A process for the enzymatic preparation of tea polyphenol palmitate

Abstract

This invention discloses a highly efficient enzymatic method for synthesizing tea polyphenol palmitate, belonging to the field of food additives and biocatalysis technology. Addressing the shortcomings of existing enzymatic methods, such as low efficiency, high cost, and poor selectivity, this invention uses tea polyphenols and palmitic acid as raw materials. First, tea polyphenols are reacted with highly active acetyl chloride to prepare a fat-soluble ethyl ester of tea polyphenols. Then, the ethyl ester of tea polyphenols and palmitic acid undergo homogeneous transesterification under the catalysis of immobilized lipase to prepare a highly stable tea polyphenol palmitate with sustained-release properties. Key technical points include: pretreatment of tea polyphenol raw materials to prepare ethyl ester of tea polyphenols to ensure a homogeneous transesterification reaction in the later stage; followed by catalytic reaction at 30-60℃ for 12-24 hours. The tea polyphenol palmitate prepared by this method exhibits excellent sustained-release properties.

Description

Technical Field

[0001] This invention belongs to the field of food additives and biocatalysis technology, specifically involving a green preparation method for tea polyphenol palmitate by using a specific lipase as a catalyst, first introducing ethyl ester groups into water-soluble tea polyphenols to solve the fat solubility problem, so that the subsequent transesterification reaction can be carried out in a homogeneous reaction solution, thereby improving the enzyme-catalyzed transesterification efficiency. Background Technology

[0002] Tea polyphenol palmitate is an oil-soluble antioxidant obtained by esterification modification of tea polyphenols. It can overcome the poor fat solubility of natural tea polyphenols and is widely used in the oil and food industries. Currently, the industrial application mainly relies on chemical synthesis, which is relatively mature, easy to operate, and low-cost. However, the entire reaction process is controlled by solvents, reagents, concentrations, and reaction temperature, making it difficult to control the degree of esterification. It also results in numerous byproducts, which are detrimental to product structure identification and other subsequent studies. Therefore, in recent years, more and more attention has been paid to lipase-catalyzed modification methods.

[0003] Enzymatic catalysis has become a research hotspot due to its mild reaction conditions, high selectivity, and environmental friendliness. However, the biggest bottleneck in existing enzymatic processes is that the entire enzyme-catalyzed reaction is a heterogeneous reaction. Although water-soluble tea polyphenols have good solubility in water, their solubility in commonly used organic solvents for enzyme-catalyzed reactions is very poor, making the entire enzyme-catalyzed reaction a heterogeneous reaction. The disadvantages are as follows: First, the activity is low, requiring highly active ester donors, such as vinyl palmitate or palmitic anhydride [Biochemical and Biophysical Research Communications 377 (2008) 1118–1122; Bioorganic & Medicinal Chemistry Letters 18 (2008) 4249–4252;]. The use of highly active vinyl palmitate or palmitic anhydride drastically increases the cost of raw materials, making industrialization impossible. Second, the yield is not high. Even with highly active donors such as vinyl palmitate or palmitic anhydride, the yield is less than 40% [Biochemical and Biophysical Research Communications 377 (2008) 1118–1122; Bioorganic & Medicinal Chemistry Letters 18 (2008) 4249–4252;]. Chinese patent CN117660560A claims that palmitic acid can be used as a donor, and the reaction takes 24-48 hours at 45°C. Under the catalysis of lipase Novozyme 435, the yield can reach 66.7%-75%. In fact, this yield should only be the utilization rate of tea polyphenols. For example, in its patent example 3, when 1.5g of tea polyphenols and 16.04g of palmitic acid are added, only 1.05g of product is obtained. Its actual yield is still low and has no industrial value.

[0004] Therefore, in response to the above shortcomings, our strategy is to first prepare fat-soluble ethyl esters of tea polyphenols by reacting them with highly active acetyl chloride, which allows for almost directional and quantitative control of the esterification degree. Then, we conduct a homogeneous transesterification reaction with inexpensive palmitic acid in a suitable organic solvent reaction system to improve the yield, reduce costs, and hopefully achieve industrialization. Summary of the Invention

[0005] To address the shortcomings of existing enzymatic heterogeneous reactions, such as low efficiency, poor selectivity, and high cost, a green preparation method is provided that uses a homogeneous reaction, with lipase as a catalyst and palmitic acid as an ester donor, to achieve controllable esterification of tea polyphenols and prepare highly stable tea polyphenol palmitate in high yield.

[0006] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0007] Raw material pretreatment: The tea polyphenols are selected from raw materials with a polyphenol content of ≥98% and an EGCG content of ≥50%, which can be adapted to this process without high-purity purification; the ester donor is food-grade palmitic acid with a purity of ≥99%; acetyl chloride can be analytical grade; the reaction solvent is dried with molecular sieve for 24 hours to remove water, which is used to control the water activity of the reaction system to 0.11~0.35 to meet the enzyme activity requirements.

[0008] Esterification reaction: Add tea polyphenols and acetyl chloride at a molar ratio of 1:1~8, add ethyl acetate at a mass of 10~20 times that of tea polyphenols, add an alkaline catalyst (molar ratio of tea polyphenols to alkali of 1~1.5:10), control the temperature at 40~60℃ and react for 4~8 hours, filter, concentrate the filtrate to obtain quantitatively substituted ethyl esters of tea polyphenols.

[0009] Transesterification reaction: The product from the previous step, ethyl tea polyphenols, was fed to palmitic acid at a molar ratio of 1:1-8. Acetonitrile or isopropanol, at a mass ratio of 10-20 times that of the tea polyphenols, was added, along with immobilized lipase (0.5%-5% by mass relative to the mass of the tea polyphenols). The reaction temperature was controlled at 40-60℃, and the stirring rate at 200-250 r / min for 12-24 h. Thin-layer chromatography (TLC) was used for real-time monitoring. After the reaction was complete, the immobilized lipase was recovered by filtration. The recovered lipase could be reused 6-8 times. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate, which was then vacuum dried to obtain a pale yellow powder.

[0010] Quality control: Structural characterization methods included: ①¹H-NMR detection, with long carbon links confirmed by characteristic peaks at δ 1.0 ppm, 1.24 ppm, and 3.44 ppm. Figure 1 ② High-performance liquid chromatography (HPLC) was used for detection, employing an Agilent Poroshell 120 EC-C18 150mm*3.0mm 2.7μm (PN:693975-302) column. Mobile phase: A pump: water: acetonitrile = 92:8, B pump: acetonitrile, C pump: methanol, gradient elution. Flow rate: 0.5mL / min, column temperature: 40℃, detection wavelength: 280nm. Detection results are as follows: Figure 2 . Attached Figure Description Figure 1 The NMR spectrum of tea polyphenol palmitate is shown in 1H NMR.

[0011] Figure 2 This is an HPLC chromatogram of tea polyphenol palmitate. Detailed Implementation

[0012] Example 1 1 kg of tea polyphenols were added to a 100 L reactor, followed by 20 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (2 molar ratio) was then added, followed by dropwise addition of acetyl chloride (2 molar ratio), and the reaction was stirred for 2 h. The reaction solution was filtered into a 100 L reactor, extracted and washed with water, separated, and the aqueous phase was discarded. The organic phase was concentrated to obtain ethyl tea polyphenols. No purification was required; after drying, it was directly proceeded to the next step of transesterification.

[0013] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:2.2. 20 kg of acetonitrile was added as a solvent, along with immobilized lipase Lipozyme TL IM (0.5% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was complete, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. This product was freeze-dried to obtain 1.4 kg of a pale yellow powder, with an overall yield of approximately 68% and a purity of 80%. Example 2

[0014] 1 kg of tea polyphenols were added to a 100 L reactor, followed by 30 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (4 molar ratio) was then added, followed by dropwise addition of acetyl chloride (4 molar ratio), and the reaction was stirred for 3 h. The reaction mixture was filtered into a 100 L reactor, extracted and washed with water, and the aqueous phase was discarded. The organic phase was concentrated to obtain ethyl tea polyphenols. No purification was required; after drying, it was directly proceeded to the next step of transesterification.

[0015] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:4.2, along with 25 kg of acetonitrile and immobilized lipase Novozym 435 (1% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was completed, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. The product was freeze-dried to obtain 2.1 kg of a light yellow powder, with an overall yield of approximately 70% and a purity of 75%. Example 3

[0016] 1 kg of tea polyphenols were added to a 100 L reactor, followed by 30 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (6 molar ratio) was then added, followed by dropwise addition of acetyl chloride (6 molar ratio), and the reaction was stirred for 4 h. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols. No purification was required; after drying, it was directly proceeded to the next step of transesterification.

[0017] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:6.2, along with 30 kg of acetonitrile and immobilized lipase Lipozyme RM (2% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was completed, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. The product was freeze-dried to obtain 3.2 kg of a pale yellow powder, with an overall yield of approximately 80% and a purity of 72%. Example 4

[0018] 1 kg of tea polyphenols were added to a 100 L reactor, followed by 30 kg of ethyl acetate. The mixture was heated to 50 °C and stirred until dissolved. Potassium acetate (4 molar ratio) was then added, followed by dropwise addition of acetyl chloride (4 molar ratio), and the reaction was stirred for 3 h. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols. No purification was required; after drying, it was directly proceeded to the next step of transesterification.

[0019] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:4.2, along with 25 kg of acetonitrile and immobilized lipase Novozym 435 (1% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 50°C, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was completed, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. The product was freeze-dried to obtain 2.0 kg of a pale yellow powder, with an overall yield of approximately 68% and a purity of 76%. Example 5

[0020] 1 kg of tea polyphenols were added to a 100 L reactor, followed by 30 kg of ethyl acetate. The mixture was heated to 50 °C and stirred until dissolved. Potassium acetate (8 molar ratio) was then added, followed by dropwise addition of acetyl chloride (8 molar ratio), and the reaction was stirred for 4 h. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols, which, without further purification, was directly introduced into the next transesterification reaction.

[0021] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:8.2, along with 30 kg of acetonitrile and immobilized lipase Lipozyme RM (1% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 50℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was completed, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized dropwise in water, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 4.25 kg of a pale yellow powder, with an overall yield of approximately 85% and a purity of 65%. Example 6

[0022] 1 kg of tea polyphenols were added to a 100 L reactor, followed by 30 kg of ethyl acetate. The mixture was heated to 50 °C and stirred until dissolved. Sodium carbonate (4 molar ratio) was then added, followed by dropwise addition of acetyl chloride (8 molar ratio), and the reaction was stirred for 4 h. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols, which, without further purification, was directly introduced into the next transesterification reaction.

[0023] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:8.2, along with 30 kg of acetonitrile and immobilized lipase Lipozyme RM (1% by mass relative to the mass of tea polyphenols). The reaction was carried out at 50°C and a stirring rate of 200 r / min for 12 h. After the reaction was complete, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized dropwise in water, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 2.8 kg of a pale yellow powder, with an overall yield of approximately 73% and a purity of 69%.

[0024] Comparative Example 1 Referring to existing technology CN117660560A, 3g of tea polyphenols and 8.04g of palmitic acid were placed in a 500mL four-necked flask, and 150mL of dioxane was added and stirred to dissolve. After the tea polyphenols and palmitic acid were completely dissolved, 1.5g of Novozyme 435 lipase and 15g of dried 5A molecular sieve were added, and the reaction was carried out at 45℃ with magnetic stirring for 36h. After the reaction was completed, the lipase and molecular sieve were recovered by solid-liquid separation, the solvent was recovered by rotary evaporation concentration, and the palmitic acid was recovered by washing with 15% ethanol aqueous solution. After vacuum drying, 6.1g of product with a content of 3% was obtained (the majority of which was unreacted palmitic acid).

[0025] Comparative Example 2 1 kg of tea polyphenols and palmitic acid were added at a molar ratio of 1:8.2, along with 30 kg of acetonitrile and immobilized lipase Lipozyme RM (1% by mass relative to the mass of tea polyphenols). The reaction was carried out at 50°C and a stirring rate of 200 r / min for 12 h. After the reaction was completed, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized dropwise in water, and centrifuged to obtain the target product, tea polyphenol palmitate. The product was then vacuum dried to obtain a pale yellow powder. The product was identified as mostly unreacted palmitic acid.

[0026] Comparative Example 3 1 kg of tea polyphenols and ethyl palmitate were added at a molar ratio of 1:8.2, along with 30 kg of acetonitrile and immobilized lipase Lipozyme RM (1% by mass relative to the mass of tea polyphenols). The reaction was carried out at 50°C and 200 r / min for 12 h. After the reaction was completed, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized dropwise in water, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 0.12 kg of a light yellow powder with a purity of 5%.

[0027] Comparative Example 4 1 kg of tea polyphenols were added to a 100 L reactor, followed by 20 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (2 molar ratio) was then added, followed by dropwise addition of propionyl chloride (2 molar ratio), and the reaction was stirred for 2 h. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols, which, without further purification, was directly introduced into the next transesterification reaction.

[0028] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:2.2. 20 kg of acetonitrile was added as a solvent, along with immobilized lipase Lipozyme TL IM (0.5% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was complete, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 1.2 kg of a pale yellow powder, with an overall yield of 60% and a purity of 75%.

[0029] Comparative Example 5 1 kg of tea polyphenols were added to a 100 L reactor, followed by 20 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (2 molar ratio) and tert-butyryl chloride (2 molar ratio) were then added, and the mixture was stirred for 2 hours. The reaction solution was filtered into a larger 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols, which, without further purification, was directly introduced into the next transesterification reaction.

[0030] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:2.2. 20 kg of acetonitrile was added as a solvent, along with immobilized lipase Lipozyme TL IM (0.5% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was complete, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 1.1 kg of a pale yellow powder, with a total yield of 58% and a purity of 73%.

[0031] Comparative Example 6 1 kg of tea polyphenols were added to a 100 L reactor, followed by 20 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (2 molar ratio) was then added, followed by dropwise addition of acetyl chloride (2 molar ratio), and the reaction was stirred for 2 hours. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols, which, without further purification, was directly introduced into the next transesterification reaction.

[0032] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:2.2. 20 kg of dioxane was added as a solvent, along with immobilized lipase Lipozyme TL IM (0.5% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was complete, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 0.8 kg of a pale yellow powder, with an overall yield of 40% and a purity of 62%.

[0033] Comparative Example 7 1 kg of tea polyphenols were added to a 100 L reactor, followed by 20 kg of ethyl acetate. The mixture was heated to 60 °C and stirred until dissolved. Potassium acetate (2 molar ratio) was then added, followed by dropwise addition of acetyl chloride (2 molar ratio), and the reaction was stirred for 2 hours. The reaction solution was filtered into a 100 L reactor, extracted, washed, and separated, discarding the aqueous phase. The organic phase was concentrated to obtain ethyl tea polyphenols, which, without further purification, was directly introduced into the next transesterification reaction.

[0034] The product from the previous step, ethyl tea polyphenols, was added to palmitic acid at a molar ratio of 1:2.2. 20 kg of tetrahydrofuran was added as a solvent, along with immobilized lipase Lipozyme TL IM (0.5% by mass relative to the mass of tea polyphenols). The reaction temperature was controlled at 40℃, the stirring rate at 200 r / min, and the reaction was carried out for 12 h. After the reaction was complete, the immobilized lipase was recovered by filtration. The filtrate was concentrated, and the crude product was dissolved in ethanol, recrystallized at low temperature, and centrifuged to obtain the target product, tea polyphenol palmitate. Vacuum drying yielded 0.9 g of a pale yellow powder, with a total yield of 45% and a purity of 70%.

Claims

1. Polyphenols are reacted with highly active alkyl acyl chlorides to prepare fat-soluble ethyl esters of tea polyphenols. Then, ethyl esters of tea polyphenols are transesterified with palmitic acid under the catalysis of lipase to prepare highly stable tea polyphenol palmitate with sustained-release properties.

2. The method according to claim 1, characterized in that: The alkyl acyl chloride is selected from acetyl chloride, propionyl chloride or tert-butyryl chloride, with acetyl chloride being preferred.

3. The method according to claim 1, characterized in that: The reaction requires the tea polyphenols to be esterified with acetyl chloride first, and then transesterified with palmitic acid under the catalysis of an enzyme.

4. The method according to claim 1, characterized in that: The molar ratio of tea polyphenols to alkyl chloride is 1:10~15.

5. The method according to claim 1, characterized in that: The tea polyphenol content is greater than or equal to 98%.

6. The method according to claim 1, characterized in that: The lipase is an immobilized lipase selected from Lipozyme TL IM, Lipozyme RM, Novozym 435, Novozym LIP01, Novozym LIP02, Novozym LIP03, Novozym mLIP04, Novozym 400250, Eversa Transform 2.0 TG, and Frontia Jade, or a combination of two of these.

7. The method according to claim 1, characterized in that, The enzyme addition amount is 0.5%-5%; the catalytic reaction temperature is 30~60℃, and the reaction time is 12~24h.

8. The method according to claim 1, characterized in that: The organic solvent for the reaction system of tea polyphenols and alkyl acyl chlorides is ethyl acetate or acetone, and the amount of organic solvent used is 10~20:1 in mass ratio with tea polyphenols.

9. The method according to claim 1, characterized in that: The organic solvent of the lipase-catalyzed transesterification reaction system is acetone, acetonitrile, isopropanol or DMF, preferably acetone, and the amount of organic solvent used is 10 to 20 times the mass of tea polyphenol ethyl ester.

10. The application of the method according to claims 1-9 in the preparation of tea polyphenol palmitate.

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