WO2017020143A1

WO2017020143A1 - Method of biosynthesizing functional lipid using nano-magnetically immobilized enzyme for continuous targeted catalysis of phospholipid in soybean oil

Info

Publication number: WO2017020143A1
Application number: PCT/CN2015/000548
Authority: WO
Inventors: 于殿宇; 江连洲; 王立琦; 刘天一; 潘明喆; 孙立斌; 李相昕; 齐晓芬; 任悦; 王文华
Original assignee: 于殿宇; 江连洲
Priority date: 2015-08-03
Filing date: 2015-08-03
Publication date: 2017-02-09
Also published as: CN107532110A

Abstract

Provided is a method of biosynthesizing a functional lipid using a nano-magnetically immobilized enzyme for continuous targeted catalysis of a phospholipid in soybean oil, comprising: applying nano-magnetically immobilized phospholipid enzymes in a three-phase magnetic fluidized bed, as a solid phase, such that the enzymes are distributed in a magnetically stable fluidized state; using soybean oil as a continuous flow phase, wherein the soybean oil passes through different types of magnetic fluidized bed reactors of nano-magnetically immobilized enzymes, and contacts and catalyzes a phospholipid without requiring mechanical stirring, generating a corresponding product; immobilizing the nano-magnetically immobilized lipase in a three-phase magnetic fluidized bed; through adding a phytosterol, esterifying FFA in a substrate with the phytosterol to form a phytosterol ester, thus changing a conventional removal step to a process of biosynthesizing a functional lipid. In the finished product, the content of DAG is approximately 1.65%, the content of phytosterol ester is approximately 1.16%, the content of phosphorous is 9.29 mg/kg and the esterification rate of the phytosterol ester is 93.4%. Continuous production is realized by re-using two enzymatic hydrolysis processes.

Description

Method for continuously catalyzing phospholipid biosynthesis of functional lipids in soybean oil by nano magnetic immobilized enzyme

Technical field:

The invention relates to the field of oil and fat processing, in particular to a method for continuously orienting catalytic phospholipid biosynthesis functional lipids in soybean hair oil by nano magnetic immobilized enzyme.

Background technique:

Oil is an indispensable nutrient for human survival. Soybean oil contains about 3% of phospholipids. Phospholipids in soy oil are a good emulsifier. They have many functional properties in human body, but phospholipids in fats and oils. Easy to absorb water and cause rancidity of oil, and phospholipid is not resistant to high temperature, it needs to be removed from the oil. This process is called degumming. The degumming method includes hydration degumming, acid degumming, Super degumming, Special degumming, ultrafiltration degumming, Top Degumming, silica gel adsorption, SOFT degumming and enzymatic degumming.

Ordinary hydration degumming is the use of phospholipids to absorb water from the soybean oil, which produces 30% neutral oil and 30% water. The hydration degumming not only causes neutral oil loss, It will also cause environmental pollution. The phospholipids in the hair oil are divided into hydrated phospholipids and non-hydrated phospholipids. The hydrated phospholipids are hydrophilic, and the non-hydrated phospholipids are suitable for bio-enzymatic degumming. Phospholipase A ₁ is an enzyme capable of specifically hydrolyzing the acyl group at the Sn-1 position in phospholipids. For example, phosphatidic acid (PA), which is the main component of enzymatic hydrolysis of non-hydrated phospholipids, is an enzymatic hydrolyzed product of Sn-2. - lysophosphatidic acid (Sn-2-HPA), Sn-1-lysophosphatidic acid (Sn-1-HPA), glycerophosphatidic acid (GPA), and free fatty acid (FFA), as shown in Figure 1, lysophosphatidic acid has The preferred hydrophilicity can rapidly swell and swell during the hydration degumming process of the oil, thereby removing the phospholipid from the hair oil. Phospholipase A ₂ is an enzyme capable of specifically hydrolyzing the acyl group at the Sn-2 position in phospholipids. For example, PA, which is the main component of enzymatic hydrolysis of non-hydrated phospholipids, is characterized by Sn-1-HPA and FFA. ,as shown in picture 2. At the same time, because the product of enzymatic hydrolysis contains FFA, and the decomposition of the oil itself will produce a certain amount of FFA, the presence of FFA in the oil will lead to an increase in the acid value of the oil, which needs to be removed by the deacidification process, which inevitably reduces the refining yield. Cause environmental pollution. Phospholipase C hydrolyzes phospholipids at the Sn-3 position of phospholipids, mainly hydrolyzing lecithin (PC) and cephalin (PE) in soybean oil, producing diglyceride (DAG) and phosphoric acid products, converting phospholipids into DAG does not require a degumming process, thereby improving the refining rate of the degumming of the hair oil. Only the phospholipase C enzymatic hydrolysis of PC is taken as an example, and the enzymatic hydrolyzed products are DAG and hydroxylamine phosphate products, as shown in FIG. The existing soybean oil rich in phytosterol esters is mainly prepared by enzymatic method. The enzyme-catalyzed synthesis has the advantages of mild reaction conditions, low energy consumption, environmental protection of products, etc. The use of lipase (EC 3.1.1.3) to use FFA and The process of esterification of plant sterols to phytosterol esters is shown in Figure 4. The above enzyme catalytic process is generally based on free enzymes, sensitive to the environment, poor stability to heat and pH, easy to inactivate, difficult to control reaction conditions, expensive free enzymes and can not be reused, limiting the efficient use of enzymes. With the development of bio-enzymatic processing technology, the emergence of immobilized enzyme technology has expanded the application range of enzymes. Ordinary immobilized enzymes are only suitable for batch production, which can increase the number of repeated use of biological enzymes.

Summary of invention:

The invention relates to a method for continuously catalyzing the phospholipid biosynthesis of functional lipids in soybean hair oil by nano magnetic immobilized enzyme. The invention is as follows: using soybean oil obtained by filtering to remove impurities; nano magnetic immobilized phospholipase is applied in each of the three In the phase magnetic fluidized bed, as a stationary phase, it is distributed in a magnetically stable fluidized state, with soybean oil as a continuous mobile phase, and the oil flows through a magnetic fluidized bed reactor of different types of magnetic immobilized enzymes, without Contacting the phospholipids in the catalyzed soybean oil under mechanical agitation to form the corresponding product; the magnetic immobilized lipase is immobilized in a three-phase magnetic fluidized bed, and the FFA and the phytosterol in the substrate are esterified into the substrate by adding phytosterols. The phytosterol ester is a process in which a conventional removal step is converted into a biosynthesis functional lipid without mechanical agitation, thereby achieving continuous production.

Specifically, the present invention relates to a nanomagnetic immobilized enzyme for continuously orienting catalytic phospholipid biosynthesis functional lipids in soybean oil, and more particularly, the present invention relates to nanomagnetic immobilized phospholipase C , nano-magnetic immobilized phospholipase A ₁ , nano-magnetic immobilized phospholipase A ₂ , nano-magnetic immobilized lipase) used in gas-liquid-solid three-phase magnetic fluidized bed reactor, three-phase three-phase magnetic fluidization The reaction system of the bed is mainly composed of a three-phase magnetic fluidized bed, a buffer tank, a oil tank and a power device. First, the nano magnetic immobilized enzyme particles are added into a three-stage three-phase magnetic fluidized bed to make it a stationary phase. The hair oil is a continuous mobile phase. By adjusting the reaction parameters, the nano-magnetic immobilized enzyme is stably fluidized in the three-stage three-phase magnetic fluidized bed reaction system, and the corresponding substrate is subjected to directed enzymatic reaction. The process of phospholipid-forming functional lipids in the continuous enzymatic hydrolysis of soybean hair oil by a magnetically immobilized enzyme in a three-phase magnetic fluidized bed is shown in FIG. 5 . According to the enzymatic hydrolysis characteristics of phospholipases, the enzymatic hydrolysate is beneficial to the formation of functional lipids by adjusting the sequence of enzymatic hydrolysis and the reaction conditions. By adding phytosterols, the FFA and the plants in the enzymatic hydrolysate are treated with magnetic lipase. The sterol ester is esterified into a phytosterol ester, and the conventional degumming and deacidification steps are converted into a process of producing functional lipids, and the nanomagnetic immobilized enzyme is directed to catalyze the continuous production of phospholipid biosynthesis functional lipids in soybean oil.

The advantage of the invention is that the nano magnetic immobilized enzyme having the advantages of high thermal stability and high tolerance to the pH range is applied in a three-phase magnetic fluidized bed as a stationary phase to achieve a fluidized state, soy wool The oil is a continuous mobile phase, achieving continuous production, and the enzymatic hydrolysis efficiency is improved. In particular, each of the three-phase magnetic fluidized bed reactors can maintain their respective optimal reaction conditions, and the three-phase magnetic fluidized bed reaction is separated by centrifugation. The enzymatic hydrolysate at the outlet of the vessel returns the buffer to the inlet of the corresponding three-phase magnetic fluidized bed reactor, enabling each nanomagnetic immobilized enzyme to catalyze the enzymatic hydrolysis of the substrate in a separate environment. The enzymatic hydrolysis process can realize the continuous production of functional lipids of phospholipid biosynthesis in the continuous directed catalyzed soybean oil, and at the same time regenerate the nano magnetic immobilized enzyme, which can be reused and reduce the production cost.

DRAWINGS

Figure 1. Process of enzymatic hydrolysis of PA in soybean oil by phospholipase A ₁

Figure 2. Process of enzymatic hydrolysis of PA in soybean oil by phospholipase A ₂

Figure 3. Phospholipase C enzymatic hydrolysis of PC to DAG in soybean oil

Figure 4. Process for esterification of FFA with phytosterols to phytosterol esters

Figure 5. Process of phospholipid-forming functional lipids in continuous enzymatic hydrolysis of soybean hair oil by nano-magnetic immobilized enzyme in a three-phase magnetic fluidized bed

Detailed description of the invention:

By applying nano-magnetic immobilized enzyme in a gas-liquid-solid three-phase magnetic fluidized bed reactor, the magnetic immobilized phospholipase is regulated by adjusting the optimal reaction conditions and enzymatic hydrolysis products of different magnetic immobilized phospholipases. In the gas-liquid-solid three-phase magnetic fluidized bed reactor, the different sequences and corresponding reaction conditions make the enzymatic hydrolysate facilitate the formation of functional lipids, and the magnetic fatty enzymes are used to convert the free fatty acids in the enzymatic hydrolysate with phytosterols. Esterification into phytosterol esters, to achieve nano-magnetic immobilized enzyme directed catalyzed continuous production of phospholipid biosynthesis functional lipids in soybean oil.

Example 1

1. Heat the soybean oil to 60 ° C, add buffer to adjust the pH to 7.0, and then transfer the oil mixture to the first-phase three-phase magnetic fluidized bed reactor. At this time, the first-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized phospholipase C in the reactor was 0.70 g/kg, and the reaction time of the oil in the first-phase three-phase magnetic fluidized bed reactor was 4.5 h. After the reaction was completed, the first-phase three-phase magnetic fluidization was carried out. The mixture at the outlet of the bed reactor is centrifuged, and the separated buffer is sent back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; The reaction substrate separated by the first-stage three-phase magnetic fluidized bed reactor is mixed with the buffer to adjust the pH to 6.5, and the reaction temperature is adjusted to 60 ° C. At this time, the nano magnetic fixed in the two-phase three-phase magnetic fluidized bed reactor The addition amount of the phospholipase A ₁ was 0.30 g/kg, and the reaction time of the mixed solution in the second-phase three-phase magnetic fluidized bed was 3.4 h. After the completion of the reaction, the mixed solution of the outlet of the two-stage three-phase magnetic fluidized bed reactor was carried out. Centrifugation, transport the separated buffer back to the secondary buffer Depositing the tank, transferring the separated reaction substrate to the three-stage three-phase magnetic fluidized bed reactor; 3. Mixing the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor with the buffer to adjust the pH value To 7.5, adjust the reaction temperature to 45 ° C, and then add a certain amount of phytosterol. At this time, the amount of nano-magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor is 1.96 g / kg, and the mixture is in three. The reaction time in the magnetic fluidized bed is 2.8h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is sent back to the tertiary buffer storage tank. The separated reaction substrate was transferred to a temporary storage tank. At this time, the phosphorus content in the hair oil was 9.29 mg/kg, and the esterification rate of the plant sterol ester was 93.34%. Other factors use the central combination design experimental method, the design scheme and results are shown in Table 1. It can be seen from Table 1 that for the three nano-magnetic immobilized enzymes, the phosphorus content gradually increases with the increase of the reaction temperature, indicating that the excessive temperature is unfavorable for the three nano-magnetic immobilized enzymes to catalyze the hydrolysis of phospholipids. At the same time, the esterification rate is gradually reduced, which is not conducive to the synthesis of functional lipids. With the increase of pH, the phosphorus content increases gradually, and the esterification rate decreases gradually, which is not conducive to the synthesis of functional lipids. With the prolongation of reaction time, Phosphorus content tends to be flat, and the esterification rate does not change significantly; the comprehensive effect is the most advantageous.

Example 2

1. Heat the soybean oil to 60 ° C, add buffer to adjust the pH to 7.0, and then transfer the oil mixture to the first-phase three-phase magnetic fluidized bed reactor. At this time, the first-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized phospholipase C in the reactor was 0.80 g/kg, and the reaction time of the oil in the first-phase three-phase magnetic fluidized bed reactor was 4.5 h. After the reaction was completed, the first-phase three-phase magnetic fluidization was carried out. The mixture at the outlet of the bed reactor is centrifuged, and the separated buffer is sent back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; The reaction substrate separated by the first-stage three-phase magnetic fluidized bed reactor is mixed with the buffer, the pH is adjusted to 5.5, and the reaction temperature is adjusted to 50 ° C. At this time, the nano magnetic immobilization in the two-phase three-phase magnetic fluidized bed reactor is performed. The addition amount of phospholipase A ₂ is 0.60 g/kg, and the reaction time of the mixed solution in the secondary magnetic fluidized bed is 5.0 h. After the completion of the reaction, the mixed solution of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged. Transfer the separated buffer back to the secondary buffer storage tank The separated reaction substrate is sent to a three-stage three-phase magnetic fluidized bed reactor; three, the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor is mixed with a buffer to adjust the pH to 7.5, The reaction temperature was adjusted to 45 ° C, and then a certain amount of phytosterol was added. At this time, the amount of nano magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor was 1.96 g/kg, and the mixed liquid was in the three-stage magnetic flow. The reaction time in the chemical bed is 2.8h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is returned to the tertiary buffer storage tank, and the separated liquid is separated. The reaction substrate was transferred to a temporary storage tank. At this time, the phosphorus content in the hair oil was 10.12 mg/kg, and the esterification rate of the plant sterol ester was 92.23%. Other factors use the central combination design experimental method, and the design scheme and results are shown in Table 2. It can be seen from Table 2 that for the three nano-magnetic immobilized enzymes, the phosphorus content gradually increases with the increase of the reaction temperature, indicating that the excessive temperature is unfavorable for the three nano-magnetic immobilized enzymes to catalyze the hydrolysis of phospholipids. At the same time, the esterification rate is gradually reduced, which is not conducive to the synthesis of functional lipids. With the increase of pH, the phosphorus content increases gradually, and the esterification rate decreases gradually, which is not conducive to the synthesis of functional lipids. With the prolongation of reaction time, Phosphorus content tends to be flat, and the esterification rate does not change significantly; the comprehensive effect is the most advantageous.

Example 3

1. Heat the soybean oil to 60 ° C, add buffer to adjust the pH to 6.5, and then transfer the oil mixture to the first-stage three-phase magnetic fluidized bed reactor. At this time, the first-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized phospholipase A ₁ in the reactor was 0.10 g/kg, and the reaction time of the hair oil in the first-stage three-phase magnetic fluidized bed reactor was 3.4 h. After the reaction was completed, the first-stage three-phase magnetic flow was obtained. The mixture of the outlet of the chemical reactor is centrifuged, and the separated buffer is sent back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; Mixing the reaction substrate separated from the first-stage three-phase magnetic fluidized bed reactor with the buffer, adjusting the pH to 7.0, and adjusting the reaction temperature to 60 ° C. At this time, the nano-magnetic in the two-phase three-phase magnetic fluidized bed reactor The immobilized phospholipase C was added in an amount of 1.0 g/kg, and the reaction time in the reaction of the mixed solution in the secondary magnetic fluidized bed was 4.5 h. After the completion of the reaction, the mixed solution of the outlet of the two-stage three-phase magnetic fluidized bed reactor was carried out. Centrifugation, transport the separated buffer back to the secondary buffer a storage tank, the separated reaction substrate is sent to a three-stage three-phase magnetic fluidized bed reactor; three, the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor is mixed with a buffer to adjust the pH value. To 7.5, adjust the reaction temperature to 45 ° C, and then add a certain amount of phytosterol, at this time the amount of nano-magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor is 1.83g / kg, the mixture in three The reaction time in the magnetic fluidized bed is 2.8h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is sent back to the tertiary buffer storage tank. The separated reaction substrate was transferred to a temporary storage tank. At this time, the phosphorus content in the hair oil was 9.98 mg/kg, and the esterification rate of the plant sterol ester was 93.48%. Other factors use the central combination design experimental method, the design scheme and results are shown in Table 3. It can be seen from Table 3 that for the three nano-magnetic immobilized enzymes, the phosphorus content gradually increases with the increase of the reaction temperature, indicating that the excessive temperature is unfavorable for the three nano-magnetic immobilized enzymes to catalyze the hydrolysis of phospholipids. At the same time, the esterification rate is gradually reduced, which is not conducive to the synthesis of functional lipids. With the increase of pH, the phosphorus content increases gradually, and the esterification rate decreases gradually, which is not conducive to the synthesis of functional lipids. With the prolongation of reaction time, The phosphorus content tends to be gentle, and the esterification change is not significant; the comprehensive effect is the most advantageous.

Example 4

The soybean oil is heated to 50 ° C, buffer is added to adjust the pH to 5.5, and then the oil mixture is transferred to the first-stage three-phase magnetic fluidized bed reactor, at this time, the first-stage three-phase magnetic fluidized bed reactor The amount of nano-magnetic immobilized phospholipase A ₂ added was 0.2g/kg, and the reaction time of the hair oil in the first-stage three-phase magnetic fluidized bed reactor was 5.0h. After the reaction was completed, the first-stage three-phase magnetic fluidized bed was used. The mixture at the outlet of the reactor is centrifuged, and the separated buffer is sent back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; The reaction substrate separated by the three-phase magnetic fluidized bed reactor is mixed with the buffer, the pH is adjusted to 7.0, and the reaction temperature is adjusted to 60 ° C. At this time, the nano magnetic immobilization in the two-phase three-phase magnetic fluidized bed reactor is performed. The addition amount of phospholipase C was 1.0g/kg, and the reaction time of the mixed solution in the secondary magnetic fluidized bed was 4.5h. After the reaction was completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor was centrifuged. Transfer the separated buffer back to the secondary buffer reservoir a tank, the separated reaction substrate is sent to a three-stage three-phase magnetic fluidized bed reactor; three, the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor is mixed with a buffer to adjust the pH value to 7.5, adjust the reaction temperature to 45 ° C, and then add a certain amount of phytosterol, at this time in the three-stage three-phase magnetic fluidized bed reactor, the amount of nano-magnetic immobilized lipase is 1.81g / kg, the mixture is in the third grade The reaction time in the magnetic fluidized bed is 2.8h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is returned to the tertiary buffer storage tank to separate. The reaction substrate was transferred to a temporary storage tank. At this time, the phosphorus content in the hair oil was 10.19 mg/kg, and the esterification rate of the plant sterol ester was 92.66%. Other factors use the central combination design experimental method, the design scheme and results are shown in Table 4. It can be seen from Table 4 that for the three nano-magnetic immobilized enzymes, the phosphorus content gradually increases with the increase of the reaction temperature, indicating that the excessive temperature is unfavorable for the three nano-magnetic immobilized enzymes to catalyze the hydrolysis of phospholipids. At the same time, the esterification rate is gradually reduced, which is not conducive to the synthesis of functional lipids. With the increase of pH, the phosphorus content increases gradually, and the esterification rate decreases gradually, which is not conducive to the synthesis of functional lipids. With the prolongation of reaction time, Phosphorus content tends to be flat, and the esterification rate does not change significantly; the comprehensive effect is the most advantageous.

The phospholipid biosynthesis functional lipids in soybean oil were continuously directed by the nanomagnetic immobilized enzymes in Examples 1-4, and the DAG content in the finished product was about 1.65%, and the phytosterol ester content was about 1.16%.

Example 5

1. Heat the soybean oil to 60 ° C, add buffer to adjust the pH to 6.5, and then transfer the oil mixture to the first-stage three-phase magnetic fluidized bed reactor. At this time, the first-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized phospholipase A ₁ in the reactor was 0.10 g/kg, and the reaction time of the mixed solution in the first-order magnetic fluidized bed was 3.4 h. After the reaction was completed, the first-phase three-phase magnetic fluidized bed reaction was carried out. The mixture at the outlet of the vessel is centrifuged, and the separated buffer is returned to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; The reaction substrate separated by the three-phase magnetic fluidized bed reactor is mixed with the buffer, the pH is adjusted to 7.5, the reaction temperature is adjusted to 45 ° C, and then a certain amount of phytosterol is added, at this time, the two-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized lipase in the reactor is 5.0g/kg, and the reaction time of the mixture in the secondary magnetic fluidized bed is 2.8h. After the reaction is completed, the mixture of the two-phase magnetic fluidized bed reactor is mixed. The liquid is centrifuged and the separated buffer is Returning to the secondary buffer storage tank, the separated reaction substrate is transported to the temporary storage tank. At this time, the phosphorus content in the crude oil is 9.92 mg/kg, and the esterification rate of the phytosterol ester is 92.72%, and the phytosterol ester The content is about 2.5%. Other factors use the central combination design experimental method, the design scheme and results are shown in Table 5. It can be seen from Table 5 that for the three nano-magnetic immobilized enzymes, the phosphorus content gradually increases with the increase of the reaction temperature, indicating that the excessive temperature is unfavorable for the enzymatic hydrolysis of the two nano-magnetic immobilized enzymes. At the same time, the esterification rate is gradually reduced, which is not conducive to the synthesis of functional lipids. With the increase of pH, the phosphorus content increases gradually, and the esterification rate decreases gradually, which is not conducive to the synthesis of functional lipids. With the prolongation of reaction time, Phosphorus content tends to be flat, and the esterification rate does not change significantly; the comprehensive effect is the most advantageous.

Example 6

1. Heat the soybean oil to 50 ° C, add buffer to adjust the pH to 5.5, and then transfer the oil mixture to the first-stage three-phase magnetic fluidized bed reactor. At this time, the first-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized phospholipase A ₂ in the reactor was 0.2g/kg, and the reaction time of the mixed solution in the first-order magnetic fluidized bed was 5.0h. After the reaction was completed, the first-phase three-phase magnetic fluidized bed reaction was carried out. The mixture at the outlet of the vessel is centrifuged, and the separated buffer is returned to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; The reaction substrate separated by the three-phase magnetic fluidized bed reactor is mixed with the buffer, the pH is adjusted to 7.5, the reaction temperature is adjusted to 45 ° C, and then a certain amount of phytosterol is added, at this time, the two-phase three-phase magnetic fluidized bed The amount of nano-magnetic immobilized lipase in the reactor was 3.80g/kg, and the reaction time of the mixture in the secondary magnetic fluidized bed was 2.8h. After the reaction was completed, the mixture of the two-phase magnetic fluidized bed reactor was mixed. The liquid is centrifuged and the separated buffer is Returning to the secondary buffer storage tank, the separated reaction substrate is transported to the temporary storage tank. At this time, the phosphorus content in the hair oil is 10.28 mg/kg, and the esterification rate of the phytosterol ester is 92.78%. The content is about 2.0%. Other factors use the central combination design experimental method, and the design scheme and results are shown in Table 6. It can be seen from Table 6 that for the three nano-magnetic immobilized enzymes, the phosphorus content gradually increases with the increase of the reaction temperature, indicating that the excessive temperature is unfavorable for the effect of the two nano-magnetic immobilized enzymes to catalyze the hydrolysis of phospholipids. At the same time, the esterification rate is gradually reduced, which is not conducive to the synthesis of functional lipids. With the increase of pH, the phosphorus content increases gradually, and the esterification rate decreases gradually, which is not conducive to the synthesis of functional lipids. With the prolongation of reaction time, Phosphorus content tends to be flat, and the esterification rate does not change significantly; the comprehensive effect is the most advantageous.

Table I

Table II

Table 3

Table 4

Table 5

Table 6

Drawing

1. The process of enzymatic hydrolysis of PA in soybean oil by phospholipase A ₁

2. The process of enzymatic hydrolysis of PA in soybean oil by phospholipase A ₂

3. Phospholipase C enzymatic hydrolysis of PC to DAG in soybean oil

4. Process of esterification of FFA with phytosterols to phytosterol esters

5. Nano magnetic immobilized enzyme continuous enzymatic hydrolysis of soybean phospholipids in a three-phase magnetic fluidized bed to produce functional lipids

In the fluidization operation of the nano-magnetic immobilized enzyme in the three-phase magnetic fluidized bed, the first-stage soybean oil in the clean oil tank is sent to the three-stage three-phase magnetic fluidized bed reaction system for self-circulation, and the DC power source is turned on. According to the design scheme, the magnetic immobilized phospholipase C, the magnetic immobilized phospholipase A ₁ , the magnetic immobilized phospholipase A ₂ and the magnetic immobilized lipase particles were respectively added to the three-phase three-phase magnetic current from the ports on the magnetic fluidized bed. The chemical bed, open the outlet valve of the air compressor, open the hot water circulation system, ensure the reaction temperature of the system, and adjust the operating parameters to achieve the stable flow state of the nano-magnetic immobilized enzyme in the three-stage three-phase magnetic fluidized bed reaction system. Chemical.

The nano magnetic immobilized enzyme granule catalyzes the phospholipid process in the soybean oil in a three-phase magnetic fluidized bed, and pumps a certain amount of soybean oil from the oil tank to the buffer tank, closes the valve of the net tank, and starts the buffer. The valve of the tank, the oil is mixed with the hot water by a constant flow pump and a certain proportion through the static mixer into the first-order magnetic fluidized bed, and the different enzymatic hydrolysis time is controlled according to the height of the magnetic fluidized bed, and the corresponding After the enzymatic hydrolysis time, it is separated from the first-order magnetic fluidized bed and then enters the secondary magnetic fluidized bed. The different enzymatic hydrolysis time is controlled according to the height of the magnetic fluidized bed. After the corresponding enzymatic hydrolysis time, the oil is obtained. After the secondary magnetic fluidized bed is separated, it enters the three-stage magnetic fluidized bed. The different enzymatic hydrolysis time is controlled according to the height of the magnetic fluidized bed. After the corresponding enzymatic hydrolysis time is reached, the separated reaction substrate is transported. To the temporary storage tank, complete a cyclic reaction, and then use a double enzymatic hydrolysis process to realize the continuous production of phospholipid-forming functional lipids in the continuous enzymatic hydrolysis of soybean hair oil by the nano-magnetic immobilized enzyme in a three-phase magnetic fluidized bed. .

Example 5-6 uses a two-phase three-phase magnetic fluidized bed to catalyze the process of phospholipids in soybean oil.

Claims

The method for continuously catalyzing the phospholipid biosynthesis of functional lipids in soybean oil by nano magnetic immobilized enzyme is characterized in that it is achieved by the following steps: using soybean oil obtained by filtering to remove impurities; magnetic immobilized phospholipase is applied in each In the three-phase magnetic fluidized bed, as a stationary phase, it is distributed in a magnetically stable fluidized state, with soybean oil as a continuous mobile phase, and the oil flows through a magnetic fluidized bed reactor of different types of magnetic immobilized enzymes. Contacting phospholipids in catalyzed soybean oil without mechanical agitation to produce corresponding products; magnetic immobilized lipase is immobilized in a three-phase magnetic fluidized bed, and FFA and phytosterol in the substrate are added by adding phytosterol Esterification into phytosterol esters enables the conventional removal process to be a process of biosynthesizing functional lipids without mechanical agitation, thereby achieving continuous production.
The method for continuously catalyzing phospholipid biosynthesis of functional lipids in soybean oil by the nanomagnetic immobilized enzyme according to claim 1, characterized in that firstly, the soybean oil which is filtered and removed is heated to 60 ° C, and buffer is adjusted. After the pH value is 7.0, the oil mixture is transferred to the first-stage three-phase magnetic fluidized bed reactor. At this time, the amount of nano-magnetic immobilized phospholipase C in the first-stage three-phase magnetic fluidized bed reactor is 0.70. g/kg, the reaction time of the oil in the first-stage three-phase magnetic fluidized bed reactor is 4.5h. After the reaction is completed, the mixture of the outlet of the first-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated liquid is separated. The buffer is transported back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; secondly, the reaction substrate separated from the first-stage three-phase magnetic fluidized bed reactor Mixing with the buffer, adjusting the pH to 6.5, adjusting the reaction temperature to 60 ° C, at this time, the amount of nano magnetic immobilized phospholipase A 1 in the two-phase three-phase magnetic fluidized bed reactor is 0.30 g / kg, the mixture In the second magnetic fluidized bed After 3.4h, after the reaction is completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated buffer is sent back to the secondary buffer storage tank to transport the separated reaction substrate. To the three-stage three-phase magnetic fluidized bed reactor; finally, the reaction substrate separated from the two-stage three-phase magnetic fluidized bed reactor is mixed with the buffer, the pH is adjusted to 7.5, the reaction temperature is adjusted to 45 ° C, and then added. A certain amount of phytosterol, at this time, the amount of nano-magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor was 1.96 g/kg, and the reaction time of the mixed solution in the three-stage magnetic fluidized bed was 2.8 h. After completion, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is sent back to the tertiary buffer storage tank, and the separated reaction substrate is transferred to the temporary storage tank. At this time, the phosphorus content in the hair oil was 9.29 mg/kg, and the esterification rate of the plant sterol ester was 93.34%.
The method for continuously catalyzing phospholipid biosynthesis of functional lipids in soybean oil by the nanomagnetic immobilized enzyme according to claim 1, characterized in that firstly, the soybean oil which is filtered and decontaminated is heated to a temperature of 60 ° C, and a buffer is added. Adjusting the pH to 7.0, and then transferring the oil mixture to the first-stage three-phase magnetic fluidized bed reactor. At this time, the amount of nano-magnetic immobilized phospholipase C in the first-stage three-phase magnetic fluidized bed reactor is 0.80g/kg, the reaction time of the oil in the first-stage three-phase magnetic fluidized bed reactor is 4.5h. After the reaction is completed, the mixture of the first-stage three-phase magnetic fluidized bed reactor outlet is centrifuged and separated. The buffer is transported back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; secondly, the reaction bottom separated by the first-stage three-phase magnetic fluidized bed reactor The substance is mixed with the buffer, the pH is adjusted to 5.5, and the reaction temperature is adjusted to 50 ° C. At this time, the amount of the nano-magnetic immobilized phospholipase A 2 in the two-phase three-phase magnetic fluidized bed reactor is 0.60 g/kg, mixed. Liquid in a secondary magnetic fluidized bed The reaction time is 5.0h. After the reaction is completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated buffer is sent back to the secondary buffer storage tank, and the separated reaction bottom is separated. The material is transported to a three-stage three-phase magnetic fluidized bed reactor; finally, the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor is mixed with a buffer to adjust the pH to 7.5, and the reaction temperature is adjusted to 45 ° C. Then, a certain amount of phytosterol was added. At this time, the amount of nano-magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor was 1.96 g/kg, and the reaction time of the mixed solution in the three-stage magnetic fluidized bed was 2.8 h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is returned to the tertiary buffer storage tank, and the separated reaction substrate is transported to the temporary storage. In the can, the phosphorus content in the hair oil was 10.12 mg/kg, and the esterification rate of the plant sterol ester was 92.23%.
The method for continuously catalyzing phospholipid biosynthesis of functional lipids in soybean oil by the nanomagnetic immobilized enzyme according to claim 1, characterized in that firstly, the soybean oil which is filtered and removed is heated to 60 ° C, and buffer is adjusted. After the pH value is 6.5, the oil mixture is transferred to the first-stage three-phase magnetic fluidized bed reactor. At this time, the amount of nano-magnetic immobilized phospholipase A 1 in the first-stage three-phase magnetic fluidized bed reactor is 0.10g/kg, the reaction time of the oil in the first-stage three-phase magnetic fluidized bed reactor is 3.4h. After the reaction is completed, the mixture of the first-stage three-phase magnetic fluidized bed reactor outlet is centrifuged and separated. The buffer is transported back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; secondly, the reaction bottom separated by the first-stage three-phase magnetic fluidized bed reactor The substance is mixed with the buffer, the pH is adjusted to 7.0, and the reaction temperature is adjusted to 60 ° C. At this time, the amount of the nano magnetic immobilized phospholipase C in the two-phase three-phase magnetic fluidized bed reactor is 1.0 g/kg, and the mixture is mixed. In the second magnetic fluidized bed The reaction time is 4.5h, and after the reaction is completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated buffer is sent back to the secondary buffer storage tank, and the separated reaction bottom is separated. The material is transported to a three-stage three-phase magnetic fluidized bed reactor; finally, the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor is mixed with a buffer to adjust the pH to 7.5, and the reaction temperature is adjusted to 45 ° C. Then, a certain amount of phytosterol is added. At this time, the amount of nano-magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor is 1.83 g/kg, and the reaction time of the mixed solution in the three-stage magnetic fluidized bed is 2.8 h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is returned to the tertiary buffer storage tank, and the separated reaction substrate is transported to the temporary storage. In the can, the phosphorus content in the hair oil was 9.98 mg/kg, and the esterification rate of the plant sterol ester was 93.48%.
The method for continuously catalyzing the phospholipid biosynthesis of functional lipids in soybean oil by the nanomagnetic immobilized enzyme according to claim 1, characterized in that firstly, the soybean oil which is filtered and decontaminated is heated to 50 ° C, and buffer adjustment is added. After the pH value is 5.5, the oil mixture is transferred to the first-stage three-phase magnetic fluidized bed reactor. At this time, the amount of nano-magnetic immobilized phospholipase A 2 in the first-stage three-phase magnetic fluidized bed reactor is 0.2g/kg, the reaction time of the oil in the first-stage three-phase magnetic fluidized bed reactor is 5.0h. After the reaction is completed, the mixture of the first-stage three-phase magnetic fluidized bed reactor outlet is centrifuged and separated. The buffer is transported back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; secondly, the reaction bottom separated by the first-stage three-phase magnetic fluidized bed reactor The substance is mixed with the buffer, the pH is adjusted to 7.0, and the reaction temperature is adjusted to 60 ° C. At this time, the amount of the nano magnetic immobilized phospholipase C in the two-phase three-phase magnetic fluidized bed reactor is 1.0 g/kg, and the mixture is mixed. In the second magnetic fluidized bed The reaction time is 4.5h, and after the reaction is completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated buffer is sent back to the secondary buffer storage tank, and the separated reaction bottom is separated. The material is transported to a three-stage three-phase magnetic fluidized bed reactor; finally, the reaction substrate separated from the two-phase three-phase magnetic fluidized bed reactor is mixed with a buffer to adjust the pH to 7.5, and the reaction temperature is adjusted to 45 ° C. Then, a certain amount of phytosterol was added. At this time, the amount of nano-magnetic immobilized lipase in the three-stage three-phase magnetic fluidized bed reactor was 1.81 g/kg, and the reaction time of the mixed solution in the three-stage magnetic fluidized bed was 2.8 h. After the reaction is completed, the mixture of the three-stage three-phase magnetic fluidized bed reactor outlet is centrifuged, and the separated buffer is returned to the tertiary buffer storage tank, and the separated reaction substrate is transported to the temporary storage. In the can, the phosphorus content in the hair oil was 10.19 mg/kg, and the esterification rate of the plant sterol ester was 92.66%.
The method for continuously catalyzing phospholipid biosynthesis of functional lipids in soybean oil by the nanomagnetic immobilized enzyme according to claim 1, characterized in that firstly, the soybean oil which is filtered and decontaminated is heated to a temperature of 60 ° C, and a buffer is added. Adjusting the pH to 6.5, and then transferring the oil mixture to the first-stage three-phase magnetic fluidized bed reactor. At this time, the amount of nano-magnetic immobilized phospholipase A 1 in the first-stage three-phase magnetic fluidized bed reactor is increased. The reaction time is 0.10g/kg, and the reaction time in the first-stage magnetic fluidized bed is 3.4h. After the reaction is completed, the mixture of the outlet of the first-stage three-phase magnetic fluidized bed reactor is centrifuged to separate the buffer. The liquid is transported back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; then the reaction substrate separated from the first-stage three-phase magnetic fluidized bed reactor is discharged. Mix with the buffer, adjust the pH to 7.5, adjust the reaction temperature to 45 ° C, and then add a certain amount of phytosterol. At this time, the amount of nano-magnetic immobilized lipase in the two-phase three-phase magnetic fluidized bed reactor is 5.0. g/kg, The reaction time of the mixed solution in the secondary magnetic fluidized bed is 2.8 h. After the reaction is completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated buffer is returned to the secondary buffer. The storage tank transports the separated reaction substrate to the temporary storage tank. At this time, the phosphorus content in the hair oil is 9.92 mg/kg, the esterification rate of the plant sterol ester is 92.72%, and the phytosterol ester content is about 2.5%.
The method for continuously catalyzing the phospholipid biosynthesis of functional lipids in soybean oil by the nanomagnetic immobilized enzyme according to claim 1, characterized in that the soybean oil which is filtered and decontaminated is first heated to a temperature of 50 ° C, and a buffer is added. Adjusting the pH to 5.5, and then transferring the oil mixture to the first-stage three-phase magnetic fluidized bed reactor. At this time, the amount of nano-magnetic immobilized phospholipase A 2 in the first-stage three-phase magnetic fluidized bed reactor is increased. 0.2g/kg, the reaction time in the reaction of the mixed liquid in the first-class magnetic fluidized bed is 5.0h. After the reaction is completed, the mixture of the outlet of the first-stage three-phase magnetic fluidized bed reactor is centrifuged to separate the buffer. The liquid is transported back to the primary buffer storage tank, and the separated reaction substrate is sent to the secondary three-phase magnetic fluidized bed reactor; then the reaction substrate separated from the first-stage three-phase magnetic fluidized bed reactor is discharged. Mix with the buffer, adjust the pH to 7.5, adjust the reaction temperature to 45 ° C, and then add a certain amount of phytosterol. At this time, the amount of nano-magnetic immobilized lipase in the two-phase three-phase magnetic fluidized bed reactor is 3.80. g/kg, The reaction time of the mixed solution in the secondary magnetic fluidized bed is 2.8 h. After the reaction is completed, the mixture of the outlet of the two-stage three-phase magnetic fluidized bed reactor is centrifuged, and the separated buffer is returned to the secondary buffer. The storage tank transports the separated reaction substrate to the temporary storage tank. At this time, the phosphorus content in the hair oil is 10.28 mg/kg, the esterification rate of the plant sterol ester is 92.78%, and the phytosterol ester content is about 2.0%.