Application of glyceryl tributyrate as extraction agent and use method of glyceryl tributyrate

Abstract

The invention provides application of triglyceride as an extracting agent and a using method of the triglyceride, and belongs to the technical field of application of triglyceride. The invention provides an application of triglyceride as an extracting agent, the triglyceride has higher solubility and selectivity, can effectively improve the extraction efficiency, has the characteristics of no toxicity and biodegradability, is environment-friendly, and is easy to recycle and reuse. Meanwhile, the invention also provides a method for extracting the vanillin, which comprises the following steps: mixing the vanillin with the triglyceride, and extracting to obtain the vanillin. According to the method, the vanillin is extracted by utilizing the triglyceride butyrate, so that a vanillin product with the purity of 98% or above is obtained.

Description

Technical Field

[0001] This invention relates to the field of glyceryl tributyrate application technology, and more particularly to the application of glyceryl tributyrate as an extractant and its usage methods. Background Technology

[0002] Vanillin, also known as vanillin, chemically named 3-methoxy-4-hydroxybenzaldehyde, is an organic compound extracted from the vanilla bean, an orchid. It has a rich, creamy aroma and is a widely used flavoring agent in food, cosmetics, and pharmaceuticals. Traditional methods for vanillin extraction include solvent extraction, distillation, and crystallization, but these methods suffer from problems such as high solvent toxicity, severe environmental pollution, and low extraction efficiency. Therefore, developing a highly efficient and environmentally friendly extractant is of great significance.

[0003] Glyceryl tributyrate (also known as glyceryl tributyrate) is a colorless to slightly yellow oily liquid with a buttery aroma and a slightly bitter taste. It is readily soluble in organic solvents and has stable chemical properties, making it a potential application in the field of extraction. Summary of the Invention

[0004] The purpose of this invention is to provide the application of glyceryl tributyrate as an extractant and its method of use. This invention obtains vanillin products with a purity of over 98% by using glyceryl tributyrate to extract vanillin.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of glyceryl tributyl ester as an extractant.

[0007] Preferably, the extractant extracts vanillin.

[0008] The present invention also provides a method for extracting vanillin, comprising the following steps:

[0009] Vanillin fermentation broth was mixed with glyceryl tributyrate and extracted to obtain vanillin.

[0010] Preferably, the volume ratio of vanillin fermentation broth to glyceryl tributate is 1-10:1-10; and the concentration of vanillin fermentation broth is 5-100 g / L.

[0011] Preferably, the method for preparing the vanillin fermentation broth includes the following steps:

[0012] (1) Escherichia coli was inoculated into a culture medium and cultured to obtain a bacterial solution;

[0013] (2) The bacterial solution obtained in step (1) is inoculated into a liquid fermentation medium for fermentation to obtain vanillin fermentation broth.

[0014] Further preferably, the *Escherichia coli* in step (1) is *Escherichia coli* strain XLS24-4; the inoculation amount is 0.2–2%; the culture medium is a seed culture medium, the formulation of which includes: 8–12 g / L peptone, 4–6 g / L yeast extract, 4–6 g / L sodium chloride, and 4–6 g / L ammonium sulfate; the culture temperature is 35–40°C, the culture rotation speed is 200–240 r / min, and the culture time is 6–8 h.

[0015] More preferably, the inoculation amount in step (2) is 2-10%; the liquid fermentation culture medium comprises: yeast powder 10-20 g / L, corn steep liquor powder 5-15 g / L, sodium dihydrogen phosphate 3-9 g / L, dipotassium hydrogen phosphate 1-3 g / L, ammonium sulfate 2-12 g / L, sodium citrate dihydrate 2-12 g / L, ammonium chloride 1-10 g / L, magnesium sulfate 0.12-2.4 g / L, and calcium chloride 0.1-1.0 g / L. The concentrations of the following components are present: ferrous sulfate 0.01–0.1 g / L, cobalt chloride 0.005–0.1 g / L, ampicillin 90–110 mg / L, and isopropyl-β-D-thiogalactoside (IPTG) 100–130 mg / L; the fermentation temperature is 25–30°C, the fermentation time is 35–40 h, the oxygen content during fermentation is controlled at 30–35%, and the pH during fermentation is controlled at 7.0–7.5.

[0016] Preferably, the mixture is stirred after mixing; the stirring temperature is 4-80℃, the stirring speed is 5-500 r / min, and the stirring time is 1-120 h.

[0017] Preferably, the stirring process is followed by centrifugation; the centrifugation temperature is 4–80°C, the centrifugation speed is 100–10000 r / min, and the centrifugation time is 10–120 min.

[0018] Preferably, the centrifugation is followed by molecular distillation or back-extraction; the molecular distillation temperature is 130–140°C, the molecular distillation pressure is 0.05–0.1 mbar, the feed flow rate during molecular distillation is 4–6 ml / min, the cooling liquid temperature during molecular distillation is 45–55°C, and the scraping speed during molecular distillation is 350–450 r / min; the extractant added during back-extraction is water, ethanol, or acetone.

[0019] The beneficial effects of this invention compared to the prior art are as follows:

[0020] This invention provides the application and method of using glyceryl tributylate as an extractant. The glyceryl tributylate possesses advantages such as high efficiency, environmental friendliness, and stability. Furthermore, this invention utilizes glyceryl tributylate to extract vanillin, obtaining a vanillin product with a purity of over 98%. Simultaneously, the extraction method provided by this invention not only improves extraction efficiency but also reduces costs and environmental pollution, showing broad application prospects. Moreover, the extraction method provided by this invention is applicable to various liquids, such as microbial fermentation broth, whole-cell catalytic reaction broth, and enzyme reaction catalytic broth, and exhibits strong adaptability to variations in vanillin content in the liquid.

[0021] Furthermore, this invention also successfully separated glycerol tributyrate from vanillin by distillation and other treatments, thus achieving the recycling of the extractant. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] This invention provides the application of glyceryl tributyl ester as an extractant.

[0028] In this invention, the extractant preferably extracts vanillin; the glyceryl tributylate has high solubility and selectivity for vanillin, effectively improving extraction efficiency. Furthermore, the glyceryl tributylate is non-toxic, biodegradable, environmentally friendly, easy to recycle and reuse, reducing waste generated during production and meeting environmental protection requirements. Compared to traditional extractants, glyceryl tributylate is chemically stable, does not readily react with other substances, is suitable for various extraction conditions, and is widely available and inexpensive, helping to reduce production costs.

[0029] The present invention also provides a method for extracting vanillin, comprising the following steps:

[0030] Vanillin fermentation broth was mixed with glyceryl tributyrate and extracted to obtain vanillin.

[0031] In this invention, the volume ratio of vanillin fermentation broth to glyceryl tributate is preferably 1-10:1-10, more preferably 2-8:2-8, even more preferably 4-6:4-6, and still more preferably 5:5; the concentration of vanillin fermentation broth is preferably 5-100 g / L, more preferably 10-80 g / L, even more preferably 20-60 g / L, and still more preferably 40-50 g / L; the preparation method of vanillin fermentation broth is preferably microbial transformation.

[0032] The preparation method of the vanillin fermentation broth includes the following steps:

[0033] (1) Escherichia coli was inoculated into a culture medium and cultured to obtain a bacterial solution;

[0034] (2) The bacterial solution obtained in step (1) is inoculated into a liquid fermentation medium for fermentation to obtain vanillin fermentation broth.

[0035] In this invention, the *Escherichia coli* in step (1) is preferably *Escherichia coli* strain XLS24-4; the construction method of the *Escherichia coli* strain XLS24-4 is preferably to treat *Escherichia coli* BW25113 (purchased from China General Microbiological Culture Collection Center (CGMCC)) according to the method described in *Yan Huayuan. Construction and Fermentation Process Optimization of Engineered Bacteria for Vanillin Production [D]. Hangzhou: Zhejiang University of Technology*; the inoculation amount is preferably 0.2-2%, more preferably 0.5-1.5%, even more preferably 0.8-1.2%, and still more preferably 1.0%; the culture medium is preferably a seed culture medium, and the formulation of the seed culture medium preferably includes: 8-12 g / L peptone, 4-6 g / L yeast extract, 4-6 g / L sodium chloride, and 4-6 g / L ammonium sulfate; more preferably including: 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 5 g / L ammonium sulfate; the culture temperature is preferably... The temperature is selected as 35-40℃, more preferably 36-38℃, and even more preferably 37℃; the rotation speed of the culture is preferably 200-240 r / min, more preferably 210-230 r / min, and even more preferably 220 r / min; the culture time is preferably 6-8 h, more preferably 6.5-7.5 h, and even more preferably 7 h; the inoculation amount in step (2) is preferably 2-10%, more preferably 4-8%, and even more preferably 35-40℃, and even more preferably 37℃. The preferred concentration is 5-6%; the preferred formulation of the liquid fermentation medium includes: yeast powder 10-20 g / L, corn steep liquor powder 5-15 g / L, sodium dihydrogen phosphate 3-9 g / L, dipotassium hydrogen phosphate 1-3 g / L, ammonium sulfate 2-12 g / L, sodium citrate dihydrate 2-12 g / L, ammonium chloride 1-10 g / L, magnesium sulfate 0.12-2.4 g / L, calcium chloride 0.1-1.0 g / L, ferrous sulfate 0.01-0.1 g / L, and cobalt chloride 0.005-0.1 g / L, ampicillin 90-110 mg / L, isopropyl-β-D-thiogalactoside (IPTG) 100-130 mg / L; the liquid fermentation medium preferably includes yeast powder 10-20 g / L, more preferably 12-18 g / L, even more preferably 14-16 g / L, and still more preferably 15 g / L; the liquid fermentation medium preferably includes corn steep liquor powder 5-15 g / L, more preferably 8-12 g / L, and even more preferably 10 g / L; the liquid fermentation medium preferably includes... The selected components include sodium dihydrogen phosphate at 3-9 g / L, more preferably 4-8 g / L, even more preferably 5-7 g / L, and still more preferably 6 g / L; the liquid fermentation medium preferably includes dipotassium hydrogen phosphate at 1-3 g / L, more preferably 2 g / L; the liquid fermentation medium preferably includes ammonium sulfate at 2-12 g / L, more preferably 5-10 g / L, even more preferably 6-8 g / L, and still more preferably 7 g / L; the liquid fermentation medium preferably includes sodium citrate dihydrate at 2-12 g / L, more preferably... Preferably, the concentration is 5-10 g / L, more preferably 6-8 g / L, and even more preferably 7 g / L; the liquid fermentation medium preferably includes 1-10 g / L of ammonium chloride, more preferably 4-8 g / L, and even more preferably 5-6 g / L; the liquid fermentation medium preferably includes 0.12-2.4 g / L of magnesium sulfate, more preferably 0.15-2.0 g / L, even more preferably 0.2-1.5 g / L, and even more preferably 0.5-1.0 g / L; the liquid fermentation medium preferably includes... The calcium content is 0.1–1.0 g / L, more preferably 0.4–0.8 g / L, and even more preferably 0.5–0.6 g / L; the liquid fermentation medium preferably includes ferrous sulfate at 0.01–0.1 g / L, more preferably 0.05–0.08 g / L, and even more preferably 0.06 g / L; the liquid fermentation medium preferably includes cobalt chloride at 0.005–0.1 g / L, more preferably 0.01–0.08 g / L, even more preferably 0.04–0.06 g / L, and even more preferably 0.The liquid fermentation medium preferably contains ampicillin at a concentration of 90-110 mg / L, more preferably 95-105 mg / L, and even more preferably 100 mg / L; the liquid fermentation medium preferably contains isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 100-130 mg / L, more preferably 110-120 mg / L, and even more preferably 115 mg / L; the fermentation temperature is preferably 25-30℃, more preferably 26-28℃, and even more preferably 27℃; the fermentation time is preferably 35-40 h, more preferably 36-38 h, and even more preferably 37 h; the oxygen content during fermentation is preferably controlled at 30-35%, more preferably 32-34%, and even more preferably 33%; the pH during fermentation is preferably controlled at 7.0-7.5, more preferably 7.2-7.4, and even more preferably 7.3.

[0036] The mixture is preferably stirred after mixing; the stirring process allows vanillin to fully dissolve in the glyceryl tributate; the stirring temperature is preferably 4–80°C, more preferably 10–60°C, even more preferably 20–50°C, and still more preferably 30–40°C; the stirring speed is preferably 5–500 r / min, more preferably 50–400 r / min, even more preferably 100–300 r / min, and still more preferably 200 r / min; the stirring time is preferably 1–120 h, more preferably 10–100 h, even more preferably 30–80 h, and still more preferably 50–60 h; the mixture is preferably centrifuged after stirring. The centrifugation temperature is preferably 4–80°C, more preferably 10–60°C, even more preferably 20–50°C, and still more preferably 30–40°C; the centrifugation speed is preferably 100–10000 r / min, more preferably 1000–8000 r / min, even more preferably 2000–6000 r / min, and still more preferably 4000–5000 r / min; the centrifugation time is preferably 10–120 min, more preferably 20–100 min, even more preferably 40–80 min, and still more preferably 50–60 min; the centrifugation can separate the vanillin-containing glycerol tributyrate phase from the vanillin fermentation broth phase. The centrifugation process preferably involves molecular distillation or back extraction. The molecular distillation temperature is preferably 130–140°C, more preferably 132–138°C, and even more preferably 134–136°C. The molecular distillation pressure is preferably 0.05–0.1 mbar, more preferably 0.06–0.08 mbar, and even more preferably 0.07 mbar. The feed flow rate during molecular distillation is preferably 4–6 ml / min, more preferably 4.5–5.5 ml / min, and even more preferably 5 ml / min. The cooling liquid temperature during molecular distillation is preferably 45–55°C. The temperature is further preferably 46–54°C, even more preferably 48–52°C, and still more preferably 50°C; the scraping speed during molecular distillation is preferably 350–450 r / min, further preferably 360–440 r / min, even more preferably 380–420 r / min, and still more preferably 400 r / min; the molecular distillation preferably involves two-stage separation; the conditions for the primary and secondary separations are the same; the extractant added during back-extraction is preferably water, ethanol, or acetone; after back-extraction, the product is preferably concentrated, crystallized, centrifuged, and dried to obtain a high-purity vanillin product.

[0037] Example 1: Preparation of vanillin fermentation broth

[0038] Vanillin fermentation broth stock solution was obtained using a microbial transformation method. The solids in the fermentation broth were then removed using a centrifuge to obtain a vanillin fermentation broth with a concentration of 8.8 g / L. The specific operating steps are as follows:

[0039] Take 10 mL of *E. coli* XLS24-4 bacterial culture stored at -80℃ and inoculate it at a volume ratio of 1% into a 5000 mL shake flask containing 1000 mL of seed culture medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 5 g / L ammonium sulfate). Incubate at 37℃ and 220 rpm for 7 h using a shaker. Then, transfer the *E. coli* XLS24-4 bacterial culture from the shake flask at a volume ratio of 5% to 20 L of XLS-3 liquid fermentation medium (…). Fermentation was carried out in a 50L fermenter containing 15g / L yeast extract, 10g / L corn steep liquor powder, 5g / L sodium dihydrogen phosphate, 2g / L dipotassium hydrogen phosphate, 8g / L ammonium sulfate, 10g / L sodium citrate dihydrate, 5g / L ammonium chloride, 2.0g / L magnesium sulfate, 0.5g / L calcium chloride, 0.05g / L ferrous sulfate, 0.05g / L cobalt chloride, 100mg / L ampicillin, and 120mg / L isopropyl-β-D-thiogalactopyranoside (IPTG) at 30℃, pH 7.0, and dissolved oxygen 30% for 36h. During fermentation, glucose and ferulic acid were continuously added, with the concentration controlled to be less than 5g / L. During fermentation, samples were diluted 50 times with dimethyl sulfoxide, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22μm organic filter membrane and transferred to HPLC sample vials. The product content was determined by HPLC.

[0040] When performing HPLC detection, a C18 column (ZORBAX SB-C18, 4.6×150mm, 3.5μm, Agilent, US) was used. The detection conditions were as follows: injection volume 5μL, mobile phase flow rate 1mL / min, mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, i.e., the concentration of mobile phase B increased from 5% to 50% from 0-15min; the concentration of mobile phase B decreased from 50% to 5% from 15-16min; and the concentration of mobile phase B was maintained at 5% from 16-20min. The detection wavelength was 281nm.

[0041] After fermentation, the temperature is raised to 60℃ and kept at that temperature for 30 minutes. Then, the mixture is centrifuged at 6000 rpm for 60 minutes using a tubular centrifuge. The supernatant is then collected, which is the vanillin fermentation broth with a vanillin content of 8-10 g / L.

[0042] Example 2 Vanillin Extraction Experiment

[0043] Take 1L of vanillin fermentation broth prepared in Example 1 and mix it with 2L of glyceryl tributyrate. Stir at 45℃ and 5-500r / min for 2h, centrifuge at 6000r / min for 30min, and take the supernatant to obtain vanillin-glyceryl tributyrate mixture.

[0044] Vanillin was extracted and purified using molecular distillation. Specifically, the process parameters for the first molecular distillation were set as follows: distillation temperature 135℃; distillation pressure 0.1 mbar; scraper rotation speed 400 rpm; cooling liquid temperature 50℃; feed flow rate 5 mL / min. The process parameters for the second molecular distillation were set as follows: distillation temperature 135℃; distillation pressure 0.05 mbar; scraper rotation speed 400 rpm; cooling liquid temperature 50℃; feed flow rate 5 mL / min. Vanillin was obtained after molecular distillation.

[0045] Example 3 Vanillin Extraction Experiment

[0046] Take 1L of vanillin fermentation broth prepared in Example 1 and mix it with 2L of glyceryl tributyrate. Stir at 45℃ and 5-500r / min for 2h, centrifuge at 6000r / min for 30min, take the supernatant to obtain vanillin-glyceryl tributyrate mixture, add 60kg of water continuously using an extraction centrifuge for back extraction to obtain 50kg of vanillin aqueous solution, concentrate to 50g / L at 60℃, cool to 8℃ for 12h for crystallization, filter through a Buchner funnel to obtain wet crystals, dry at 50℃ for 12h to obtain the vanillin product.

[0047] Example 4: Determination of the purity of vanillin product

[0048] The purity of the vanillin products obtained in Examples 2 and 3 was determined according to the high performance liquid chromatography method described in GB / T 32732-2016 Vanilla Test Method.

[0049] The results showed that the purity of the vanillin products obtained in Examples 2 and 3 was above 98%, indicating good product quality.

[0050] As can be seen from the above embodiments, the present invention provides a highly efficient and environmentally friendly method for extracting vanillin. This method has the advantages of high extraction efficiency, low cost, environmental friendliness, and wide applicability. The vanillin product prepared by the method provided by the present invention has high purity and stable quality, which can meet the needs of the food, beverage, fragrance, and pharmaceutical industries.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of glyceryl tributyrate as an extractant.

2. The application according to claim 1, characterized in that, The extractant extracted vanillin.

3. A method for extracting vanillin, characterized in that, Includes the following steps: Vanillin fermentation broth was mixed with glyceryl tributyrate and extracted to obtain vanillin.

4. The method according to claim 3, characterized in that, The volume ratio of vanillin fermentation broth to glyceryl tributyrate is 1-10:1-10; the concentration of vanillin fermentation broth is 5-100 g / L.

5. The method according to claim 3, characterized in that, The preparation method of the vanillin fermentation broth includes the following steps: (1) Escherichia coli was inoculated into a culture medium and cultured to obtain a bacterial solution; (2) The bacterial solution obtained in step (1) is inoculated into a liquid fermentation medium for fermentation to obtain vanillin fermentation broth.

6. The method according to claim 5, characterized in that, The *Escherichia coli* in step (1) is *Escherichia coli* strain XLS24-4; the inoculation amount is 0.2-2%; the culture medium is a seed culture medium, the formula of which includes: 8-12 g / L peptone, 4-6 g / L yeast extract, 4-6 g / L sodium chloride, and 4-6 g / L ammonium sulfate; the culture temperature is 35-40℃, the culture rotation speed is 200-240 r / min, and the culture time is 6-8 h.

7. The method according to claim 5, characterized in that, The inoculation amount in step (2) is 2-10%; the formula of the liquid fermentation medium includes: yeast powder 10-20 g / L, corn steep liquor powder 5-15 g / L, sodium dihydrogen phosphate 3-9 g / L, dipotassium hydrogen phosphate 1-3 g / L, ammonium sulfate 2-12 g / L, sodium citrate dihydrate 2-12 g / L, ammonium chloride 1-10 g / L, magnesium sulfate 0.12-2.4 g / L, calcium chloride 0.1-1.0 g / L. Ferrous sulfate 0.01–0.1 g / L, cobalt chloride 0.005–0.1 g / L, ampicillin 90–110 mg / L, isopropyl-β-D-thiogalactoside (IPTG) 100–130 mg / L; the fermentation temperature is 25–30°C, the fermentation time is 35–40 h; the oxygen content during fermentation is controlled at 30–35%; the pH during fermentation is controlled at 7.0–7.

5.

8. The method according to claim 3, characterized in that, The mixture is then stirred; the stirring temperature is 4–80°C, the stirring speed is 5–500 r / min, and the stirring time is 1–120 h.

9. The method according to claim 8, characterized in that, The stirring process is followed by centrifugation; the centrifugation temperature is 4–80°C, the centrifugation speed is 100–10000 r / min, and the centrifugation time is 10–120 min.

10. The method according to claim 9, characterized in that, After centrifugation, molecular distillation or back-extraction is performed; the temperature of molecular distillation is 130-140℃, the pressure of molecular distillation is 0.05-0.1 mbar, the feed flow rate of molecular distillation is 4-6 ml / min, the temperature of the cooling liquid of molecular distillation is 45-55℃, and the scraping speed of molecular distillation is 350-450 r / min; the extractant added during back-extraction is water, ethanol, or acetone.