How to Get Natural Vanillin from Ferulic Acid?

Abstract: Vanillin is the third largest edible spice in the world, and with the improvement of people's health consciousness, the dosage of natural vanillin is increasing year by year. In this paper, Streptomyces psammoticus OMK-4 was used as the fermenting bacterium for vanillin production, and the fermentation conditions were mapped out and optimized in a 30-L automatic replenishment fermenter. The optimal fermentation conditions were determined through the optimization of inoculum amount, incubation temperature, incubation pH, dissolved oxygen and other fermentation conditions, and the potency was as high as 25.3 g/L. The results were as follows.

Vanillin, scientifically known as 4-hydroxy-3-methoxybenzaldehyde, is found in Peruvian balsam, clove bud oil, vanilla, coffee, grapes, and brandy, and has a vanilla odor and sweet taste [1-3]. Vanillin is an important flavor that can be used as an aroma enhancer, flavoring agent, harmonizer, and aroma booster in food, tobacco, cosmetics, and agriculture.   Because of its wide range of applications, the annual demand for vanillin is growing at a rate of 10%, but the domestic production is still not enough to meet the domestic and international market demand [4-5].

Vanillin can be produced by chemical synthesis, extraction, biotransformation and enzymatic methods [6]. With the increase of people's health consciousness, the demand and price of natural vanillin have been rising continuously, and it has become a hot research topic in recent years [7] . However, the limited amount of natural vanillin extracted from plants is far from meeting the market demand, and the environmental burden caused by chemical synthesis is getting heavier and heavier, so bioconversion and enzyme methods have become the main force in the production of vanillin [8-9].

This paper adopts the bioconversion method to produce vanillin through microbial metabolism with ferulic acid as the substrate, which has short cycle, high yield and less pollution, and after optimizing the fermentation conditions, the conditions for industrialization are available.

1 Materials and Methods

1.1 Test strains

Streptomyces psammoticus OMK-4 was deposited in the Strain Room of Xiamen Omicron Biotechnology Co.

1.2 Main instruments

30 L automatic refill fermenter, Shanghai Baoxing Biotechnology Co., Ltd; high performance liquid chromatography (HPLC), Agilent, 1260 Ⅱ; biosensor, Shandong Academy of Sciences, SBA-40E; spectrophotometer, Shimadzu, UV-1780; pH meter, Mettler, S-210S.

1.3 Culture media

1.3.1 Seed media

Soluble starch 1.0~3.5 g/L, potassium dihydrogen phosphate 0.1~0.5 g/L, urea 0.1~0.3 g/L, magnesium sulfate 0.05~0.10 g/L, calcium carbonate 0.1~0.3 g/L, yeast leaching powder 0.1~1.0 g/L, corn syrup 0.1~1.0 g/L, ammonium sulfate 0.1~0.6 g/L, ferulic acid 0.2 g/L.

1.3.2 Fermentation media

Soluble starch 2.0~5.0 g/L, potassium dihydrogen phosphate 0.1~0.3 g/L, urea 0.1~0.5 g/L, magnesium sulfate 0.05~0.1 g/L, calcium carbonate 0.5~2.0 g/L, yeast extract 0.1~1.0 g/L, ammonium sulfate 0.1~0.5 g/L, and avic acid 2.0 g/L. The following are some examples of the soluble starch used in the preparation of the sample.

1.4 Cultivation methods

1.4.1 Seed culture

Under aseptic conditions, the inoculation spatula was used to insert a full ring of well-grown strains from the cultured solid slant into the sterile seed medium, the initial pH of the seed medium was 5~8, and the organisms were cultivated to the logarithmic growth stage under the conditions of incubation temperature of 28~35 ℃ and rotational speed of 200~500 r/min.

1.4.2 Fermenter cultures

The seed liquid cultured to logarithmic growth stage was added to the fermentation medium under aseptic conditions at a volume of 5%~15%; the initial pH of the fermentation medium was 7.2~7.8, and the fermentation was carried out for 70~120 h under the conditions of temperature of 30~40 ℃, churning speed of 200~500 r/min, and aeration rate of 1︰0.5. The fermentation was carried out under the conditions of 1︰0.5, and the fermentation was carried out under the conditions of 1︰0.5.

1.5 Optimization of fermentation conditions

1.5.1 Effect of pH on vanillin fermentation

The pH of the fermentation process not only affects the existence of the substrate, but also correlates with the osmotic pressure of the cells, which affects the entry and exit of the substrate into and out of the cells. In order to obtain the optimal fermentation pH, the effect of different pH control on the potency of vanillin was investigated, and the specific methods were as follows: (1) the pH of the whole process of fermentation was controlled to be 7.5; (2) the pH of the fermentation process was controlled to be 7.5 for 0-12 h, and then the pH of the subsequent fermentation process was controlled to be 8.0;

(3) The pH was controlled at 8.0 throughout the fermentation process.

1.5.2 Effect of dissolved oxygen on vanillin fermentation

Experiments have shown that the control of dissolved oxygen in the fermentation broth has a great influence on the growth of the bacteria and the accumulation of the products in the later stage, therefore, it is necessary to control the dissolved oxygen in the fermentation process. In order to obtain the optimal control of dissolved oxygen in fermentation, the effects of different dissolved oxygen controls on the growth of bacteria and the accumulation of products were investigated. The following experiments were carried out: 0%, 10%, 20%, 30%, 40% and 50% of the dissolved oxygen was controlled by rotational speed and aeration, respectively.

1.5.3 Effect of incubation temperature on vanillin fermentation

Temperature has an important effect on microbial production, metabolite synthesis and accumulation. Elevated temperature can accelerate the growth and metabolism of microorganisms, but too high temperature can also lead to the premature decline of the organism and affect the accumulation of products. Therefore, it is important to optimize the temperature of the strain [10]. In order to get the optimal fermentation culture temperature, we investigated the effect of different culture temperatures on the growth of the bacteria, and compared the growth of the bacteria and the accumulation of vanillin by controlling the temperatures at 25 ℃, 30 ℃, 35 ℃ and 40 ℃, so as to get the optimal temperature control method.  

1.5.4 Effect of inoculum amount on vanillin fermentation

Different inoculum amounts have important effects on the normal growth and metabolism of the bacteria, too little inoculum will lead to slow growth of the bacteria, the delay period of the growth of the bacteria will be longer, and the complexity of secondary metabolites is not conducive to the accumulation of the later products; too much inoculum will lead to the overgrowth of the bacteria, and the medium will increase the nutrient depletion, and the dissolved oxygen will not be able to meet the control requirements [11]. Therefore, it is important to control the amount of fermentation inoculum. In order to obtain the optimal fermentation inoculum level, the effects of different inoculum levels on the growth of bacteria were investigated, and the growth of bacteria and the accumulation of vanillin were compared by five gradients of inoculum levels of 1%, 3%, 5%, 7% and 9%, so as to obtain the optimal inoculum level.

1.6 Methods of analysis

Determination of bacterial concentration: Take the culture solution and dilute it with distilled water for a certain number of times, then mix it well and measure the absorbance at 620nm using a spectrophotometer.

Determination of residual sugar: Ferrin's reagent titration was used [12]. Determination of vanillin content: HPLC [13].

2 Results and Discussion

2.1 Effect of pH on vanillin fermentation

As shown in Fig. 1, the growth of the bacteria was better at pH 7.5, with the maximum OD620 of 0.62; the growth of the bacteria was worse at pH 8.0, with the maximum OD620 of 0.41; and the growth of the bacteria was better at pH 7.5 for 0-12 h, with the maximum OD620 of 0.64 at the subsequent control of 8.0. Therefore, the growth of bacteria was facilitated by the pH 7.5, and the growth of bacteria was slower when the pH was higher. The higher the pH, the slower the growth of the bacteria. From the segmented pH control, it can be seen that the growth of the bacteria was not affected when the pH was adjusted to 8.0 at the later stage, so the bacteria had basically reached the stabilization stage at about 12 h of fermentation.

From Fig. 2, it can be seen that the vanillin concentration was 13.200 g/L, 16.988 g/L and 14.500 g/L under different pH control, and the vanillin conversion rate was the highest at pH 7.5 from 0 to 12 h, and then at pH 8.0, followed by pH 8.0, and was poorer at pH 7.5, which may be due to the fact that enzyme activity was better than that of pH 7.5 at pH 8.0. This may be due to the fact that the enzyme activity at pH 8.0 was better than that at pH 7.5.

Combining Figures 1 and 2, it can be summarized that the growth of the bacteria was better at pH 7.5, and the enzyme conversion rate was higher at pH 8.0. Therefore, it was chosen that the segmentation control during fermentation would help the growth of the bacteria and obtain better enzyme activity.

2.2 Effect of dissolved oxygen on vanillin fermentation

As can be seen from Fig. 3, with the increasing of dissolved oxygen, the concentration of bacteria was increasing, in which the growth of bacteria was the worst at 0% of dissolved oxygen, basically no growth of bacteria; at 40%~50% of dissolved oxygen, it was the optimal growth condition for bacteria, and the growth of bacteria was the highest, and the OD620 was 0.75. In the case of increasing dissolved oxygen, the time for the bacteria to enter the logarithmic growth period gradually decreases, and when the dissolved oxygen reaches 40%~50%, the adaptive period for the growth of the bacteria is the shortest, and the bacteria can enter the logarithmic growth period quickly, thus shortening the fermentation cycle. Therefore, it can be judged that the growth of the bacteria needs a large amount of oxygen supply, and the growth of the bacteria is best when the dissolved oxygen level is 40%~50%.

From Fig. 4, it can be seen that the accumulation of vanillin showed a gradual increase and then a slow decrease with the increase of dissolved oxygen, and the best accumulation of vanillin was found at about 30% of dissolved oxygen, and the highest accumulation was about 17.5 g/L. The low accumulation of vanillin at low dissolved oxygen might be due to the poor production of the bacteria, the insufficient enzyme or the low dissolved oxygen condition affecting the enzyme vitality. With the gradual increase of dissolved oxygen, the accumulation of vanillin gradually showed a decreasing trend, especially at 50% of dissolved oxygen, the decrease of vanillin occurred in the late stage of fermentation, and it was found that the accumulation of vanillic acid as a by-product gradually increased, which was suspected to be due to the excessive dissolved oxygen that led to the metabolism flow biased towards the production of vanillic acid. It is suspected that the dissolved oxygen level is too high, which leads to the bias of the metabolic flow towards vanillin production. Therefore, the dissolved oxygen level should not be too high in the vanillin production stage to avoid affecting the overall conversion rate.

As can be seen from Figs. 3 and 4, the control of dissolved oxygen in the fermentation process was selected to be controlled in stages, in which the dissolved oxygen was controlled at 40%~50% in the stage of bacterial growth to ensure the accumulation of the volume of bacteria and to shorten the growth cycle of the bacteria, and the dissolved oxygen was controlled at 30% in the stage of vanillin production in order to ensure the rapid accumulation of the product.

2.3 Effect of incubation temperature on vanillin fermentation

From Fig. 5, it can be seen that the growth of the bacteria was slow when the temperature was low, and the concentration of the bacteria was higher and higher with the increase of the incubation temperature, and the concentration of the bacteria was the highest when the temperature reached about 35 ℃, and the OD620 reached 0.73; however, the concentration of the bacteria began to decrease with the increase of the temperature to 40 ℃, which was probably due to the destruction of the normal metabolism of the bacteria by the high temperature.

As can be seen from Fig. 6, with the gradual increase of temperature, the conversion rate of vanillin was higher and higher, and the conversion effects were 35 ℃, 30 ℃, 40 ℃ and 25 ℃ in the order of magnitude; according to the calculation of the conversion rate of each bacterium, the optimal temperatures were 40 ℃, 35 ℃, 30 ℃ and 25 ℃ in the order of magnitude. The optimum temperatures were 40 ℃, 35 ℃, 30 ℃ and 25 ℃. It can be seen that the conversion rate of vanillin was higher and higher with the gradual increase of temperature, and the conversion rate of vanillin at higher temperatures was better than that of vanillin at lower temperatures.

Comparing Figures 5 and 6, it can be seen that the effect of temperature on the fermentation and vanillin conversion of OMK-4 strain was greater, and the temperature of 35 ℃ was chosen for the culture of the strain and the conversion of vanillin.

2.4 Effect of inoculum amount on vanillin fermentation

From Fig. 7, we can see the effects of different inoculum amounts on the final concentration of bacteria, five different inoculum amounts can reach a concentration of about 0.7, among which 1% of the inoculum has a worse effect, the highest concentration is only about 0.65.   In the first 10 h, with the increase of strain amount, the delay period of fermentation was obviously shortened, comparing with 1% and 9%, the delay period was obviously shortened by about 6 h. From Fig. 8, it can be seen that 9% of the inoculum was more effective than 1% of the inoculum. From Fig. 8, it can be seen that the inoculum amount of 9% was obviously worse than the other four inoculum amounts, probably due to the fact that the bacteria were mainly supplied in the process of cultivation and the enzyme vitality would be reduced with the increase of the concentration of the bacteria. Therefore, the inoculum amount should not be too large in the inoculation process. Comparing the inoculum amount of 1%, 3%, 5% and 7%, the conversion rate of vanillin was slightly higher than the other four inoculum amounts, and the highest conversion amount was 17.21 g/L, 17.23 g/L, 18.05 g/L and 17.27 g/L. Considering the results of Figs. 7 and 8, the optimal inoculum amount of 5% was finally selected.

3 Conclusion

The optimal fermentation conditions obtained from the optimization of the 30L auto-recharging fermenter were: pH 7.5 and dissolved oxygen 40%~50% from 0 to 12 h, and then pH 8.0 and dissolved oxygen 30%; the fermentation temperature was 35 ℃ throughout the fermentation, and the inoculum quantity was 5%. The fermentation temperature was 35 ℃, and the inoculum quantity was 5%. The fermentation was carried out under the optimal conditions for 36 h, and the final potency of vanillin was as high as 25.3 g/L, which was a good result in the pilot experiment.

References:

[1] Ou Shiyi , Li Aijun , Bao Huiyan , et al. Progress of biological production of vanillin [J]. Guangzhou Food Industry Science and Technology ,2000,20(2):119-121.

[2] Cuizhu Wang . Safe, Healthy, Delicious , Make Better Vanillin Products and Solutions--Interview with Liu Yang, General Manager of Asia-Pacific Region, and Huang Haobo, Director of Global Strategy and Marketing, Solvay Flavors and Performance Chemicals Division [J]. Food Safety Journal ,2019,238(13):28-30.

[3] YANG Wenwen . "Progress of biosynthesis of natural vanillin, the queen of spices [J]. Microbiology Bulletin ,2013,40(6):1087-1095.

[4] Fan Y. Research Progress of Microbial Conversion of Ferulic Acid as Substrate for the Production of Vanillin [J]. Grain and Food Industry ,2017,24(6):43-47.

[5] Xu Congwu . Countermeasures to improve the profitability of vanillin export in China [J]. Journal of Chifeng College (Natural Science Edition),2014(2):82-84.

[6] Zhen Da , Wu Xiaogang , Deng Bei , et al. Application of microbial synthesis of vanillin [J]. Food Research and Development ,2012(3):144-149.

[7] Lv Xiaojie . Development status of vanillin industry [J]. Modern Food ,2019,7(5):14-16.

[8] Zhang Jianbin . Research progress and prospect of vanillin preparation technology [J]. Guangdong Chemical Industry ,2018(12):163-166.

[9] Horvat M, Fiume G, Fritsche S, et al. Discovery of carboxylic acid reductase (CAR) from Thermothelomyces thermophila and its evaluation for vanillin synthesis[J].Journal of biotechnology,2019,8(7):44-51.

[10] Nida K, Aamer A S, Sadia Q. Optimization of pH and temperature for degradation of tyre rubber by Bacillus sp. strain S10 isolated from sewage sludge[J]. .International Biodeterioration & Biodegradati on,2015,5(9):154-160.

[11] WANG Haopeng , YANG Meng , WU Liming , et al. Optimization of fermentation conditions and pilot test of fumonisin 5L tank [J]. China Brewing ,2019,38(2):98-103.

[12] Zhang Shengzhen , Ma Yanzhi . Comparison of methods for the determination of total sugar content in apples [J]. Jiangsu Agricultural Science 2009(2):259-260.

[13] HUANG Yuhuan , LIU Hongzhou , YU Ruichen . Determination of vanillin by high performance liquid chromatography [J]. Subtropical Plant Science ,1998,27(2):17-20.