Under alkaline conditions, guaiacol made from pyrocatechol reacts with glyoxylic acid to generate 3-methoxyhydroxyphenylacetic acid, and then undergo oxidative decarboxylation and acidification reactions to synthesize vanillin.
Obtain a vanillin salt by reacting eugenol with oxygen in the presence of a strong base and a catalyst, and then convert the salt to vanillin by reacting it with an acid 5. Lignin is a renewable resource that can be obtained from various sources. It has a high content in waste wood, straw and pulp waste liquid, mainly in the form of sodium lignosulfonate. Sodium lignosulfonate is hydrolyzed under alkaline conditions and then goes through oxidative acidification to obtain vanillin.
By the way, eugenol is also used as raw material to biosynthesis vanillin. Keep reading and it will be mentioned later. The natural sources of eugenol ex clove oil and ferulic acid are commonly used as substrates, and microorganisms such as yeasts, fungi, and bacteria as production hosts to achieve the bioconversion.
As its quality aroma is close to natural vanillin, it is favored by the top food and beverage manufacturers. The global market demand for vanillin made from these two sources continues to grow and has become more and more popular in the high-end market.
The current annual demand is to tons, and its demand is mainly from the world Well-known fragrance and flavor companies. Eugenol naturally exists in a variety of essential oils. Its content is high in clove oil, bay leaf oil and clove basil oil.
Eugenol from clove oil is mainly used as the raw material in the market. Ferulic acid is one of the most abundant phenolic compounds in nature. It can be found in the cell walls of rice bran, wheat bran, corn bran, and sugar beet crops. Image Source. It has a chemical structure similar to vanillin, and involves fewer reactions in bioconversation, which is beneficial to increase the conversion rate. Therefore, ferulic acid is an ideal raw material for microbial fermentation.
An organic compound, a white crystalline powder with a creamy scent of vanilla in smell and taste. Easily affected by light, and gradually oxidized in the air. It is a phenolic aldehyde, its functional groups include aromatic ring, aldehyde group, hydroxyl phenolic, and methoxy group. Vanillin is often used as a flavoring agent to boost milk flavor in sweet food, such as in the following food list:.
Vanillin is also used to formulate a variety of flavors and fragrances, for example, in the flavors of pineapple, cherry, banana, cocoa, fruit blends and etc. Since the flavor of ethyl vanillin is stronger than vanillin and can last for a long time, vanillin is often used together with ethyl vanillin. Under the same conditions, using a 70 mM phosphate buffer pH 9. To evaluate, in more detail, the effect of the initial substrate concentration on vanillin production, we carried out time course experiments at ferulic acid concentrations of 14 and 20 mM batch mode or using a fixed volume fed-batch approach in which ferulic acid was entrapped and released from an agarose-gel matrix.
Results reported in Figure 7 showed that, during the first 6 h of incubation, the increase in the initial ferulic acid concentration from 14 to 20 mM had no significant effect on ferulic acid consumption and vanillin accumulation rate in batch mode, whereas, in agreement with RSM data presented before, a significant difference in the vanillin concentration was detected after 24 h incubation.
Increasing the initial ferulic acid concentration from 14 to 20 mM, the maximal level of vanillin decreased from 8. In contrast, significant differences in the bioconversion rates and vanillin accumulation profile were observed comparing experiments carried out in batch and fed-batch mode.
The use of the sol-gel technology to encapsulate ferulic acid and modulate its release in the liquid phase delayed the exposure of the cells to high substrate concentrations and allowed their adaptation to ferulic acid burden.
Interestingly, using ferulic acid-adapted cells in fed-batch operation mode, ferulic acid consumption and vanillin production rate increased between 2 and 6 h of incubation and the total amount of vanillin that accumulated in the medium after 24 h increased 2. Using the 70 mM phosphate buffer pH 9. Figure 7. Effect of the initial ferulic acid concentration on the production of vanillin. Experiments were carried out in triplicate in saline phosphate buffer at pH 9.
Bioconversions were carried out in batch mode, in the presence of 14 circle or 20 mM square ferulic acid, or in fed-batch mode triangle , using 12 agarose-ferulic acid cylinders 0. Ferulic acid: filled symbols; vanillin: empty symbols. Although feruloyl-CoA synthetase Fcs is a key enzyme for the coenzyme-A-dependent conversion of ferulic acid into vanillin, the effect of temperature and pH on this enzyme activity has not been described before.
Analyzing the catalytic activity profile of the enzyme produced by the ferulic acid-degrader P. Pseudomonas genes responsible for the conversion of ferulic acid to vanillin can be constitutively expressed in E.
Fcs activity is dependent on the size and composition of the intracellular CoA pool and might compete with the major E. In this regard, the growth of recombinant E. An alternative strategy that could be pursued to reduce problems associated to accumulation of this enzyme activity is the modulation of fcs transcript levels by decreasing the copy number of the ferulic catabolic operon.
Single-copy integration of ech - fcs gene cassette into E. The same strategy was successfully applied to integrate the ferulic catabolic operon in different E. These results may reflect differences in cellular metabolism and physiology between E. Results reported in Table 5 indicated that the use of E. The latter result is in agreement with our previous observations that vanillin production in E.
Another remarkable result that was achieved through the use of non-proliferating cells was the demonstration that bioconversion of ferulic acid to vanillin is strongly affected by extracellular and intracellular pH. Mitra et al. Combining this information with our evidence that Fcs activity can be enhanced, about 1. Using resting cells of recombinant E. It can be predicted that extracellular acid-base disturbance of intracellular pH can influence transport and reactivity of specific compounds such as phenolic acids.
At pH 9. At the same time, as reported for ketosteroid isomerase, the model system used for enzymatic proton-transfer chemistry Kraut et al. An additional factor that can positively impact vanillin production is ATP availability. ATP is an essential cofactor for the activation of ferulic acid to the corresponding CoA thioester Figure 1 and, as demonstrated by Padan et al. In agreement with this hypothesis, we observed that the pH-dependent increase in ferulic acid consumption rate Figure 3 was consistent with changes in the level of intracellular ATP, which increased 1.
It can also be postulated that deprotonation of vanillin could negatively affect its reduction to the corresponding alcohol by E. Combining the information on the effect of pH on enzymatic activities involved in the conversion of ferulic acid to vanillin, on protonation of the substrate and the final product and on intracellular pH of E. Therefore, incubation temperature and pH affect the efficacy of the whole ferulic acid to vanillin biotransformation process through different mechanisms, which include modulation of Fcs activity, availability of the substrate ferulic acid , impact of the protonation state of the substrate on the enzyme-substrate interaction and inhibitory effects on enzymes involved in byproduct vanillyl alcohol formation.
Bioconversion experiments carried out at pH 9. In agreement with Gray and Jakob , which demonstrated that, in E. As reviewed by Korneberg , inorganic polyphosphate can act as a substitute for ATP, which is required for the activity of feruloyl-CoA synthetase Figure 1 and can be a buffer against alkali ions. In our experiments with resting cells, these properties of polyphosphates can explain the direct correlation that was observed between Poly-P hydrolysis and consumption rates of the substrate.
Schurig-Briccio et al. Vanillin is toxic to microorganisms Fitzgerald et al. It should be noted that, under our experimental conditions, vanillin yield and product selectivity decreased independently by Poly-P level Figures 4A,B. Based on these observations we can conclude that Poly-P level can have a positive effect on Fcs activity and, probably, as a buffer against alkali ions, but the response elicited by Poly-P is not sufficient to alleviate vanillin-induced stress and stimulation of detoxifying enzymes responsible for the conversion of vanillin to vanillyl alcohol.
In order to determine the optimal operating conditions of the bioconversion process, maximization of the vanillin yield and product selectivity is required. By using response surface methodology, we demonstrated that the catalytic activity of E. The elliptical contour of the response surface Figure 6A indicated that there was a perfect interaction between both independent variables.
At the extreme values of stirring speed and initial ferulic acid concentration, vanillin yield was low. These observations indicated that bioconversion of ferulic acid into vanillin is affected by several parameters: dissolved oxygen DO level that regulates the oxidation-reduction potential ORP and activity of enzymes involved in conversion of vanillin to vanillyl alcohol stirring speed ; substrate toxicity ferulic acid concentration ; shortage of acetyl-CoA and ATP both variables.
The highest predicted vanillin concentration 7. A further decrease in the stirring speed from to rpm, which resulted in a reduction of ORP and DO levels, had a negative effect on vanillin yield and led to an increase in vanillyl alcohol concentration, determining a reduction in the product selectivity Table 6.
The experimental results indicated a minimum vanillyl alcohol concentration of 0. The verification experiments carried out under the optimum conditions obtained from RSM studies ferulic acid concentration of Moreover, these results clearly indicated that in the bioconversion process of ferulic acid into vanillin with E. Interestingly, the sol-gel technology allowed us to demonstrate that the catalytic activity of E. In the fed-batch operation mode, the vanillin production rate referred to the first 6 h, increased significantly and remained high up to 24 h 2.
Interestingly, the use of saline phosphate buffer with a high concentration of potassium ions m , which reduces the alkaline stress on E. To the best of our knowledge, this is the first report in which vanillin is produced from ferulic acid using a plasmid-free E. The use of this strain under resting cell conditions allowed us to improve the vanillin yield and selectivity minimize the toxic effect of ferulic acid and vanillin.
The maximum amount of vanillin that accumulated in the liquid phase under optimized conditions was FR13 can be used as a platform strain to test metabolic engineering strategies to further improve vanillin production in E. MR and FL conceived the project and wrote the manuscript. All authors contributed to the analysis of results, read and approved the manuscript and significantly to the work. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Vanilla is also an important component of flavors such as chocolate, strawberry, caramel, and coconut. It rounds out the taste by adding creaminess, balancing sweetness, and toning or masking bitterness and acidity. A flavor wheel is how the food community tracks the specific attributes of an ingredient, food, or beverage.
The vanilla wheel used by the flavor company Fona International measures fully 29 distinct flavor characteristics.
They are grouped into 10 main categories: smoky, spicy, botanical, sulfury, sweet, creamy, medicinal, cooked, fatty, and floral. Like wine, natural vanilla grown in different places—Madagascar, Mexico, or Tahiti—has different taste and potency profiles. Madagascar vanilla, typically called Bourbon vanilla, is highly sought for its rummy taste and sweet aroma.
Food and flavor firms rely on highly trained tasters to help with the transition to natural. Potential matches are then tested with consumers. The process can take months or even years.
Food companies that abandon synthetic vanillin can turn to natural vanillin from sources other than vanilla beans. For example, Solvay makes Rhovanil Natural vanillin by fermenting ferulic acid, a by-product of rice bran oil, using a proprietary strain of yeast. The French flavor company Mane uses a different raw material, eugenol from clove oil, to make natural vanillin. Plant , DOI: High concentrations of both ferulic acid and eugenol are toxic to most microbes, as is vanillin.
Indeed, all three compounds are made by plants as antimicrobials. Obtaining a vanillin yield of more than a few grams per liter of fermentation broth requires specialized or mutated strains and often lengthy incubation periods. The biotechnology firm Evolva developed a process to get around high feedstock costs and toxicity problems by feeding glucose to a genetically modified microbe that produces vanillin glucoside. The sugar group makes vanillin much less toxic to the production organism but must be removed to get vanillin.
It is not yet clear whether vanillin made from genetically modified organisms will be adopted or marketed. GMO labeling requirements and could lend itself to no-artificial-ingredient claims. On the other hand, the Non-GMO Project says foods containing ingredients made with synthetic biology will not be allowed to carry its voluntary label. Heat and pressure in the refinery convert the lignin to vanillin.
But the process fell out of favor as papermakers changed their methods to reduce waste. That left Borregaard the only major firm to make vanillin from wood. There is also a taste difference, Byholt says, thanks to tiny amounts of other aromatic components that come from lignin.
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