Enzyme activity detection reagent for synthesizing OPO lipase and detection method thereof

Abstract

The invention discloses an enzyme activity detection reagent for synthesizing OPO lipase and a detection method thereof, and relates to the field of chemical detection.The detection reagent is prepared from, by mass, 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.01 part of OPO lipase, 8-12 parts of normal hexane, 5-7 parts of acetone, 3-5 parts of water, 1 mL of phenolphthalein indicator and 2.6-3.0 mL of 1 mol / L KOH; the method comprises the following steps: preparing oleic acid and 1, 3-dioleic acid-3-palmitic acid triglyceride from a detection reagent and synthesized OPO lipase; and detecting and synthesizing the OPO lipase through liquid chromatography. The method can accurately reflect the OPO synthesis capability of the enzyme preparation, and can rapidly and accurately determine the OPO synthesis enzyme activity.

Description

Technical Field

[0001] The invention relates to the detection field, in particular to an enzyme activity detection reagent for synthesizing OPO lipase and a detection method thereof. Background Art

[0002] Lipase is a biological enzyme that is activated at the oil-water interface and can catalyze a variety of reactions such as hydrolysis, esterification, transesterification, acidolysis, alcoholysis, and aminolysis. Lipase is one of the most commonly used biocatalysts in oil modification, especially for modifying fatty acids at specific positions of triglycerides, which has greater advantages than chemical transesterification, and the enzymatic reaction has three recognized advantages: mild reaction conditions, few by-products, and environmental friendliness. Based on safety, reliability, and environmental friendliness, GB 30604 stipulates that OPO is allowed to be produced and manufactured as a food nutritional enhancer only by lipase catalyzed by edible industrial lipase, which makes the production and supply of OPO-specific lipase extremely important. The national standard GB / T 23535 takes triglyceride hydrolysis reaction as the research object, which is quite different from the transesterification reaction in OPO synthesis and is not suitable for the determination of OPO synthetic lipase activity. Therefore, it is very necessary to develop a set of standardized methods suitable for OPO synthase activity, and the present invention solves such problems. Summary of the invention

[0003] To address the deficiencies of the prior art, the present invention aims to provide an enzyme activity detection reagent and a detection method for synthesizing OPO lipase. The method developed by the present invention accurately reflects the ability of the enzyme preparation to synthesize OPO and can quickly and accurately determine the enzyme activity of OPO synthesis.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: A synthetic OPO lipase enzyme activity detection reagent comprises, by weight: 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.010-0.011 parts of OPO lipase, 8-12 parts of n-hexane, 5-7 parts of acetone, 3-5 parts of water, 1 mL of phenolphthalein indicator, and 2.6-3.0 mL of 1 mol / L KOH.

[0005] The aforementioned synthetic OPO lipase enzyme activity detection reagent includes, by mass, 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.01 parts of OPO lipase, 10 parts of n-hexane, 6 parts of acetone, 4 parts of water, 1 mL of phenolphthalein indicator, and 2.8 mL of 1 mol / L KOH.

[0006] A method for detecting a synthetic OPO lipase enzyme activity detection reagent comprises the following steps: Step 1: prepare oleic acid and 1,3-dioleoyl-3-palmitoylglycerol triesters by using a detection reagent and OPO lipase; the detection reagent includes, by weight: 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.01 parts of OPO lipase, 8-12 parts of n-hexane, 5-7 parts of acetone, 3-5 parts of water, 1 mL of phenolphthalein indicator, and 2.6-3.0 mL of 1 mol / L KOH; Step 2, determining the content of 1,3-dioleyl-3-palmitoyl triglyceride in the sample by liquid chromatography; Step 3, calculating enzyme activity; The calculation formula is shown in (1): Enzyme activity (U / g) = (1) in, M: initial amount of oleic acid; C: amount of oleic acid converted to 1,3-dioleo-2-palmitoyl triglyceride; W: quality of enzyme preparation; t: reaction time.

[0007] The aforementioned detection method for synthesizing an enzyme activity detection reagent for OPO lipase, step 1, the specific steps of preparing oleic acid and 1,3-dioleyl-3-palmitoyl triglyceride by using the detection reagent and OPO lipase include: (1) Solution preparation: Preparation of oleic acid-palm oil substrate solution: Accurately weigh 2.5 parts of palm oil and 7.5 parts of oleic acid in a beaker, stir thoroughly, seal the beaker with a sealing film, and place the beaker in a 60℃ water bath for 30 minutes to completely dissolve the substrate. The substrate needs to be stored in a 60±1℃ water bath, otherwise precipitation will precipitate. Take 10g of substrate for each sample during the reaction; (2) Determination steps: Accurately weigh 0.01 portion of enzyme sample into a 100 mL stoppered flask, and weigh 3 parallel samples for each sample; Add 10g of substrate to a stoppered Erlenmeyer flask, cover the flask tightly, and immediately place it in a (60±1)℃ water bath for ultrasonic reaction for 20min, then in a 90℃ water bath for 15min, and filter (filter paper dripping) to obtain a sample; add a certain volume of n-hexane to the prepared sample, stir for 5min to dissolve it completely, add a certain volume of acetone aqueous solution, drop 1ml of phenolphthalein indicator, and slowly drop 1mol / L KOH solution while stirring until the solution turns pink and does not fade after stirring for 3min; transfer the solution to a separatory funnel, let it stand, separate the layers, and separate the lower layer; add n-hexane to the lower layer solution for extraction and separation, combine the upper layer liquid, and wash with acetone aqueous solution until the solution is clear, transparent, and free of bubbles; distill the upper layer solution under reduced pressure to remove n-hexane to obtain the purified sample.

[0008] The detection method of the enzyme activity detection reagent of the aforementioned synthetic OPO lipase, step 2, determines the content of 1,3-dioleyl-2-palmitylglycerol triester in accordance with the method "GB30604-2015 National Food Safety Standard Food Nutrition Fortifier 1,3-dioleyl-2-palmitylglycerol triester", in terms of %.

[0009] The aforementioned detection method of a synthetic OPO lipase enzyme activity detection reagent, step three, the specific method for calculating the enzyme activity is: The calculation formula is shown in (1): Enzyme activity (U / g) = (1) in, M: initial amount of oleic acid, 8.85 mol, i.e. 8850 mmol; C: The amount of oleic acid converted into 1,3-dioleoyl-2-palmitoylglycerol triester, which should be in the range of 0.05<C<0.20. If it exceeds the range, the mass of the immobilized enzyme weighed should be changed; W: mass of enzyme preparation, g; t: reaction time, 20min.

[0010] The present invention is beneficial in that: The present invention prepares oleic acid and 1,3-dioleyl-3-palmitoyl triglyceride by using a detection reagent and synthetic OPO lipase; and then detects the synthetic OPO lipase by liquid chromatography; such an original method can accurately reflect the ability of the enzyme preparation to synthesize OPO, can quickly and accurately determine the enzyme activity of OPO synthesis, and has simple operation steps, good parallelism and repeatability, greatly improved accuracy, and a stable measurement process; The detection time of the present invention is short, only 3 hours, and the required sample amount is small, only 30g; the method of the prior art requires two days to complete the entire process (synthesis reaction, molecular distillation), and more than 2kg of samples must be synthesized for molecular distillation and deacidification. DETAILED DESCRIPTION

[0011] The present invention is described in detail below with reference to specific embodiments.

[0012] The technical effect of the present invention is verified by the following experiments: Example 1 (1) Solution preparation Oleic acid-palm oil substrate solution Accurately weigh 60.0g of palm oil and 180.0g of oleic acid in a beaker, stir thoroughly, seal the beaker with a sealing film, and place the beaker in a 60℃ water bath for 30 minutes to completely dissolve the substrate. The substrate must be stored in a 60±1℃ water bath, otherwise precipitation will occur. Take 10g of substrate for each sample during the reaction.

[0013] (2) Measurement steps Accurately weigh 0.0100 g of enzyme sample 1 (Lipozyme® TL), enzyme sample 2 (Lipozyme® RMC), enzyme sample 3 (immobilized lipase RIM-03), enzyme sample 4 (immobilized lipase RM), enzyme sample 5 (immobilized lipase-1), and enzyme sample 6 (immobilized lipase-2) into a 100 mL stoppered conical flask, and weigh 3 parallel samples for each sample.

[0014] Add 10g of substrate to a stoppered conical flask, cover the conical flask tightly, immediately put it in a water bath, and start timing. Immediately put it in a (60±1)℃ water bath for ultrasonic reaction for 20min, 90℃ water bath for 15min, and filter (filter paper dripping) to obtain the sample. Add a certain volume of n-hexane to the prepared sample, stir for 5min to dissolve it completely, add a certain volume of acetone aqueous solution, add 1ml phenolphthalein indicator, and slowly add 1mol / L KOH solution while stirring until the solution turns pink and does not fade after stirring for 3min. Transfer the solution to a separatory funnel, let it stand, separate the layers, and separate the lower layer; add n-hexane to the lower layer solution for extraction, separate the layers, combine the upper liquid, and wash with acetone aqueous solution until the solution is clear, transparent, and free of bubbles. The upper solution is distilled under reduced pressure to remove n-hexane to obtain the purified sample.

[0015] Liquid chromatography determination of 1,3-dioleo-3-palmitoyl triglyceride content in samples: Determine the content of 1,3-dioleo-2-palmitoyl triglyceride in accordance with the method "GB30604-2015 National Food Safety Standard Food Nutrition Fortifier 1,3-dioleo-2-palmitoyl triglyceride" in %.

[0016] (3) Calculation Enzyme activity (U / g) = in, M: initial amount of oleic acid, 8.85 mol, i.e. 8850 mmol C: The amount of oleic acid converted into 1,3-dioleoyl-2-palmitoylglycerol triester, which should be in the range of 0.05<C<0.20. If it exceeds the range, the mass of the immobilized enzyme weighed should be changed.

[0017] W: mass of immobilized enzyme preparation, g t: reaction time, 20min Comparative Example 1 The enzyme activities of enzyme sample 1 (Lipozyme® TL), enzyme sample 2 (Lipozyme® RMC), enzyme sample 3 (immobilized lipase RIM-03), enzyme sample 4 (immobilized lipase RM), enzyme sample 5 (immobilized lipase-1) and enzyme sample 6 (immobilized lipase-2) were tested in accordance with GB / T 23535-2009 Lipase Preparations.

[0018] Synthesis Experiment Palm oil and oleic acid were added in a mass ratio of 1:3, and the amount of enzyme preparation was 10% of the total mass of the feed. Samples were taken at 50℃, 55℃, and 60℃ for 3h, 4h, 5h, 6h, 7h, and 8h, respectively. The samples were molecularly distilled and deacidified twice at 170℃ to reduce the acid value of the samples to below 1mgKOH / g. The content of 1,3-dioleyl-2-palmitylglycerol triester was determined in accordance with the method "GB30604-2015 Food Safety National Standard Food Nutrition Fortifier 1,3-dioleyl-2-palmitylglycerol triester", in %. It takes two days to complete the overall process (synthesis reaction, molecular distillation), and more than 2L of samples must be synthesized for molecular distillation and deacidification.

[0019] The enzyme activity data of different enzyme preparations tested according to Example 1 and Comparative Example 1 are as follows: Table 1 Enzyme activities of different enzyme preparations Synthetic experimental data: (1) Lipozyme® RMC enzyme Table 2 Effect of Lipozyme®RMC enzyme on the synthesis of OPO (2) Lipozyme® TL Table 3 Effect of Lipozyme®TL enzyme on the synthesis of OPO (3) Immobilized lipase RIM-03 Table 4 Effect of immobilized lipase RIM-03 on the synthesis of OPO (4) Immobilized lipase RM Table 5 Effect of immobilized lipase RM on the synthesis of OPO (5) Immobilized lipase-1 Table 6 Effect of immobilized lipase 1 on the synthesis of OPO (6) Immobilized lipase-2 Table 7 Effect of immobilized lipase 2 on the synthesis of OPO The data synthesized from the above experiment are consistent with those in Example 1, and the enzyme activity data can more accurately reflect the conversion rate of enzyme synthesis of OPO: Lipozyme®RMC>Lipozyme®TL>immobilized lipase RIM-03>immobilized lipase RM>immobilized lipase-2>immobilized lipase-1, which is quite different from the experimental data of the control group. "GB / T 23535-2009 Lipase Preparations" is to determine the ability of enzyme preparations to hydrolyze triglycerides into fatty acids, diglycerides, monoglycerides and glycerol, while the main function of enzyme preparations for synthesizing OPO is to undergo ester exchange reaction with oleic acid, so the conventional test of lipase activity cannot accurately reflect the ability of the enzyme to synthesize OPO.

[0020] In summary, the present invention prepares oleic acid and 1,3-dioleoyl-3-palmitoyl triglyceride by detecting reagents and synthetic OPO lipase; and then detects the synthetic OPO lipase by liquid chromatography; such an original method can accurately reflect the ability of the enzyme preparation to synthesize OPO, with a small sample amount (30g), rapid (detection time 3 hours), and accurate determination of the enzyme activity of OPO synthesis.

[0021] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A synthetic OPO lipase enzyme activity detection reagent, characterized in that: The components include, by weight: 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.010-0.011 parts of OPO lipase, 8-12 parts of n-hexane, 5-7 parts of acetone, 3-5 parts of water, 1 mL of phenolphthalein indicator, and 2.6-3.0 mL of 1 mol / L KOH.

2. The enzyme activity detection reagent for synthetic OPO lipase according to claim 1, characterized in that: The components include, by mass: 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.01 parts of OPO lipase, 10 parts of n-hexane, 6 parts of acetone, 4 parts of water, 1 mL of phenolphthalein indicator, and 2.8 mL of 1 mol / L KOH.

3. A method for detecting a synthetic OPO lipase enzyme activity detection reagent, characterized in that: The steps include: Step 1: prepare oleic acid and 1,3-dioleoyl-3-palmitoylglycerol triesters by using a detection reagent and a synthetic OPO lipase; the detection reagent comprises, by weight: 2.5 parts of palm oil, 7.5 parts of oleic acid, 0.010-0.011 parts of OPO lipase, 8-12 parts of n-hexane, 5-7 parts of acetone, 3-5 parts of water, 1 mL of phenolphthalein indicator, and 2.6-3.0 mL of 1 mol / L KOH; Step 2, determining the content of 1,3-dioleyl-3-palmitoyl triglyceride in the sample by liquid chromatography; Step 3, calculating enzyme activity; The calculation formula is shown in (1): Enzyme activity (U / g) = (1) in, M: initial amount of oleic acid; C: amount of oleic acid converted to 1,3-dioleo-2-palmitoyl triglyceride; W: quality of enzyme preparation; t: reaction time.

4. The method for detecting the enzyme activity detection reagent of a synthetic OPO lipase according to claim 3, characterized in that: The specific steps of preparing oleic acid and 1,3-dioleyl-3-palmitoyl triglyceride by using a detection reagent and OPO lipase include: (1) Solution preparation: Preparation of oleic acid-palm oil substrate solution: accurately weigh 2.5 parts of palm oil and 7.5 parts of oleic acid in a beaker, stir thoroughly, seal the beaker with a sealing film, and place the beaker in a 60℃ water bath for 30 minutes to completely dissolve the substrate; the substrate must be stored in a 60±1℃ water bath, otherwise precipitation will precipitate; take 10g of substrate for each sample during the reaction; (2) Determination steps: Accurately weigh 0.010-0.011 parts of OPO lipase sample into a 100 mL stoppered conical flask, and weigh 3 parallel samples for each sample; Add 10 g of substrate to a stoppered Erlenmeyer flask, cover the flask tightly, and immediately place it in a 60±1°C water bath for ultrasonic reaction for 20 min, then in a 90°C water bath for 15 min, and filter to obtain a sample; add n-hexane to the prepared sample, stir for 5 min to completely dissolve it, add an acetone aqueous solution, drop 1 ml of phenolphthalein indicator, and slowly drop 1 mol / L KOH solution while stirring until the solution turns pink and does not fade after stirring for 3 min; transfer the solution to a separatory funnel, let it stand, separate the layers, and separate the lower layer; add n-hexane to the lower layer solution for extraction, separate the layers, combine the upper layer liquid, and wash with acetone aqueous solution until the solution is clear, transparent, and free of bubbles; distill the upper layer solution under reduced pressure to remove the n-hexane to obtain the purified sample.

5. The method for detecting the enzyme activity detection reagent of a synthetic OPO lipase according to claim 3, characterized in that: The specific method for determining the content of 1,3-dioleoyl-3-palmitoyl triglyceride in the sample by liquid chromatography in step 2 is: determining the content of 1,3-dioleoyl-2-palmitoyl triglyceride in accordance with the method "GB30604-2015 National Food Safety Standard Food Nutrition Fortifier 1,3-dioleoyl-2-palmitoyl triglyceride", expressed in %.

6. The method for detecting the enzyme activity detection reagent of a synthetic OPO lipase according to claim 3, characterized in that: In step 3, the specific method for calculating the enzyme activity is: The calculation formula is shown in (1): Enzyme activity (U / g) = (1) in, M: initial amount of oleic acid, 8.85 mol, i.e. 8850 mmol; C: The amount of oleic acid converted into 1,3-dioleoyl-2-palmitoylglycerol triester, which should be in the range of 0.05<C<0.

20. If it exceeds the range, the mass of the immobilized enzyme weighed should be changed; W: mass of enzyme preparation, g; t: reaction time, 20min.

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