Enzymatic pretreatment process of deodorized distillate and its application in purification of natural vitamin e

Abstract

The present application relates to the technical field of biological chemical industry, and particularly relates to an enzyme pretreatment process of deodorized distillate and application thereof in natural vitamin E purification. The enzyme pretreatment process comprises the following steps: firstly, adding lipase into the deodorized distillate, and adding a certain mass of methanol through a constant flow pump, and then, when the acid value of the system is reduced to 6-8 mgKOH / g, vacuum water removal is performed to promote the forward reaction, and then, methanol is continuously added to perform methyl esterification reaction until the acid value of the system is reduced to less than 3 mgKOH / g. The process has mild reaction conditions, low requirement on equipment conditions, good acid resistance of the used lipase, no need for carrier immobilization, greatly reduced reaction cost, simple operation steps, no need for introducing a second lipase to achieve the natural vitamin E purification pretreatment standard, no acid wastewater discharge, green environmental protection, and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to an enzymatic pretreatment process for deodorized distillates and its application in the purification of natural vitamin E. Background Technology

[0002] Deodorized distillate is an important raw material for extracting natural vitamin E. However, since the boiling points and solubility of various components in deodorized distillate are similar, it is difficult to extract natural vitamin E directly from deodorized distillate. Pretreatment of the raw material is required. Generally, the free fatty acids and glycerides in the deodorized distillate are subjected to transesterification and esterification reactions to convert them into fatty acid methyl esters with lower boiling points. At the same time, the viscosity of the entire system is reduced, which helps to improve product purity and reduce production costs.

[0003] Acid catalysis is a commonly used pretreatment process for deodorized distillates, characterized by short reaction times and high esterification efficiency. Common acid catalysts include sulfuric acid, superphosphoric acid, and sulfonic acid. However, acid catalysis places high demands on the reactor, requiring strong corrosion resistance. After the reaction, multiple cleaning steps are needed to remove unreacted catalyst, and the catalyst cannot be separated and reused, leading to environmental pollution after discharge.

[0004] Enzymatic pretreatment of deodorized distillates offers advantages such as mild conditions, high efficiency and specificity, low energy consumption, high yield, and easy product separation and purification. However, current enzymatic pretreatment has a lower conversion rate than chemical acid catalysis, and the acid value of the pretreated product exceeds 5 mg KOH / g. In the purification and enrichment process of natural vitamin E, the final acid value after esterification is generally controlled below 3 mg KOH / g; otherwise, it will affect the efficiency of subsequent molecular distillation steps. Therefore, enzymatic pretreatment is often combined with chemical methods or a process involving the addition of lipase for secondary methyl esterification. Although this significantly reduces the amount of chemical catalysts such as sulfuric acid, it still presents problems such as acid wastewater discharge, cumbersome procedures, and high costs. Therefore, there is an urgent need to develop a one-step enzymatic pretreatment process for the purification of natural vitamin E that is highly efficient, easy to operate, and low-cost. Summary of the Invention

[0005] In order to solve the problems of the prior art, the present invention provides an enzymatic pretreatment process for deodorized distillates, which can reduce the acid value of deodorized distillates and thus promote their application in the purification of natural vitamin E.

[0006] The enzymatic pretreatment process for deodorized distillate used in this invention mainly includes the following steps: (1) First stage of enzyme reaction: Deodorized distillate and lipase are added to the reaction vessel, and methanol is added by constant flow pump at a certain rate. The temperature is controlled at 30-40℃ and the reaction is carried out for 6-8 hours. (2) Stop adding methanol and dehydrate the reaction solution under vacuum for 30 minutes using a vacuum pump; (3) Second stage enzyme reaction: The reaction solution after dehydration in step (2) is continuously fed with methanol by vacuum pump at a certain rate, and the temperature is controlled at 30-40℃ for 2 hours.

[0007] The amino acid sequence of the lipase described in step (1) is SEQ ID NO:7.

[0008] The deodorized distillate mentioned in step (1) is preferably any one or more of the following: soybean deodorized distillate, corn deodorized distillate, peanut deodorized distillate, rapeseed deodorized distillate, flaxseed deodorized distillate, and sesame deodorized distillate.

[0009] The mass ratio of the deodorized distillate to the lipase in step (1) is 100:1.5-3.

[0010] The amount of methanol added in step (1) is 15-20% of the amount of the deodorized distillate.

[0011] In step (2), the temperature of the reactor is adjusted to 60°C and the vacuum degree is -0.1 MPa during vacuum dehydration.

[0012] The amount of methanol added in step (3) is 2-5% of the amount of the deodorized distillate.

[0013] More preferably, the present invention provides an enzymatic pretreatment process for deodorized distillates, comprising the following steps: (1) First stage of enzyme reaction: Add deodorized distillate to the reactor, adjust the temperature to 40°C, turn on the stirrer for preheating, and after preheating for 10 min, add lipase at 1.5% of the mass of deodorized distillate; close the reactor, and add methanol at a certain rate through a constant flow pump within 6 h, with the amount of methanol added being 15% of the mass of deodorized distillate; control the temperature at 40°C and react for 6 h; (2) Stop adding methanol, and dehydrate the reaction solution under vacuum using a vacuum pump at a temperature of 60°C and a vacuum degree of -0.1 MPa for 30 min. (3) Second stage enzyme reaction: The reaction solution after dehydration in step (2) is continuously fed with methanol at a certain rate through a vacuum pump. The amount of methanol added is 5% of the amount of deodorized distillate. The temperature is controlled at 40℃ and the reaction is carried out for 2 hours.

[0014] After the deodorized distillate is pretreated by the above-mentioned enzymatic method, the acid value is reduced to below 3 mg KOH / g. After further sedimentation, cold precipitation and distillation purification processes, purified natural vitamin E can be obtained.

[0015] The beneficial effects of this invention are as follows: Compared with traditional chemical methods, the enzymatic pretreatment process provided by this invention has mild reaction conditions, simple steps, low requirements for equipment conditions, no acid wastewater discharge, reduces the difficulty of concentrating and refining natural vitamin E, and gives full play to the green and environmentally friendly characteristics of enzymatic methods.

[0016] Compared with existing enzymatic processes, the lipase mutant used in this invention has high acid tolerance and can react without immobilization, greatly reducing reaction costs. Furthermore, vacuum dehydration removes water generated during the reaction, promoting the forward enzymatic reaction. This eliminates the need for combining with chemical methods or introducing a second lipase, resulting in a final acid value below 3 mg KOH / g, meeting the standard requirements for natural vitamin E pretreatment. Detailed Implementation

[0017] The present invention will be further described in conjunction with specific implementation examples, which will enable those skilled in the art to better understand and master the invention, rather than limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0018] The method for determining lipase activity in this embodiment of the invention is as follows: (1) Definition of enzyme activity unit According to GB / T23535-2009, the amount of enzyme required to generate 1 μmol of fatty acid per minute under the conditions of 40℃ and pH 7.5 is defined as one enzyme activity unit, denoted by U.

[0019] (2) Measurement method Olive oil and 4% (w / v) polyvinyl alcohol (PVA) were mixed at a ratio of 1:3 (v / v) and homogenized twice using a high-speed homogenizer, with an interval of 5 min and each treatment lasting 3 min, to obtain a milky white PVA emulsion. The emulsified olive oil was used as the substrate for lipase hydrolysis. Two 100 mL beakers were prepared. 4 mL of olive oil emulsion and 5 mL of citrate-phosphate buffer were added to each beaker (B), one blank beaker (A) and one sample beaker (B). 15 mL of 95% ethanol was added to beaker A. The mixture was preheated in a 40℃ water bath for 5 min. Then, 1 mL of the enzyme solution to be tested was added to each beaker (A) and B. The mixture was immediately mixed and the reaction time was set. After 15 min of reaction time, 15 mL of 95% ethanol was immediately added to beaker B to terminate the reaction. The beakers were then removed. The beakers were placed on a magnetic stirrer and titrated with 0.05 mol / L sodium hydroxide standard solution while stirring until a faint red color appeared and remained unchanged for 30 s. The volume of sodium hydroxide standard solution consumed was recorded.

[0020] Enzyme activity calculation formula:

[0021] In the formula: X D V1 represents the enzyme activity of the sample (U / mL); V2 represents the volume of sodium hydroxide standard solution consumed during sample titration (mL); V3 represents the volume of sodium hydroxide standard solution consumed during blank titration (mL); c represents the concentration of sodium hydroxide standard solution (mol / L); 50 represents the equivalent of 50 μmol of fatty acid in 1 mL of 0.05 mol / L sodium hydroxide solution; n1 represents the dilution factor of the sample; 0.05 represents the conversion factor for sodium hydroxide standard solution concentration; and 1 / 15 represents the reaction time (15 min). Example 1: Screening of lipase mutants The amino acid sequence of the lipase TLS is SEQ ID NO: 1, and its encoding nucleotide sequence is SEQ ID NO: 2. It was artificially synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0022] To improve the acid resistance of lipase TLS, the applicant screened a large number of mutation sites in the gene without destroying the protein's secondary structure and active site. Using lipase TLS as a template, PCR amplification was performed using the aforementioned primers with the GeneMorphII random mutagenesis PCR kit (Stratagene). The PCR product was recovered from the gel, digested with enzymes, and ligated into the pET-28a vector digested with the same enzymes. The transformed cells were then transformed into E. coli BL21(DE3) and plated on LB+Kana plates (0.5% yeast extract, 1% peptone, 1% NaCl, 50 μg / mL kanamycin, pH 7.0). The plates were incubated upside down at 37°C. After the transformants appeared, they were picked one by one into a 96-well plate with a toothpick. 150 μL of LB+Kana medium containing 0.1 mM IPTG was added to each well. The plates were incubated at 37°C and 220 rpm for about 6 hours. The supernatant was discarded by centrifugation, and the cells were resuspended in buffer. The cells were repeatedly freeze-thawed to break up the cell walls and obtain E. coli cell lysate containing lipase.

[0023] 40 μL of lysis buffer was transferred to two new 96-well plates, and the lipase activity was measured after reacting at pH 3.0 and pH 5.0 for 15 min, respectively. The results showed that some mutants did not exhibit any change in enzyme activity under acidic conditions; some mutations even worsened their acid tolerance or enzyme activity; and some mutations, while improving the pH tolerance of lipase TLS, significantly altered the enzyme's active properties, which did not meet the requirements. Finally, the mutation sites that significantly improved the acid tolerance of lipase TLS without affecting its enzyme activity and original enzymatic properties were obtained: G113V, T136W, and I277P.

[0024] The lipase mutant containing the T136W single-point mutation was named TLS-M1, and its amino acid sequence is SEQ ID NO:3. Based on this sequence, a coding nucleotide sequence was synthesized as SEQ ID NO:4.

[0025] The lipase mutant containing the two-point mutation G113V / T136W was named TLS-M2, and its amino acid sequence is SEQ ID NO:5. Based on this sequence, a coding nucleotide sequence was synthesized as SEQ ID NO:6.

[0026] The lipase mutant containing the G113V / T136W / I277P three-point mutation was named TLS-M3, and its amino acid sequence is SEQ ID NO:7. Based on this sequence, a coding nucleotide sequence was synthesized as SEQ ID NO:8.

[0027] Example 2 Construction of Pichia pastoris engineered strains 2.1 Construction of Recombinant Plasmids Using the genes of the aforementioned lipase TLS and its mutants as templates, the PCR products were amplified, recovered by gel electrophoresis, digested with EcoRI and NotI, and ligated into the pPIC-9k vector digested with the same enzymes overnight at 16°C. The ligation was then performed on E. coli DH5α, plated on LB+Amp plates (0.5% yeast extract, 1% peptone, 1% NaCl, 100 μg / mL ampicillin, pH 7.0), and incubated upside down at 37°C. After the transformants appeared, positive clones were verified by colony PCR, and the correct recombinant plasmid was finally obtained after sequencing verification.

[0028] 2.2 Preparation of yeast competent cells Pichia pastoris strain GS115 was activated on YPD plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar) and cultured at 30℃ for 48 h. Afterward, activated GS115 single clones were inoculated into 5 mL of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) and cultured at 30℃ and 220 rpm for approximately 18 h. The bacterial culture was then transferred to Erlenmeyer flasks containing 50 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for approximately 5 h. Cell density was measured using a UV spectrophotometer. Once the OD600 value was in the range of 1.1–1.3, the cells were centrifuged at 4℃ and 6000 rpm for 3 min, and 5 mL of the cells were collected into sterile EP tubes. The supernatant was gently discarded, and the remaining supernatant was blotted dry with sterile filter paper. The cells were then resuspended in 2 mL of pre-cooled sterile water and incubated at 4℃. Centrifuge at 6000 rpm for 3 min, gently discard the supernatant, and resuspend the bacterial cells in 2 mL of pre-cooled sorbitol (1 mol / L); centrifuge at 6000 rpm for 3 min at 4℃, gently discard the supernatant, and gently resuspend the bacterial cells in 100-150 μl of pre-cooled sorbitol (1 mol / L).

[0029] 2.3 Conversion and Screening The recombinant plasmids of the constructed lipase TLS and its mutants were linearized with Sac I. After purification and recovery, the linearized fragments were transformed into Pichia pastoris GS115 by electroporation. The plasmids were then processed on MD plates (2% glucose, 1.34% YNB, 4×10⁻⁶ oz.). -5 Recombinant Pichia pastoris strains were obtained by screening on 2% biotin and 2% agar. Single transformants were then transferred to BMGY medium (2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4×10⁻⁶ ppm). -5 After incubating in a solution of 1% biotin and 1% glycerol at 30°C with shaking at 250 rpm for 1 day, the culture was transferred to BMMY medium (2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 × 10⁻⁶ ppm). -5 The cells were cultured in a solution of 1% biotin and 0.5% methanol at 30°C with shaking at 250 rpm, with 0.5% methanol added daily. After 3 days of induction, the cells were removed by centrifugation to obtain the supernatant, and the lipase activity was measured.

[0030] The results showed that the enzyme activity of lipase in the fermentation supernatant of the recombinant Pichia pastoris strains expressing lipase TLS and its mutants constructed in this invention was 176-225 U / ml.

[0031] Example 3: Acidity Resistance Analysis of Lipase Mutants The supernatant of Pichia pastoris fermentation of the recombinant lipase and its mutant obtained above was added to citrate-phosphate buffer at pH 3.0. After 15 min, the residual viability of the lipase was detected. The relative enzyme activity was calculated with the initial enzyme activity as 100% relative enzyme activity (see Table 1).

[0032] Table 1. Acidity analysis of lipase mutants Wild TLS - 3.20% mutant TLS-M1 T136W 30.20% mutant TLS-M2 G113V / T136W 38.80% mutant TLS-M3 G113V / T136W / I277P 44.33% As shown in Table 1, the lipase mutants provided by this invention exhibit significantly enhanced tolerance to acidic conditions compared to the wild-type lipase TLS. After treatment at pH 3.0 for 15 minutes, the residual enzyme activity of the mutants still reached 30.20%-44.33%, far exceeding that of the wild type. Among them, the lipase mutant containing the G113V / T136W / I277P three-point mutation had the highest residual enzyme activity, reaching 44.33%, achieving an unexpected technical effect.

[0033] Example 4: Fermentation Production Method of Lipase The Pichia pastoris strain that produced the recombinant lipase mutant TLS-M3 obtained in Example 2 was fermented in a 20-liter fermenter.

[0034] The fermentation medium formula is as follows: calcium sulfate 1.2 g / L, potassium sulfate 20.5 g / L, magnesium sulfate 13 g / L, phosphate 40 g / L, potassium hydroxide 2 g / L, and defoamer 0.05%. Fermentation production process: temperature 30℃, pH value 4.7, stirring speed 400rpm, ventilation volume 1.0-1.5 (v / v), dissolved oxygen controlled above 20%.

[0035] The entire fermentation process is divided into three stages: the first stage is the cell culture stage, where seed culture is introduced at a ratio of 7% and cultured at 30℃ for 24 hours, marked by the depletion of glucose; the second stage is the starvation stage, where no carbon source is added after glucose is depleted, and the stage ends when dissolved oxygen rises above 60%, lasting approximately 30 minutes; the third stage is the induction expression stage, where methanol is added for induction, and dissolved oxygen is maintained above 20%, with a culture time of 180 hours. After fermentation, the fermentation broth is processed through a plate and frame filter press to obtain crude enzyme solution.

[0036] The enzyme activity test results showed that the enzyme activity of the lipase mutant TLS-M3 in the crude enzyme solution obtained from the above fermentation production was 2170 U / mL.

[0037] Example 5 Enzymatic pretreatment process for deodorized distillate Accurately weigh 100g of soybean deodorized distillate (acid value 90 mgKOH / g) and add it to a 250mL reactor. Adjust the temperature to 40℃ and start stirring for preheating. After 10 minutes of preheating, add 2.5mL of crude lipase TLS1 solution. Close the reactor and add 18g of methanol over 6 hours using a constant flow pump at a controlled rate. Determine the acid value of the reaction solution using the medium-heat ethanol method according to national standard GB 5009.229-2016. The acid value is 7.2 mgKOH / g. Pause the reaction and connect the reactor to a vacuum pump. Adjust the temperature to 60℃ and perform vacuum dehydration for 30 minutes. After dehydration, adjust the temperature to 40℃ and add 2g of methanol over 2 hours using a constant flow pump at a controlled rate. The reaction is then complete, and the acid value of the reaction system is determined to be 2.9 mgKOH / g.

[0038] Example 6 Enzymatic pretreatment process for deodorized distillate Accurately weigh 100g of soybean deodorized distillate (acid value 91.5 mgKOH / g) and add it to a 250mL reactor. Adjust the temperature to 40℃ and start stirring for preheating. After 10 minutes of preheating, add 2.5mL of crude lipase TLS2 solution. Close the reactor and add 15g of methanol at a constant flow rate over 6 hours. Determine the acid value of the reaction solution using the medium-heat ethanol method according to national standard GB 5009.229-2016. The acid value is 5.9 mgKOH / g. Pause the reaction and connect the reactor to a vacuum pump. Adjust the temperature to 60℃ and perform vacuum dehydration for 30 minutes. After dehydration, adjust the temperature to 40℃ and add 5g of methanol at a constant flow rate over 2 hours. The reaction is then complete, and the acid value of the reaction system is determined to be 2.5 mgKOH / g.

[0039] Example 7 Enzymatic pretreatment process for deodorized distillate Accurately weigh 100g of soybean deodorized distillate (acid value 96.5mgKOH / g) and add it to a 250mL reactor. Adjust the temperature to 40℃ and start stirring for preheating. After 10 minutes of preheating, add 3mL of crude lipase TLS3 solution. Close the reactor and add 15g of methanol over 6 hours using a constant flow pump at a controlled rate. Determine the acid value of the reaction solution using the medium-heat ethanol method according to national standard GB 5009.229-2016. The acid value is 6.5 mgKOH / g. Pause the reaction and connect the reactor to a vacuum pump. Adjust the temperature to 60℃ and perform vacuum dehydration for 30 minutes. After dehydration, adjust the temperature to 40℃ and continue adding 2g of methanol over 2 hours using a constant flow pump to continue the reaction. The reaction is then complete, and the acid value of the reaction system is measured to be 2.9 mgKOH / g.

Claims

1. An enzymatic pretreatment process for deodorized distillates, characterized in that, The process includes the following steps: (1) First stage of enzyme reaction: Deodorized distillate and lipase with amino acid sequence SEQ ID NO:7 were added to the reaction vessel, and methanol was added by constant flow pump at a certain rate. The temperature was controlled at 30-40℃ and the reaction was carried out for 6-8 hours. (2) Stop adding methanol and dehydrate the reaction solution under vacuum for 30 minutes using a vacuum pump; (3) Second stage enzyme reaction: The reaction solution after dehydration in step (2) is continuously fed with methanol by vacuum pump at a certain rate, and the temperature is controlled at 30-40℃ for 2 hours.

2. The pretreatment process as described in claim 1, characterized in that, The deodorized distillate mentioned in step (1) is any one or more of the following: soybean deodorized distillate, corn deodorized distillate, peanut deodorized distillate, rapeseed deodorized distillate, flaxseed deodorized distillate, sesame deodorized distillate, and cottonseed deodorized distillate.

3. The pretreatment process as described in claim 1, characterized in that, The mass ratio of the deodorized distillate to the lipase in step (1) is 100:1.5-3.

4. The pretreatment process as described in claim 1, characterized in that, The amount of methanol added in step (1) is 15-20% of the amount of the deodorized distillate.

5. The pretreatment process as described in claim 1, characterized in that, In step (2), the temperature of the reactor is adjusted to 60°C and the vacuum degree is -0.1 MPa during vacuum dehydration.

6. The pretreatment process as described in claim 1, characterized in that, The amount of methanol added in step (3) is 2-5% of the amount of the deodorized distillate.

7. The pretreatment process as described in claim 1, characterized in that, Includes the following steps: (1) First stage of enzyme reaction: Add deodorized distillate to the reactor, adjust the temperature to 40°C, turn on the stirrer for preheating, and after preheating for 10 min, add lipase at 1.5% of the mass of deodorized distillate; close the reactor, and add methanol at a certain rate through a constant flow pump within 6 h, with the amount of methanol added being 15% of the mass of deodorized distillate; control the temperature at 40°C and react for 6 h; (2) Stop adding methanol, and dehydrate the reaction solution under vacuum using a vacuum pump at a temperature of 60°C and a vacuum degree of -0.1 MPa for 30 min. (3) Second stage enzyme reaction: The reaction solution after dehydration in step (2) is continuously fed with methanol at a certain rate through a vacuum pump. The amount of methanol added is 5% of the amount of deodorized distillate. The temperature is controlled at 40℃ and the reaction is carried out for 2 hours.

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