Vanillin What Is It?

Vanillin, 3-methoxy-4-hydroxybenzaldehyde, is the main ingredient of vanilla beans, originally from Mexico, is a broad-spectrum high-level flavor. Vanillin is widely found in nature, e.g. in many essential oils and plants such as Java lemongrass, benzoin, Peruvian balsam, clove buds, vanilla pods, etc. It is also used as an ingredient of vanillin, which has been found in many other essential oils and plants. Due to the small quantity and high price of natural vanillin extracted from plants, it is difficult to meet the market demand. Therefore, vanillin became the first fragrance synthesized by human beings, which was successfully synthesized by Dr. M. Hallmann and Dr. G. Thiemann of Germany in 1874. Molecular formula is C 8H 8 O3, molecular weight is 152.15, melting point 81~83 ℃ (lit.), white or yellowish needle-like crystals, with the aroma of vanilla and rich milky flavor, the chemical structure is:

As the most important edible flavor, vanillin is a kind of edible flavoring agent with the aroma of vanilla bean and rich milky flavor, which is an indispensable important raw material in the food additive industry and the largest synthetic flavor in the world. It is widely used in all kinds of food, tobacco, toothpaste, perfume, cosmetics and daily chemicals, and it can also be applied to rubber, plastics and pharmaceuticals. Vanillin is widely used in the production of pharmaceutical intermediates in foreign countries. At present, the annual consumption of vanillin in the world market is 16000~20000 tons [1], which is widely used in a variety of food products that need to increase the aroma of milk, especially the main source of vanilla flavor, in accordance with the FCCIV standard.

The application of vanillin in China is mainly as food additives, but in recent years, the application in the field of medicine is also broadening, which has become the most potential field of vanillin application. At present, the distribution of domestic vanillin consumption is as follows: food industry accounts for 55%, pharmaceutical intermediates account for 30%, feed, flavoring agents account for 10%, cosmetics and so on account for 5%, and the annual consumption of vanillin in China is in the range of 2,000~2,500 tons[2]. This paper mainly reviews the three types of vanillin preparation technology and gives an overview of the future direction of vanillin technology development.

1 Overview of vanillin preparation technology

There are two types of vanillin available in the market - synthetic vanillin and natural vanillin.  Synthetic vanillin is vanillin obtained by chemical synthesis of common fossil raw materials. There are nearly 10 methods of chemical synthesis of vanillin, with stable raw material sources and production technology, sufficient supply and low market price of about US$15 per kilogram, it has become the main method of production of vanillin in the market, and 90% of the market share is synthetic vanillin at present. The production process of synthetic vanillin is stable, the raw materials and reaction mechanism are clear, and the main impurities can be detected and controlled; of course, the product has a single aroma and lacks the compound aroma of natural vanillin, and the production process is prone to environmental pollution and other problems.

Natural vanillin, due to the differences in international and domestic regulations and terms for natural grade flavors in recent years, for example, the United States defines natural flavors as those obtained from plants, animals or microorganisms as raw materials by physical methods, biotechnological methods or soft chemical methods using natural raw materials. For example, in the United States, natural flavors are defined as those obtained from plants, animals or microorganisms by physical, biotechnological or soft chemical means. In China and the European Union, natural flavors are only defined as those obtained from plants, animals or microorganisms by physical or biotechnological means or by traditional food processes.

For this reason, there are two kinds of natural vanillin production technologies according to the methods defined in the regulations of China and the EU: (1) extraction method; (2) biological method (which can be divided into microbial fermentation method, plant cell culture method and enzyme method). In addition to the above two types of natural vanillin production technology defined by the U.S. regulations, there is also a third type: (3) soft chemistry (production of natural equivalent vanillin) with natural raw materials. By soft chemistry, it is meant that no biocatalysts or hard or caustic chemicals are used in the preparative reactions; the chemical reactions that can be carried out include: hydrolysis, oxidation, condensation, addition, rearrangement, and Meladic reactions. Reaction conditions such as pH can also be adjusted, and reactions can be carried out in organic solvents, oxygen, or other atmospheres. In general terms, natural vanillin, as defined by the US regulations, is recognized as natural vanillin when its naturalness reaches 95% as determined by analysis of the unstable isotope 14C.

The most traditional production technology for natural vanillin is extraction and mono-isolation. Vanilla bean is called "queen of spices", natural vanilla bean contains about 2% vanillin, which has a unique aroma that cannot be compounded by artificial methods, and high value natural vanillin can be obtained after extraction with solvent (usually ethanol). Natural vanillin can be obtained by solvent extraction (usually ethanol). Natural vanillin can also be obtained by the technical means of physical mono-isolation after enrichment of natural raw materials containing vanillin. The extraction of isolated vanillin from natural vanillin is extremely expensive due to the limited area of vanilla cultivation, climate-dependent yield, and labor-intensive nature of the natural vanillin, which is sold at a price of up to 4,000 US dollars per kilogram, which is about 300 times higher than that of synthetic vanillin [3].

Another recognized technology for the production of natural vanillin is the biological method (which can be divided into microbial fermentation, plant cell culture and enzyme method). Biological method can prepare natural vanillin, and has the advantages of less pollution, clean production and safety. In recent years, people's healthy consumption concept of returning to nature has promoted the diversification of the consumer market, which makes the preparation of natural vanillin by biotechnology become a hot spot of academic research at home and abroad. The production of vanillin by biotechnology has the advantages of raw materials being natural, cheap and easy to obtain, the process of production being clean and pollution-free, and fast and efficient, so that the use of biotechnology for the production of natural vanillin has become a new channel worthy of being promoted [4]. The use of biotechnology to produce natural vanillin has become a new channel worth promoting [4]. However, how to achieve the high yield required for industrial production, and how to make the downstream product separation and purification process simpler and more economical, so as to achieve higher economic benefits, are also the main factors affecting the high price of natural vanillin.

2 Synthetic Chemical Preparation Techniques for Vanillin

At present, the following synthetic chemical preparation techniques can be used to produce vanillin, including guaiacol method (which can be divided into guaiacol-dimethylaniline method and guaiacol-glyoxylate method), lignin method, safrole method, eugenol method, p-hydroxybenzaldehyde method, p-cresol method, and electrochemical method; the early synthetic vanillin production process was mainly guaiacol-dimethylaniline method, due to the pollution and toxicity of the production process, now the main production process has been changed to guaiacol-glyoxylate method, whose reaction conditions are easier to control, with high yield, less waste treatment and convenient post-treatment. Due to the pollution and toxicity of the production process, the main production process has been changed to guaiacol-glyoxalic acid method, which is easier to control the reaction conditions, with high yield, fewer three wastes and convenient post-processing, and is currently used by three foreign vanillin producers, namely, Rhodia of France, Bollinger of Norway and UBE of Japan.

2.1 Guaiacol method

The synthesis of vanillin from guaiacol has always been the main industrial production technology for vanillin, and there are two successful industrialized synthesis routes, namely, the nitrosyl method and the glyoxylate method.

2.1.1 Nitroso route

Guaiacol and formaldehyde (or urotropin) in the presence of p-nitrosodimethylaniline hydrochloride condensation, hydrolysis to produce vanillin, benzene extraction condensation products, the first distillation, toluene crystallization, secondary distillation, water crystallization to obtain vanillin finished products, the reaction equation is as follows Figure 1.

The raw materials used in this method are complicated, and the product quality is unstable, the yield is low (generally 55%~60%), and the production process requires a large number of highly toxic auxiliary raw materials, such as dimethyl aniline, formaldehyde, sodium nitrite, benzene, etc. The waste liquid discharged contains nitroso compounds, amino compounds, aldehyde compounds and polymers. The wastewater discharged contains nitroso compounds, amino compounds, aldehyde compounds and polymers, etc. The wastewater cannot be treated biochemically and there is no economic and effective treatment measure, so the environmental pollution is extremely serious. It has long been eliminated in foreign countries, and only a small amount of domestic production capacity is being used, facing elimination.

2.1.2 Glyoxalate route

Guaiacol and glyoxylate can generate 3-methoxy-4-hydroxymandelic acid under alkaline heating conditions, and the acid is oxidized to generate 3-methoxy-4-hydroxyacetophenone in the presence of catalyst and oxygen, and then acidification and decarboxylation can obtain crude vanillin. The reaction equation is shown in Figure 2.

The process of preparing vanillin by glyoxalic acid method was firstly successfully industrialized by French Rhodia Company in 1970s, the raw materials used in this method are few, the conditions are easy to control, the quality of products is stable and the yield is high (generally around 70%), the raw materials and auxiliary materials used in the production process are mainly the low-toxicity and non-volatile glyoxalic acid, the production conditions are good, and the waste liquid can be treated by biochemical treatment. With the successful industrialization of guaiacol synthesis by catechol method in China and the decrease of guaiacol price, the research and development of catalyst preparation and selection, oxidation method and extraction method of the method are also increased in China. In recent years, the process of glyoxalic acid has replaced the nitroso method in the domestic three major domestic vanillin production enterprises--Zhejiang Jiaxing Zhonghua Chemical Factory and Jilin Petrochemical Vanillin Factory as the main production process for the synthesis of vanillin in China [2]. In recent years, the glyoxylate process has replaced the nitroso method in the three major domestic vanillin production enterprises - Zhejiang Jiaxing Zhonghua Chemical Factory and Jilin Petrochemical Vanillin Factory to become the main production process for the synthesis of vanillin in China [2].

2.2 Lignin method

Lignin comes from a wide range of sources, and its content is huge in waste wood, mud ash, pulp waste liquid and wine lees, and it mainly exists in the form of lignosulfonate. Lignosulfonate is hydrolyzed under alkaline condition, then oxidized by high temperature and high pressure, part of lignin is transformed into vanillin, then extracted into vanillin sodium salt by acidification and extraction, and then passed into a series of reactions, such as acidification of SO2 and recrystallization of water, to obtain pure vanillin. The specific reaction equation is shown in Figure 3 below.

Lignin method has been used to produce vanillin for a long time in two large paper enterprises in the United States and Canada, but the disadvantages of this method are obvious: low yield (10%~15%), large emission of three kinds of wastes, and serious pollution; the quality of vanillin products is on the low side, and the vanillin produced contains a large amount of heavy metals, which can not be used in the food and pharmaceutical industries, and the lignin method of vanillin was drastically shrunk in the 1990's. The lignin method of vanillin has been widely used in the paper industry in the United States and Canada.

2.3 Safrole method

The natural safrole from various camphor oils and camphor oils was isomerized and oxidized to jasmonic aldehyde under alkaline conditions, and then interacted with PCl5 to obtain protocatechuic aldehyde[5] , and finally methylated by dimethyl sulfate ((CH3 )2 SO4 ) or halomethane to obtain the mixture of vanillin and isocarboxylin, and then separated by using the different solubility of the two in alkali. The specific reaction equation is shown in Figure 4 below.

The yield of this method is limited because the source of safrole is very limited, and the synthesis route is relatively long and the process is complicated, and the by-products such as isovanillin and isoethylvanillin are generated, and the yield of the product is low.

2.4 Eugenol method

Eugenol is the main component of clove oil (85%~90%). Vanillin can be produced by isomerizing eugenol to isoeugenol with a strong base, then oxidizing and acidolysis[6] , and the specific reaction equation is shown in Fig. 5.

Clove oil is also expensive and limited in production due to its limited availability and price, but as a natural flavor equivalent under the soft chemistry concept, it is a good complement to natural vanillin.

2.5 p-Cresol, p-Hydroxybenzaldehyde method

This method is a newly developed process route[7] , initially developed by Dalian University of Technology, the p-hydroxybenzaldehyde route, the subject of more in-depth research in China, the focus of the research has been from the raw material of p-cresol, p-hydroxybenzylaldehyde is obtained from p-hydroxybenzoaldehyde after oxidizing the readily available p-hydroxybenzylaldehyde, p-hydroxybenzylaldehyde is brominated to become 3-bromo-4-hydroxybenzylaldehyde in chloroform, and then reacted with sodium methanol at high temperature (100~110 ℃) under CuO-catalyzed conditions for 1.5~2 h to obtain vanillin, yield can be generally obtained. Then, using N,N-dimethylformamide (DMF) as solvent and CuO catalyzed condition, the reaction with sodium methanol at high temperature (100~110 ℃) was carried out for 1.5~2 h to obtain vanillin, and the yield could reach 90%, and the equation of the reaction is shown in Fig. 6 below.

This route has the advantages of short process, less waste, small investment, simple and easy to obtain raw materials, bromine can be recycled and not consumed, etc. Meanwhile, the domestic production of p-cresol accounts for about 80% of the world's production, and more than 20 well-known universities and research institutes in China have been very active in researching this process, and believe that it is more in line with the domestic situation. In comparison with the glyoxalic acid process, some experts believe that the p-cresol process only consumes toluene, sulfuric acid, caustic soda, methanol (less than 0.2 tons) and oxygen, and that the bromine is recycled and only needs to be replenished in small amounts, and that these raw materials are easy to come by and inexpensive. On the other hand, there are more than ten kinds of raw materials used in the glyoxalate process, many of which are difficult to source, and only glyoxalic acid accounts for a considerable portion of the cost. Meanwhile, the total amount of three kinds of wastes generated by the process is only about 1/20 of that of the glyoxalate process, which really realizes green production. However, there are still many difficulties in this method: complicated operation, pressure reactor and harsh reaction conditions; and safety problems in oxidation operation, all these bottlenecks affect the promotion of the p-cresol process, and at present, the production of vanillin by this method has not been industrialized.

2.6 Organic electrochemical synthesis methods

Organic chemical synthesis of vanillin is also based on guaiacol and glyoxylate as raw materials, the first two condensation synthesis of 3-methoxy-4-hydroxyphenylglycolic acid, the difference is that the intermediate is not oxidized by dangerous oxidants, but electrolytic oxidation to obtain 3-methoxy-4-hydroxyphenylglycolic acid, and then acidification and decarboxylation to obtain vanillin [8], the reaction equation is as follows Figure 7.

There is no need to add catalyst and oxidant in this method, and there is no need to separate the guaiacol carbonyl carboxylic acid (salt) generated in the reaction, after adding strong acid to adjust the reaction solution to weak acidity, the decarboxylation will get the vanillin crude, and then the high purity product will be refined. Electrolytic oxidation method has high reaction selectivity, high purity and quality of products, high yield, easy to obtain raw materials, simple equipment, greatly reducing the impact of strong oxidant heavy metal pollution on the environment, if we can continue to reduce power consumption, the industrial value of this method will be greatly highlighted.

3 Natural Plant Extraction and Preparation Technology of Vanillin

Vanillin is widely found in natural plants in free form and as glucosides, especially in primary processed vanilla beans, with a content of about 20 g/kg (dry weight). The world production of vanilla beans is mainly concentrated in Madagascar, Indonesia, Comoros Islands, etc. The annual production of vanillin can reach 2000-2400 tons, which accounts for about 2% of the total production of vanillin in the world [9].

Natural vanillin is usually used in the form of vanilla bean tincture, which is obtained by chopping primary processed and aged vanilla beans into an extractor, extracting them with 95% ethanol at 50~60 ℃, and then filtering them to obtain the bean tincture. In order to avoid hydrolysis, oxygenation and esterification of vanillin during the process, some scholars [10] used supercritical CO2 extraction technology to extract vanillin from vanilla beans, the process does not contact with the air, no solvent residue, and higher yields.

The vanillin extracted from natural plant - vanilla bean has unique aroma that cannot be compounded by artificial methods, and its status cannot be replaced, but due to the limited area of vanilla planting, the production is greatly affected by climate, the crop planting needs artificial pollination and processing is too labor-intensive, the supply of pure natural vanillin is far from meeting the market demand. The supply of pure natural vanillin is far from meeting the market demand.

4 Technical study on the preparation of vanillin by bioconversion method

In recent years, with the great influence of international and domestic natural and healthy market consumption concepts, the demand for natural vanillin is growing rapidly, and vanillin produced by biotechnology is defined as "natural" by the food regulations in Europe, the United States, Japan and other countries around the world[11] , therefore, the study of natural vanillin produced by biotechnology has become a hot topic for researchers from all over the world. Therefore, the research on the preparation of natural vanillin by biotechnology has become a hot topic for researchers in different countries. The main types of biotechnological methods for the preparation of natural vanillin are microbial fermentation, plant cell culture and enzymatic methods.  4.1 Preparation of natural vanillin by microbial transformation of natural raw materials as substrates Microbial transformation of vanillin is the use of microbial fermentation to simulate the secondary metabolic process of plants to produce vanillin.

Microbial fermentation is based on natural eugenol, isoeugenol, ferulic acid, glucose and other compounds as raw materials, through the fermentation of bacteria, molds and so on, so as to synthesize vanillin [12]; its advantage is that there is no need to purify the enzyme, so the cost is low, and can be further reduced through cell immobilization and other measures to reduce the cost of production and the reduction of by-products, so microbial fermentation has become a trend of production of bio-vanillin in recent years [13]. Therefore, microbial fermentation has become a trend for the production of bio-vanillin in recent years. Ferulic acid, eugenol or isoeugenol are the most studied substrates for vanillin production by microbial transformation.

4.1.1 Biotransformation with eugenol or isoeugenol as substrates

Eugenol is mainly derived from essential oils such as clove leaf oil, however, eugenol has an effective bactericidal effect and is toxic to microorganisms, inhibiting normal growth and metabolism[13] Therefore, screening for microorganisms tolerant to eugenol or isoeugenol is one of the keys to this method.

Ashengroph et al. isolated a strain of Pseudomonas resinovorans SPR1, which was able to obtain 0.24 g/L vanillin by fermentation for 30 h using eugenol as the sole carbon source and energy without further optimization [14]. Zhao Li-Qing et al. screened and obtained a spindle bacillus Bacillus fusifornis CGMCC134 from soil, which could tolerate high concentration of isoeugenol and efficiently convert vanillin, and this bacterium could be converted in a two-phase system of isoeugenol-water, under the conditions of 60% isoeugenol v/v, initial pH=4.0, temperature 37 ℃, rotational speed 180 r/min, and 72 h. The results of vanillin were obtained under the condition of 72 h, and the results were summarized as follows Under the condition of 72 h, the mass concentration of vanillin was as high as 46.1 g/L [15].

In view of the various research reports, it has been difficult to make a breakthrough in the bioconversion of eugenol as a substrate for the preparation of vanillin.

4.1.2 Biotransformation by Ferulic Acid as a Substrate for Fermentation Preparation

Ferulic acid is a derivative of cinnamic acid, which is one of the components of the cell wall, and it is widely found in cereals, wheat and corn bran, sugar beet crops or grain residues, and it is abundant in nature and has no toxic effect on the bacterium, and it has a similar chemical structure with vanillin, and it involves fewer reactions in biotransformation, which is conducive to the improvement of the conversion rate [16], therefore, ferulic acid is an ideal microbial fermentation feedstock. Therefore, ferulic acid is an ideal raw material for microbial fermentation. Therefore, ferulic acid is an ideal raw material for microbial fermentation. The transformation pathway is shown in Figure 8.

In 2000, Rabenhorst et al. obtained 11.5 g/L of vanillin from Streptomyces sp. HR167 using ferulic acid as substrate by optimizing the fermentation conditions and reducing the toxicity of vanillin to the bacterium by adsorption of resin[21] . In 2000, Rabenhorst et al. used Amycolatopsis sp. HR167 as a substrate, and obtained 11.5 g/L vanillin by optimizing the fermentation conditions and reducing the toxicity of vanillin to the bacterium with the help of adsorbent resin[21] . In 2007, Hua et al. utilized Streptomyces sp. V-1 to obtain 19.2 g/L vanillin with ferulic acid as substrate and adsorbent resin after 55 h of fermentation, with a total molar yield of 54.5 %[22] . This is the highest yield ever achieved for the production of natural vanillin by microbial transformation using ferulic acid as substrate.

The above-listed high vanillin-producing strains, Amy-colatopsis and Streptomyces (with vanillin yields higher than 10 g/L) [23], are Gram-positive bacteria, which can be optimized to obtain considerable yields of vanillin, but the most difficult challenge for industrial application is the isolation and purification of the downstream products. The biggest challenge in fermentation with these strains in industrial applications is the downstream product isolation and purification technology. Due to the dense mycelium of actinomycetes, the fermentation broth is very viscous, which makes the product purification very difficult, resulting in high cost of the downstream process and low overall economic efficiency.

4.1.3 Preparation of vanillin by fermentation with glucose as substrate

Glucose can be obtained from starch hydrolysis, which is a sufficient raw material and has a low production cost.Li et al[24] used a genetic engineering method to genetically recombinant Escherichia coli (Escherichia coli KL7/pKL5.26A or KL7/3KL5.97A) so that the bacterium synthesized vanillic acid from glucose via the pentose phosphate pathway and the mangiferic acid pathway, and then vanillin was reduced and produced by the arylaldehyde dehydrogenase isolated from Neurospora crassa (Neurospora crassa). Then, vanillic acid was reduced by aromatic aldehyde dehydrogenase isolated from Neurospora crassa to produce vanillin.

In 2009, Hansen et al. genetically engineered two common yeast strains, Schizosaccharomyces pombe and Saccharomyces cerevisiae, using glucose as the initial substrate, and introduced three and four exogenous genes of different origins (molds, bacteria, and humans) into the two strains, respectively. At the same time, the gene for vanillin degradation in the original strain was knocked out, and 65 mg/L and 45 mg/L vanillin were obtained without other optimizations [25].

Compared with the expensive ferulic acid, glucose as fermentation substrate is extremely abundant and economical, and the metabolic pathway is simple and controllable, which makes it possible to realize industrial production, and this is also the most important topic for the production of natural vanillin in the U.S.A. Certainly, in order to make glucose as substrate bioconversion method become a practical technology for the industrialization of the production of natural vanillin, it is necessary to continue to improve its yield.

4.2 Preparation of vanillin by plant cell culture method

Plant tissue culture is a technique that enables plant cells to synthesize metabolites in the culture medium along with the development of biotechnology.In 1989, Knuth et al.[26] used vanilla as the material for high-density cell culture, and the mass concentration of vanillin in the fermentation broth was up to 1.9 g/L, and the yield could be regulated by phytohormones.In 1991, Knuth et al. showed that a complex vanillin scented substance was secreted from a vanilla guiding tissue cell suspension culture, which was extracted with activated charcoal for 14 days without adding precursors, and 0.099 g/L of vanillin could be obtained. In 1991, Knuth et al. showed that vanilla healing tissue cell suspension cultures secreted a complex vanillin-flavored substance, and 0.099 g/L vanillin was obtained after continuous extraction with activated charcoal for 14 d without the addition of precursors [27].

Westcott et al [28] used the roots of Vanilla planifolia in tissue culture and found that vanillin accumulated in the tissues at a rate of 0.4 g/(kg-d), and the highest concentration of vanillin in the tissues reached 7.0 g/kg . Later, some researchers found a new method to convert isoeugenol, protocatechuic aldehyde and caffeic acid into vanillin in Cap-sicum frutescens cells and Haematococcus pluvialiscell cells[29] , but the process of vanillin preparation in these cells was affected by the inhibitory effect of vanillin itself. However, in the process of vanillin preparation from these cell cultures, the yield is very low due to the influence of the bacteriostatic effect of vanillin itself, the substrate and the environment of cell growth, which makes it difficult to break through the state of laboratory research in the cell culture method.

4.3 Preparation of vanillin by enzymatic conversion

At present, the microbial transformation method has not been formally reported for industrialization, the main reason is that vanillin itself is a kind of microbial inhibitor, when the content of vanillin is increased, it can inhibit and kill microorganisms, and this is the biggest challenge faced by the microbial transformation method. To solve this problem, an effective technical solution is to select and breed some suitable microorganisms that are more tolerant to vanillin and less capable of converting vanillin into vanillic acid; at present, there has only been significant progress in the conversion of vanillin with ferulic acid, which is an expensive raw material.

Domestic scholars [30] believe that the most effective way to solve this problem should be to fully understand the characteristics of the relevant enzymes, if we can isolate the appropriate enzymes and form a large-scale production of these enzymes, and take advantage of the specificity and high efficiency of the enzymatic reaction, then the production of vanillin can be more direct and efficient.

All biometabolic conversion techniques of vanillin are enzymatic reactions, and although the mechanism of action of the various biotransformation preparations is not yet fully understood, it is certain that they are all produced by the action of one or more enzymes [31]. In the technical study of enzymatic synthesis of vanillin, van den Heuvel Robert HH et al. from the University of Wagneingne, the Netherlands [34] found that vanillin can be produced by vanillin oxidases (VAOs) through two routes. One is the production of vanillin from wood tar alcohol via vanillyl alcohol in the presence of VAO. Vanillyl amine is converted by VAO under alkaline conditions to an intermediate that can be hydrolyzed directly to vanillin.

In 2001, Gatfield I-L et al [32] proposed the enzymatic conversion of vanillin using lipases Chirazyme L-2, c-f, and C2 lyo as catalysts with isoeugenol and pine aldehyde as substrates, which resulted in vanillin quality fractions of 30.4 and 83.1 %, respectively. In 2004, Sun Zhihao et al [33] investigated the preparation of vanillin by conversion of isoeugenol with soybean lipoxygenase. In 2004, Sun ZH et al. [33] investigated the conversion of isoeugenol by soybean lipoxygenase to vanillin. The molar conversion was 13.27 %, and 24.53 % was achieved by the addition of the macroporous cationic resin HD-8 for the adsorption of vanillin.

Conversion of vanillin by enzyme method has become a promising research direction because of its easy accumulation of products, few by-products, easy purification, mild reaction conditions, low energy consumption and low pollution. Of course, how to make use of the theory of enzymology, chemical engineering and modern biotechnology to carry out directional transformation and modification of the existing enzymes, research on their immobilization technology and develop suitable multi-enzyme reactors will be the main tasks facing the manufacturers in the production of vanillin by enzyme method.

5 Prospects

The vanillin produced by chemical synthesis will still occupy the dominant position in the market for a long time due to its rich and stable raw material source, high yield and low cost, etc. Of course, chemical synthesis uses toxic and harmful raw materials such as guaiacol, p-cresol, strong acid and strong alkali, heavy metal and so on, and many by-products of the process pollute the environment and the three wastes are serious.

At a time when China attaches great importance to environmental protection, it is undoubtedly a major issue for practitioners to research and develop a synthesis process with high conversion rate and zero emission of three wastes to realize green production. Throughout the synthesis process routes, with the continuous breakthrough of domestic guaiacol and glyoxylate production technology, and the supply of raw materials with high quality and low price is basically realized, the synthesis of vanillin by organic electrochemical method will become the green process with the most promising development prospects. The synthesis of vanillin by organic electrochemical method will become the most promising green process; it needs no oxidizer, simple equipment, safe operation, few by-products, high purity of products, little sewage and easy to be treated. Once the problem of unit power consumption is solved, the current guaiacol-glyoxylate method will be quickly replaced for the production of vanillin.

The production of natural vanillin by phytoextraction method is limited by the difficulty of vanilla cultivation, and more research on its extraction technology will always be a half-measure with twice the effort. However, as natural vanillin exists in a wide range of plants, exploring the process of enriching the components from various vanillin-rich natural plant extracts and then isolating and purifying the natural vanillin will naturally become a promising technology option with the advancement of bioengineering technology.

With the rise of "natural" fever, the preparation of natural vanillin by bioconversion method has undoubtedly become the most popular topic of vanillin research. In the past decade, the technical research on the production of natural vanillin by microbial conversion method has been extensive and in-depth, and a lot of important research results have been achieved from the metabolic pathway to the study of key genes at molecular level. In China, combining the advantages of natural resources such as rice bran and sugar beet, the research and development of microbial fermentation of ferulic acid and isoeugenol as substrate for the production of vanillin has made a great breakthrough in the past few years.

However, in the past two years, the microbial fermentation of glucose as substrate for vanillin production has made obvious progress in the United States and Europe, and this method is extremely abundant and inexpensive, with a simple and controllable metabolic pathway, and the isolation and enrichment of vanillin is simpler than that of ferulic acid. By choosing suitable enzymes and fermenting with glucose as substrate, the production cost of vanillin is expected to be close to that of synthetic technology, which can satisfy the needs of consumers and producers, and it is a practical technology with more application prospects. It is reported that the development project of yeast-based glucose fermentation for natural vanillin production between IFF and Evolva Holding SA will enter the stage of small batch trial production. Although substantial progress has been made in the biosynthesis of vanillin, there are still many scientific issues that need to be explored and researched in depth, and the industrial production of vanillin with high efficiency needs to be further optimized. Of course, with the help of advanced molecular biology and genetic engineering, cheaper natural vanillin is expected to be realized in the near future.

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