CN114763404A - Method for preparing polylactic acid from fruit peel - Google Patents

Abstract

The invention provides a method for preparing polylactic acid from fruit peel, which sequentially includes sample preparation, sugar extraction, lactic acid preparation, and polymerization processes. Polylactic acid turns waste into treasure. Further, the present invention does not need to rely on enzymes for hydrolysis, but uses lye hydrolysis, which can better extract the glucose in the peel. Compared with starch-based materials, peels have higher yields of lactic acid from fermentative degradation of peels. In addition, the lactic acid polymerization process obtained by the optimized design of the present invention can make the prepared polylactic acid with high purity.

Description

A kind of method for preparing polylactic acid from peel

technical field

The invention belongs to the field of kitchen waste recycling, and in particular relates to a method for preparing polylactic acid from fruit peel.

Background technique

Polylactic acid, also known as polylactide, is a new type of biodegradable material. Polylactic acid has good thermal stability and good solvent resistance, and can be processed in various ways, such as extrusion, spinning, biaxial stretching, and injection blow molding. In addition to being biodegradable, products made of polylactic acid have good biocompatibility, gloss, transparency, hand feel and heat resistance, as well as certain bacteria resistance, flame retardancy and UV resistance, so they are very useful. Widely used as packaging materials, fibers and nonwovens, etc., it is currently mainly used in clothing, industry (construction, agriculture, forestry, papermaking) and medical and health fields.

The production of polylactic acid uses lactic acid as the raw material, and traditional lactic acid fermentation mostly uses starchy raw materials, such as corn and cassava. In recent years, turning waste into profit has been one of the focuses of attention in recent years. The transformation of kitchen waste into materials such as bio-alcohol that can be used has received a lot of attention. Among them, the transformation of kitchen waste into polylactic acid is an effective use One of the ways of kitchen waste. At present, potato peels in kitchen waste have been fully developed as polylactic acid materials, while fruit peels, another major type of kitchen waste, are rarely reported to be developed into polylactic acid.

The existing technology of preparing polylactic acid from potato peels generally requires adding water and an appropriate amount of α-amylase to make pulp, then cooking, and then transferring it to a fermenter, and after fermentation, the lactic acid is polymerized to obtain polylactic acid. However, this method first needs to rely on amylase for hydrolysis. Due to the large amount of starch in potato peel, it can be hydrolyzed with amylase, while the large amount of carbohydrates contained in peel is mostly cellulose, hemicellulose and pectin, etc. It is not suitable to be hydrolyzed with amylase, and the peel contains more and more complex components. Due to the specificity of enzymatic hydrolysis, it is impossible to completely hydrolyze various sugars in the peel by adding a single enzyme for hydrolysis. Causes the problem of low sugar extraction rate. Secondly, it will also result in a low rate of lactic acid from fermentation and degradation. Finally, enzymatic hydrolysis also introduces impurities, resulting in low product purity.

Therefore, the existing method for preparing polylactic acid from potato peel cannot be used in peel. Therefore, it will be of great practical significance if a method for developing the peel into a polylactic acid material can be developed.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects of the prior art, the present invention proposes a method for preparing polylactic acid from the peel by designing the method for how to extract saccharides from the peel and how to prepare polylactic acid.

Specifically through the following technical solutions:

A method for preparing polylactic acid from pericarp, comprising sample preparation, carbohydrate extraction, lactic acid preparation, polymerization process, specifically comprising the following steps:

S1. Sample preparation: the peel is pretreated, and the pretreatment process includes drying the sample;

S2, carbohydrate extraction: hydrolyze the dried pericarp in step S1 with lye, and obtain glucose solution after filtering;

S3, lactic acid preparation: prepare substratum with the glucose solution obtained in step S2, inoculate the lactic acid bacteria of simple culture in substratum and hatch; Use starter to inoculate in substratum, produce lactic acid through fermentation;

S4, prepolymer formation: the lactic acid obtained in step S3 is distilled, put into the prepolymer reactor after removing moisture, and polymerize to obtain the prepolymer;

S5. Polymerization process: the prepolymer obtained in step S4 is subjected to a polymerization reaction to obtain polylactic acid.

Further, the polymerization process is condensation polymerization or ring-opening polymerization.

Further, the condensation polymerization process includes:

S511: in the first reactor with catalyzer, lactic acid and organic solvent are azeotropically dehydrated to form polymer; The polymer is recovered in the second reactor, and azeotropic dehydration is obtained to leave solution;

S512: Concentrating the remaining solution, adding an extractant to obtain a mixed solution;

S513: remove the catalyst by filtration or extraction, pour the mixed solution into an organic solvent, and separate out crystals; the crystals are collected by suction filtration, washed, and dried under reduced pressure to obtain polylactic acid.

Further, in the step S511, the polymer is recovered into the reactor through molecular sieves.

Further, the ring-opening polymerization process includes:

S521: inject the prepolymer obtained in step S4 into the first reactor to obtain thick L-lactide, then extract and purify the thick L-lactide;

S522: take the second reactor and remove the oxygen in the second reactor; mix the purified L-lactide, tin octoate and lauryl alcohol obtained in step S521 to form a mixed solution, and seal the mixed solution in the second reactor and heated with stirring under nitrogen atmosphere;

S523: keep the mixed solution at the same temperature, reduce the pressure in the second reactor, and carry out vacuum distillation; after the distillation of the mixed solution is stopped, poly(L-lactide) is discharged from the bottom of the container in the form of a chain.

Preferably, in step S2, the alkali solution is NaOH.

Preferably, in step S2, the hydrolysis time is 45 minutes. If the hydrolysis time is less than 45 minutes, the efficiency is low and complete hydrolysis into glucose cannot be achieved.

Preferably, in step S3, the fermentation time is 72h. If the time is shorter than 72h, the fermentation is incomplete, and when the fermentation time is 72h, a better yield of lactic acid fermentation can be obtained.

Preferably, in step S513, the organic solvent is methanol.

Preferably, the reactor in the ring opening polymerization process is equipped with a Dean-Stark trap.

Compared with the existing use of kitchen waste to prepare polylactic acid, the present invention has the following advantages:

1. Compared with starchy raw materials such as potato peel, the fruit peel has a high yield of lactic acid from fermentation and degradation;

2. It does not need to rely on enzymes for hydrolysis, and the sugars in the peel have a high degree of complete hydrolysis and high utilization;

3. The polylactic acid obtained by the polymerization of lactic acid has high purity.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the drawings that are used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the synthetic schematic diagram of polylactic acid (PLA);

Fig. 2 is polylactic acid structural representation;

Fig. 3 is the schematic flow sheet that takes fruit peel as raw material to prepare polylactic acid;

Fig. 4 is the influence of different fermentation time on lactic acid yield;

Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram of polylactic acid;

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

As shown in Figure 1, polylactic acid (PLA) can be made of two monomer polymers, one is lactide and the other is lactic acid. If the monomer is lactide, polylactic acid is formed by ring-opening polymerization, that is, process A in Figure 1; if the monomer is lactide, polylactic acid is formed by condensation polymerization, that is, process B in Figure 1.

The specific reaction conditions required for the synthesis of polylactic acid by two different routes are different. The basic process of the condensation polymerization of lactic acid includes: vacuum distillation of lactic acid at 130 ° C for 2 to 3 hours, and then under the action of a catalyst, the lactic acid is linked together by dehydration to form oligomers (usually with a weight average molecular weight of less than 1000g mol ^-1 ), the water produced by the condensation reaction is removed by high temperature and vacuum, which is beneficial to the formation of oligomers. A catalyst (eg, tin octoate) and a suitable solvent (eg, diphenyl ether) are added and heated at 130° C. for an additional 30-40 hours to convert the oligomers to polymers. This process can be carried out in a closed system and the water formed from this reaction stage can be removed by molecular sieves. The polymer can be isolated as is or dissolved and precipitated for further purification.

For the ring-opening condensation reaction of lactide, compared with condensation polymerization, the ring-opening condensation reaction can usually obtain polylactic acid with better monodispersity and higher molecular weight, and the polymerization conditions are also broader. In industry, a typical catalyst used for ring-opening polymerization is tin octoate. In addition, many different types of catalysts are used, mainly including: enzymatic catalysis, organic catalysts, and metal-based catalysts. It is suitable for commercial polylactic acid products, such as cups and bottles, and usually has a molecular weight M greater than 100,000 g mol-1, while the ring-opening polymerization of lactide can usually obtain polymers with a molecular weight greater than 100,000 g mol ^-1 .

The lactic acid molecule contains an asymmetric carbon atom. The atoms around carbon have two different three-dimensional arrangements (R or S). When two lactic acid molecules combine to form lactide (dimer), three different monomers are produced: L-lactide, D-lactide, and meso-lactide. From these lactides can be polymerized to produce five kinds of polylactic acid, as shown in Figure 2. Among them, L represents meso-lactide, M represents L-lactide, and N represents D-lactide; P1 represents isotactic polylactic acid, which is crystalline at room temperature and has a melting point of 180°C; P2 represents vertical Structural block polylactic acid, the form is crystalline at room temperature, the melting point is greater than 200 ° C; P3 represents atactic polylactic acid, amorphous form at room temperature; P4 represents heteromeric polylactic acid, amorphous form at room temperature; P5 represents syndiotactic polylactic acid Lactic acid, in the form of crystals at room temperature, has a melting point of 152°C.

The ratio of monomers also affects some key polymer properties such as crystallinity, melting temperature and ease of processing. Crystallinity is a very important property that affects many properties of polymers, including hardness, tensile strength, stiffness and melting point. Some mechanical properties of PLA are shown in Table 1. These values indicate that PLA prepared with L-lactide monomer has stronger toughness than D,L-PLA prepared with racemic lactide. Commercial PLA resins are usually produced from L-lactide, and the resulting polymer, L-polylactic acid (L-PLA), is a semi-crystalline material with high melting temperature and glass transition temperature.

Table 1. Effects of stereochemistry and crystallinity on mechanical properties

The mechanical properties of PLA can also be improved by the formation of composite materials, such as blending with cornstarch. Table 2 summarizes the mechanical properties of some composites. The higher the content of cornstarch in the composite, the lower the onset temperature of thermal degradation. In addition, the L-PLA/Cornstarch composite has a higher water leaching rate (5-7% increase in moisture compared to L-PLA). Additives can also be added along with polycaprolactone, polyethylene glycol, glycerol and lauryl alcohol. The results show that with the addition of L-polylactic acid and polyvinyl alcohol blend, its tensile strength and Young's modulus decreased. A PLA/PHB blend is a typical biopolymer blend with the best properties of both polymers. In addition, the PLA/PHB blend improved the mechanical properties of the two neat polymers. Poly(butadiene-terephthalate) (PBAT) and PLA blends have a positive effect on the elongation and strength of PLA and make the viscosity of PLA more constant, resulting in a wider processing temperature window. width. See Table 2, which lists the use of L-polylactic acid (L-PLA), corn starch (CS), high amylose corn starch (HACS), polyhydroxybutyrate (PHB), polycaprolactone (PCL) , Polyvinyl alcohol (PVA), poly (butene adipate-co-terephthalate) (PBAT) as raw materials to synthesize the mechanical properties of composites.

Table 2. Mechanical properties of composites

As shown in Table 3, the peel contains a large amount of carbohydrates, including sugars, cellulose, hemicellulose, and pectin. The preparation of polylactic acid from peel as raw material mainly includes extracting sugars from peel, converting sugars into lactic acid by microbial fermentation, then purifying and concentrating lactic acid, and finally obtaining corresponding polylactic acid by condensation polymerization or ring-opening polymerization . The focus of the present invention is how sugars are extracted from the peel and converted into lactic acid.

Table 3. Water and sugar content of some fruits (per 100g of raw fruit)

The present invention provides a method for preparing polylactic acid from fruit peel, including sample preparation, sugar extraction, lactic acid preparation, and polymerization processes in sequence, as shown in Fig. 3, and the details are as follows:

The peel is pretreated, and the pretreatment process includes drying the sample; under the condition of heating and stirring, the dried peel is hydrolyzed with lye, filtered to obtain a glucose solution, and the lye can hydrolyze the ester bond on the polysaccharide to degrade, The polysaccharides in the peel are hydrolyzed into glucose.

The culture medium is prepared with the obtained glucose solution, and the lactic acid bacteria of simple culture are inoculated into the culture medium and incubated; the starter is inoculated into the culture medium, and lactic acid is produced by fermentation; In the polymer reactor, polymerization is performed to obtain a prepolymer; and then a polymerization reaction is performed to obtain polylactic acid.

The polymerization process also includes condensation polymerization or ring-opening polymerization.

The process of condensation polymerization includes:

In the first reactor with catalyst, azeotropic dehydration of lactic acid and diphenyl ether in a volume ratio of 2:3 is carried out to form a polymer; the polymer is recovered into the reactor through molecular sieve, and azeotropic dehydration is carried out to obtain the remaining solution. Molecular sieves can improve overall efficiency while being recyclable for reuse. The remaining solution was concentrated to half of the original volume, and chloroform or ethyl acetate was added to obtain a mixed solution; the catalyst was removed by filtration or extraction, the mixed solution was poured into an organic solvent, and crystals were precipitated; the crystals were collected by suction filtration, washed with methanol, After drying under reduced pressure, polylactic acid was obtained.

The process of ring-opening polymerization includes:

Inject the obtained prepolymer into the first reactor to obtain crude L-lactide, and then extract and purify the crude L-lactide; under vacuum conditions, use nitrogen to remove oxygen in the reactor; take the purified L-lactide Lactide, tin octoate, lauryl alcohol to form a mixed solution, the mixed solution is sealed in the second reactor, and heated with stirring under a nitrogen atmosphere; keep the mixed solution at the same temperature, reduce the pressure in the second reactor, Carry out vacuum distillation; after the distillation of the mixed liquid is stopped, the poly(L-lactide) is discharged in the form of chains from the bottom of the container.

Example 1

Peel the fruit, then wash the peel with clean water to remove dirt particles. The peel was then cut into small pieces and dried at 60°C for 24 hours. The moisture content of the peel was calculated by measuring the weight of the peel before and after drying, which was used to calculate the amount of reagents required for the subsequent fermentation process. It is better if the water content is below 3%, and if it is higher than 3%, the error will be large. The peel is then blended into a powder with a blender.

Weigh the dried peel powder, hydrolyze the dried peel with sodium hydroxide under heating conditions, cool to room temperature, neutralize with acid, and the neutralized pH value range is 5-7; A glucose solution was obtained; simply cultured lactic acid bacteria were inoculated into MRS broth at pH 5.5, and incubated at 37°C for 24 hours. 15% MRS broth was prepared with glucose extracted from fruit. 10% of the LAB standard starter was inoculated and incubated in a temperature-controlled shake flask at 37°C for up to 72 hours to produce lactic acid. 10% LAB standard starter was inoculated in the MRS medium to produce lactic acid; using a reaction vessel equipped with a Dean-Stark trap, in the presence of 0.1 g methanesulfonic acid (catalyst), 40.2 g L-lactic acid was azeotropically dehydrated in 400 ml of diphenyl ether for 2 h at a temperature of 140 °C. After the distilled water in the Dean-Stark trap was removed, a test tube with 40 g of molecular sieve was installed above the reactor, and the distilled solvent was recovered into the reactor through the molecular sieve. Azeotropic dehydration was carried out at 130°C for 20-40h. After the solvent passes through the molecular sieve, the moisture content is below 3ppm. After the reaction mixture was concentrated to about half volume, 300 ml of chloroform was added. The catalyst was removed by filtration or extraction, and the resulting mixture was poured into 900 mL of methanol. The precipitated crystals were collected by suction filtration, washed with methanol, and dried under reduced pressure. The yield of polylactic acid white powder is 80-85%.

Example 2

Compared with Example 1, the present embodiment differs in that the condensation process is ring-opening condensation, and the concrete steps are:

0.5 g of oxazolidine was added, and in a 25 mL round bottom flask, L-lactic acid (1.60 g of 50 wt% lactic acid) was added, followed by 10 mL of solvent (toluene or o-xylene) and a magnetic stir bar. A solvent reflux trap is installed on top of the round bottom flask, this setup ensures that the lighter solvent phase is refluxed and water can be recycled. At the start of the reaction, the flask was immersed in a preheated, stirred, temperature-controlled oil bath. The bath is usually kept at 140°C (or 130°C) for the reaction in toluene and 170°C for the reaction in ortho-xylene, slightly above the respective boiling points to ensure that the solvent can be refluxed. The mixture was continuously stirred under reflux of the solvent, and the reaction was carried out for 3 hours to obtain L-lactide.

In a thick-walled cylindrical stainless steel polymerization vessel equipped with a stirrer, 216 g (1.5 mol) of L-lactide, 0.01% by mass of tin octoate and 0.01% by mass of L-lactide were sealed (calculated relative to the amount of lactide) 0.03% lauryl alcohol. The polymerization vessel was deoxygenated with nitrogen for 2 hours under vacuum. The mixture was heated with stirring at 200°C for 3 hours under nitrogen atmosphere. While keeping the mixture at the same temperature, the polymerization vessel was gradually pumped to a lower pressure. Distillation of monomers and low molecular weight volatiles ceased 1 hour after the start of extraction. The vessel was filled with nitrogen and the polymer was discharged in chains from the bottom of the vessel. The chains were pelletized to obtain poly(L-lactide) in 96% yield. See Figure 5 for the 1H NMR spectrum of polylactic acid.

Example 3: Glucose concentrations obtained by hydrolysis of banana peels of different qualities by different concentrations of NaOH On the basis of Example 1, the peels of this example were selected from banana peels.

5 grams of dried peels were weighed separately with an analytical balance and placed in three different beakers. Dried banana peels were subjected to alkaline pretreatment hydrolysis with 1%, 2% and 3% sodium hydroxide. The weighed samples in the beaker were then marked, mixed with 1%, 2%, and 3% NaOH, respectively, to start hydrolysis. Then put a magnetic stirrer into the beaker and stir well. The temperature of the three hot plates was set at 80°C, the speed was adjusted to approximately 700 rpm, and the treatment was carried out for 45 minutes. After all the solutions in the beaker have cooled to room temperature, record the initial pH of all samples. 1M HCl solution was then added dropwise to each sample until the sample was neutralized.

The contents of the solution were filtered through filter paper into test tubes and then analyzed for glucose content. Weigh 1g of the filtered sample and dilute it with distilled water in a 50ml volumetric flask. The purpose is to dilute the color of the filtered sample to colorless, because colored samples cannot be well detected in the UV-Vis spectrum, as this will affect obtained concentration results. Absorbance values were obtained in a UV-Vis spectrometer at 265 nm. Absorbance should be a linear function of concentration, according to the Lambert-Beer law, under ideal conditions, the concentration of a substance is proportional to the absorbance of the solution.

The extraction results of glucose concentration in banana peel are shown in Table 4.

Table 4. Glucose concentrations obtained by hydrolysis of banana peels of different qualities by different concentrations of NaOH

Embodiment 4: different concentrations of NaOH handle the glucose concentration that different quality apple peel hydrolysis obtains

The difference between this embodiment and Embodiment 3 is that the peel of this embodiment is selected from apple peel. The extraction results of glucose concentration in apple peel are shown in Table 5.

Table 5. Glucose concentrations obtained by hydrolysis of apple peels with different concentrations of NaOH

Embodiment 5: different concentrations of NaOH handle the glucose concentration that different quality mango peels are obtained by hydrolysis

The difference between this example and Example 3 is that the peel of this example is mango peel. The extraction results of glucose concentration in mango peel are shown in Table 6.

Table 6. Glucose concentrations obtained by hydrolysis of mango peels with different concentrations of NaOH

As can be seen from the experimental data in Tables 4-6, under the condition of 3% NaOH concentration, whether it is banana peel, apple peel or mango peel, the glucose concentration obtained after the treatment is all higher. This proves that higher alkali concentration can extract higher glucose concentration.

Example 6: Effects of different fermentation times on lactic acid yield

On the basis of embodiment 1, select these 5 kinds of bacterial classifications of Bacillus lactis, Streptococcus lactis, Lactobacillus delbrueckii, Lactobacillus plantarum, Pediococcus beer, and the fermentation time of lactic acid bacteria is set to 36h, 48h, 72h, 96h respectively. 25 ml of the culture medium of the solid residue after fermentation was taken and transferred to a 100 ml flask, and the content of lactic acid in the fermentation broth was measured. One milliliter of phenolphthalein indicator (0.5% in 5% alcohol) was added to the flask. Titrate with 0.25M NaOH until the surface is pink. Titratable acidity was calculated as lactic acid % w/v, equal to 90.08 mg lactic acid per milliliter of 1 M NaOH. The titrated acidity is then calculated. The results are shown in Figure 4. It can be seen from Figure 4 that the lactic acid yield is generally higher after the fermentation time is 72h.

To sum up, the present invention provides a method for preparing polylactic acid from fruit peel, which sequentially includes sample preparation, sugar extraction, lactic acid preparation, and polymerization processes. Polylactic acid is extracted from the peel to realize turning waste into treasure. Compared with the existing extraction of polylactic acid from starchy raw materials such as potato peel, the yield of lactic acid obtained by peeling fermentation and degradation is high; and it is not necessary to rely on enzymes to hydrolyze, and the complete hydrolysis degree of carbohydrates in the peel is high; finally this method The polylactic acid obtained by the lactic acid polymerization process has high purity.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. A method for preparing polylactic acid from pericarp comprises the processes of sample preparation, saccharide extraction, lactic acid preparation and polymerization, and is characterized in that:

s1, sample preparation: pretreating the peel, wherein the pretreatment process comprises drying the sample;

s2, saccharide extraction: hydrolyzing the dried pericarp in the step S1 with alkali liquor, and filtering to obtain glucose solution;

s3, lactic acid preparation: preparing a culture medium with the glucose solution obtained in the step S2, inoculating a single-purity cultured lactic acid bacterium into the culture medium and incubating; inoculating a starter in the culture medium, and fermenting to produce lactic acid;

s4, prepolymer formation: distilling the lactic acid obtained in the step S3, removing water, putting the lactic acid into a prepolymer reactor, and polymerizing to obtain a prepolymer;

s5, polymerization process: and (4) carrying out polymerization reaction on the prepolymer obtained in the step S4 to obtain polylactic acid.

2. The method as claimed in claim 1, wherein the polymerization process is condensation polymerization or ring-opening polymerization.

3. The method as claimed in claim 2, wherein the condensation polymerization process comprises:

s511: azeotropically dehydrating the lactic acid with an organic solvent in a first reactor in the presence of a catalyst to form a polymer; recovering the polymer into a second reactor, and performing azeotropic dehydration to obtain a remaining solution;

s512: concentrating the remaining solution, and adding an extracting agent to obtain a mixed solution;

s513: removing the catalyst by filtration or extraction, and pouring the mixed solution into an organic solvent to separate out crystals; and carrying out suction filtration, collection, washing and reduced pressure drying on the crystals to obtain the polylactic acid.

4. The method of claim 3, wherein in step S511, the polymer is recycled back to the second reactor through molecular sieves.

5. The method of claim 2, wherein the ring-opening polymerization process comprises:

S521: injecting the prepolymer obtained in the step S4 into a first reactor to obtain crude L-lactide, and extracting and purifying the crude L-lactide;

s522: taking a second reactor, and removing oxygen in the second reactor; mixing the purified L-lactide obtained in the step S521, tin octylate and lauryl alcohol to form a mixed solution, sealing the mixed solution in the second reactor, and stirring and heating the mixed solution in a nitrogen atmosphere;

s523: keeping the mixed solution at the same temperature, reducing the pressure in the second reactor, and carrying out reduced pressure distillation; after the distillation of the mixed liquid is stopped, the poly (L-lactide) is discharged in the form of chains from the bottom of the container.

6. The method of claim 1, wherein in step S2, the alkali solution is NaOH.

7. The method of claim 1, wherein the hydrolysis time in step S2 is 45 minutes.

8. The method of claim 1, wherein the fermentation time of step S3 is 72 hours.

9. The method as claimed in claim 3, wherein the organic solvent in step S513 is methanol.

10. The method as claimed in claim 3, wherein said reactor is equipped with a dean-stark trap.

Images