is 750-810 mg / kg; and the oryzanol content is 8800-9150 mg / kg.

10. The functional rice bran oil composition according to claim 9, characterized by, ​