A comprehensive review of eclectic approaches to the biological synthesis of vanillin and their application towards the food sector

Abstract

Vanillin, a highly regarded flavor compound, has earned widespread recognition for its natural and aromatic qualities, piquing substantial interest in the scientific community. This comprehensive review delves deeply into the intricate world of vanillin synthesis, encompassing a wide spectrum of methodologies, including enzymatic, microbial, and immobilized systems. This investigation provides a thorough analysis of the precursors of vanillin and also offers a comprehensive overview of its transformation through these diverse processes, making it an invaluable resource for researchers and enthusiasts alike. The elucidation of different substrates such as ferulic acid, eugenol, veratraldehyde, vanillic acid, glucovanillin, and C₆–C₃ phenylpropanoids adds a layer of depth and insight to the understanding of vanillin synthesis. Moreover, this comprehensive review explores the multifaceted applications of vanillin within the food industry. While commonly known as a flavoring agent, vanillin transcends this role by finding extensive use in food preservation and food packaging. The review meticulously examines the remarkable preservative properties of vanillin, providing a profound understanding of its crucial role in the culinary and food science sectors, thus making it an indispensable reference for professionals and researchers in these domains.

Graphical abstract

Introduction

Vanillin stands as a notable aromatic flavor constituent found in substantial quantities within the pods of the Vanilla planifolia, a type of vanilla orchid plant. Vanillin stands as a crucial ingredient in the food, beverage, and fragrance sectors, renowned for its unique vanilla odor and flavor associated with vanilla beans (Peña-Barrientos et al., 2023). Vanillin, an essential component of natural vanilla, has been regarded as one of the most significant and frequently employed flavoring agents globally. Vanillin possesses several biological properties, serving as an antioxidant, anti-inflammatory agent, and antimicrobial agent, all of which are depicted in Fig. 1. The earliest documented utilization of cultivated vanilla can be traced back to the Aztec civilization of Mexico. The cultivation of V. planifolia andrews, a particular species of vanilla orchid, was undertaken among 110 other species and the cultivation was done primarily for its aromatic properties and its use as a flavoring agent in coffee and chocolate (Teoh, 2019). Based on recent research, the worldwide bio-vanillin market is poised to exhibit strong growth, with a projected annual growth rate of 7.4% spanning from 2017 to 2025 (Luziatelli et al., 2019).

Fig. 1.

Illustration of various biological characteristics exhibited by vanillin and its application

The vanillin market has often surpassed the supply of natural vanilla beans, prompting research into alternative methods and sources for its production (Fache et al., 2016). The situation has been influenced by multiple factors, including the limited availability of natural vanilla, its high cost, and the increasing market demand for products with vanilla flavor (Sinha et al., 2008). Diverse varieties of lignin are employed in the manufacturing of vanillin, yet the resulting yield of vanillin from these sources consistently fails to meet the desired standards (Aarabi et al., 2017; Zhang et al., 2017). Several methods have been devised to meet the demand for vanillin, namely physical, chemical, and biological methods. Although physical methods of producing vanillin offer certain advantages, they also entail certain drawbacks (Tarabanko and Tarabanko, 2017).

With a market demand for approximately 20,000 tons of vanillin and a natural supply of less than 2000 tons, there is growing interest in exploring alternative methods for the production of vanillin (Braga et al., 2018). Chemical production of vanillin involves synthesizing the compound from various precursor compounds. The substrates frequently employed for vanillin biosynthesis include ferulic acid, eugenol, and guaiacol (Banerjee and Chattopadhyay, 2019). Most vanillin (95%) is currently synthesized chemically from lignin and guaiacol. Bio-based vanillin, derived from sources like eugenol and ferulic acid, is gaining popularity, particularly eugenol-based bio-vanillin in the high-end food and beverage market for its robust aroma and safety, despite its limited production yield (< 1 g/L). Indonesia, a major clove oil producer, excels in this method. Ferulic acid can efficiently convert to vanillin with low environmental impact and robust food safety standards, reaching impressive titers, up to 22.30 g/L. The utilization of synthesized vanillin is subject to strict limitations in the food and pharmaceutical sectors under UK and EU regulations, being classified as an artificial substance (Zamzuri and Abd-Aziz, 2013).

There is a greater emphasis on the production of vanillin through biotechnological methods involving microbial, enzymatic, or immobilized system from a range of raw materials (Kumar and Pruthi, 2014). The enzymatic approach entails utilizing enzymes in vanillin biosynthesis through various substrates (Klaus et al., 2019). Using microorganisms to produce vanillin from agricultural byproducts is commonly known as the microorganism-based approach (Shaaban et al., 2016). The agricultural byproducts function as substrates for microorganisms, thereby enabling the generation of vanillin as an intermediate product (Paul et al., 2021). Another notable challenge to implementing technology for vanillin production from lignin pertains to the separation of structurally analogous byproducts resulting from lignin depolymerization (Tarabanko et al., 2013).

Regarding microbial biosynthesis, the lignin-degrading white rot fungi Pycnoporus cinnabarinus was investigated explicitly as a potential alternative to traditional chemical and physical methods. This fungus produces ligninolytic enzymes that facilitate the breakdown of lignin into smaller aromatic compounds, including vanillin (Miyauchi et al., 2020). The utilization of Pycnoporus cinnabarinus for vanillin production offers numerous benefits, such as the employment of renewable resources, selectivity, potential for sustainable processes, and biodegradability (Nayak et al., 2022). In accordance with research findings, capsaicin has the capacity to activate genes that are responsible for the biosynthesis of vanillin in vanilla orchids, leading to increased levels of vanillin production (Olatunde et al., 2022). Vanillin can be synthesized by various substrates found in lignocellulosic biomass, which include ferulic acid, eugenol, glucovanillin, and verateraldehyde (Fig. 1).

Roughly 16,000 metric tons of vanillin are manufactured annually, with a value of around $650 million. Nevertheless, less than 1% of this yearly output stems from the extraction of natural vanilla from vanilla pods. As a result, there remains a consistent and significant market demand for nature-identical vanillin. In vanillin bioproduction, economic viability and technical efficiency are pivotal, as companies with lower costs and larger production capacities gain a competitive edge. Achieving this advantage involves using cheaper materials and improving bioconversion rates. Solvay, a prominent player in the vanillin industry, initiated a biotechnology platform in May 2022 with the aim of offering eco-friendly vanillin solutions. Through the innovative utilization of a rice bran byproduct, they achieved a significant milestone in September 2022 by introducing cost-effective, naturally derived vanillin flavors. This groundbreaking approach underscored the viability of commercial-scale microbial vanillin production. This review presents a comprehensive overview of the literature on the application of diverse techniques in the production of bio-vanillin from various substrates. Furthermore, the review also delves into the preservative attributes of vanillin, offering an in-depth comprehension of its vital contribution to the realms of culinary and food science.

Various methods of vanillin synthesis

The industrial-level vanillin production, approximately 12,000 tons annually, relies on chemical synthesis techniques such as glyoxylic and nitrose (Furuya et al., 2014; Hu et al., 2012). The chemical synthesis of vanillin is associated with environmental pollution and exhibits inadequate substrate selectivity, leading to decreased process efficiency and increased downstream processing costs (Xu et al., 2007). Consequently, the biotechnological production of vanillin through microbial conversion of lignin caffeic acid, veratraldehyde, eugenol, and primarily ferulic acid holds significant importance (Nayak et al., 2022). Using microorganisms in the biotransformation of natural raw materials offers an economically viable approach for synthesizing vanillin. Furthermore, the rapid development of these biocatalysts and their accessibility through molecular genetics has augmented their significance. Therefore, vanillin can be synthesized by biological synthesis (enzymatic, microbial, and immobilized processes). Different precursors used in the production of vanillin and the accompanying lignocellulosic waste are shown (Fig. 2).

Fig. 2.

Various precursor employed for the production of vanillin and its concurrent lignocellulosic waste

Ferulic acid

Ferulic acid is a naturally occurring compound that can be produced through the metabolic processes of phenylalanine and tyrosine in the cell wall and can be obtained via chemical or biological/enzymatic extraction methods (Di Gioia et al., 2011). Ferulic acid exhibits a conformation similarity to vanillin and is widely used as a precursor substrate for vanillin production.

Biological synthesis

The domain of synthetic biology has undergone substantial expansion, resulting in an increased focus on the microbial fermentation-driven production of vanillin from sustainable sources (Ciriminna et al., 2019). Enhancing the value of bamboo shoots (dried) by utilizing the novel microorganism in producing biovanillin at a high yield of 11.43 µg/mL to meet the demand of the market (Subramani and Manian, 2023). Utilizing agricultural waste as the primary source offers an alternative method to access ferulic acid cost-effectively, thereby establishing the foundation for an economically sustainable production system. By initiating the bioconversion of ferulic acid into vanillin with corncob hydrolysate biomass concentration of 1.5 g/L resulted in a notable 29.5-fold increase in the vanillin yield, reaching 477.4 mg/g (Nirwana et al., 2023).

Microbial vanillin production

The utilization of microorganisms for vanillin production is increasingly recognized for its cost-effectiveness, accessibility, streamlined process, and metabolic adaptability (Havkin-Frenkel and Belanger, 2016). Several microorganisms, such as Pseudomonas sp., Amycolatopsis and Streptomyces sp., and the various fungi were employed for the production of vanillin. Pycnoporus cinnabarinus, white rot fungi, has been suggested as a potential candidate for the production of vanillin using ferulic acid (Berger, 2009).

An experiment utilized pomegranate peels as a potential source of ferulic acid for converting into biovanillin through submerged fermentation with Enterobacter hormaechei yielding 4.2 g/L (Saeed et al., 2022). Fungal cultures of Pycnoporus cinnabarinus  demonstrated high efficiency in producing vanillin from ferulic acid in a two-step process, yielding 237 mg/L of vanillin and the addition of cellobiose increased vanillin accumulation to over 500 mg/L. Using a mixture of fungal strains, a maximum of 2.2 g/L of vanillic acid was achieved when utilizing the ferulic acid concentration of 4g/L. Transforming vanillic acid into vanillin in A. niger culture filtrate, with the aid of glucose and HZ802 resin, led to high vanillin production (Zheng et al., 2007). Amycolatopsis sp. ATCC 39116 metabolized 42 mM of ferulic acid into 12.36 mM vanillin and 2.18 mM vanillic acid via non-oxidative deacetylation and β-oxidation pathways. However, inducing ferulic acid catabolism with pre-culture treatment using ferulic, p-coumaric, and caffeic acids resulted in an over eightfold decrease in vanillin productivity in submerged culture (Contreras-Jácquez et al., 2020). The biological synthesis of ferulic acid for the vanillin production was represented in Table 1.

Table 1.

Biological synthesis of vanillin using ferulic acid via microbial and immobilized methods

System	Time	pH	Substrate concentration	Vanillin yield	References
Microbial
High-density cultures of Pycnoporus cinnabarinus in glucose-phospholipid medium	360 h	–	300 mg/ L everyday	760 mg/L	Stentelaire et al. (2000)
Using Oenococcus oeni or Lactobacillus sp.	–	5.0	3 mg/L	0–1%	Bloem et al. (2007)
Streptomyces sp. strain with V-1 Resin DM11	55 h	8.2 to 9.3	45 g/L	19.2 g/L	Hua et al. (2007)
Recombinant Pseudomonas fluorescens	1 h	–	4.5 g/L	2.5 g/L	Barghini et al. (2007)
Biotransformation of ferulic acid (rice bran oil) by Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115	36 h	–	4 g/L	2.2 g/L	Zheng et al. (2007)
Ferulic acid is biotransformed by Aspergillus niger, Pycnoporus cinnabarinus and resin	72 h	5.0	FA-4 g/L; HZ802 resin-25 g	2.8 g/L	Zheng et al. (2007)
Amycolatopsis sp. ATCC 39116	18 h	8.0	15 g/L	9.18 g/L	Ma and Daugulis (2014)
Streptomyces sannanensis biotransforms agro residue ferulic acid esters	120 h	7.5	0.7 mM	708 mg/L	Chattopadhyay et al. (2018)
Biotransformation of ferulic acid (pomogranate peels) by Enterobacter hormaechei	8 h	7.0	100 g of peels-162.5 mg of Ferulic acid	4.2 g/L	Saeed et al. (2022)
Immobilized
Immobilized plant cell cultures of Capsicum frutescens	360 h	–	2.5 mM	315 μg/culture	Sudhakar Johnson et al., 1996)
Amycolatopsis sp. ATCC 39116 transforms ferulic acid into vanillin in a fed-batch solid–liquid two-phase partitioning bioreactor	18 h	8.0	48 g/L	19.5 g/L	Ma and Daugulis (2014)
Bacillus subtilis biotransforms ferulic acid into vanillin in packed bed-stirred fermenters	20 h	9.0	1.5 g/L	0.047 g/L/h	Yan et al. (2016)
FDC and CSO2 immobilized on Sepabeads EC-EA	24 h	9.0	50 mg	1st cycle-2.8 mM; 10 cycles-1.6 mM	Furuya et al. (2017)
Other methods
Roots of Capsicum frutescens in medium containing naphthalene acetic acid, ferulic acid and b-cyclodextrin complex	144 h	–	2 mM	24.7 µM	Suresh et al. (2009)
Coenzyme-independent decarboxylase/oxygenase two-stage process	24 h	10.5	75 mM	52 mM	Furuya et al. (2015)
Enhancing biocatalyst performance and optimizing the process in E. coli JM109 to maximize vanillin production efficiency	24 h	10.0	14.94 mM	8.51 mM	Luziatelli et al. (2019)
Prosopis juliflora biomass subjected to hydrothermal liquefaction for the production of ferulic acid and bio-oil	1 h	6.0	Biomass-2.5 to 12.5 g	0.3 g	Arun et al. (2021)

Enzymatic vanillin production

The pivotal enzyme in this process is feruloyl esterase, and its primary function is to catalyze the hydrolysis of the ester bond in ferulic acid, releasing vanillin and other associated byproducts (Xu et al., 2020). Feruloyl esterase is an enzyme that liberates aromatic residues, including ferulic acid, from diverse esterified substances and plant cell wall polymers (Ou and Kwok, 2004). The study investigated the transformation of ferulic acid into vanilla flavor compounds, particularly vanillin, by Capsicum frutescens roots, noting that the highest vanillin levels of 12.3 and 16.4 mM were observed on the sixth day with the addition of 1 and 2 mM of ferulic acid, respectively (Suresh et al., 2009). By exploring the ideal quantity of vanillin biosynthetic enzymes within a cell-free system, researchers achieved the development of an improved recombinant E. coli strain. This enhanced whole-cell catalyst exhibited the capability to convert ferulic acid (20 mM) into vanillin (15 mM) (Chen et al., 2023). Extracellular enzymes from Neosartorya spinosa NRRL185 were used to extract ferulic acid from corn residues, which was then converted into vanillin while yielding the notable amount of reducing sugars, ranging from 76 to 100% (Shin et al., 2006).

Immobilized systems for vanillin production

 The inhibitory action of vanillin on free microbial cells and the genetic instability of the recombinant strains during the transformation were the primary causes of the low yield of vanillin (Di Gioia et al., 2011). Furthermore, employing free microorganisms for biotransformation can incur high costs due to expensive microbial cultures, prolonged operation, and intricate subsequent treatments (Feng et al., 2015). The immobilization of microbial cells has garnered global interest due to their outstanding biological compatibility and ability to achieve stability across wide range of conditions (Yan et al., 2016). A novel immobilized technique has for synthesizing vanillin from ferulic acid has been recently developed, eliminating the need for coenzymes. The artificial pathway comprises a coenzyme-independent decarboxylase and coenzyme independent oxygenase responsible for the conversion of ferulic acid to vanillin. The immobilized enzymes yielded a concentration of 2.8 mM vanillin and 2.5 mg after 10 reaction cycles (Furuya et al., 2017). Capsicum frutescens were immobilized for the conversion of ferulic acid to vanillin. The highest observed levels of vanillin (315 μg/culture) were found in cultures treated with ferulic acid (2.5 mM) on the 15th day. Similarly, the highest levels of capsaicin (190 μg/culture) were observed in cultures treated with vanillylamine (2.5 mM) on the 6th day. After ten reaction cycles, the co-immobilization of FDC and CSO₂ enabled the system in producing 2.5 mg of vanillin from ferulic acid, marking the pioneering instance of vanillin production through immobilized enzyme biotechnology (Furuya et al., 2014).

Eugenol/isoeugenol as substrates

Eugenol (4-allyl-2-methoxyphenol), an active ingredient in clove tree oil extracted from Syzygium aromaticum, is one of the most potential raw materials for the production of vanillin (Cassiana Frohlich et al., 2022). Numerous research studies have been conducted to elucidate the process by which eugenol transforms into ferulic acid.

Microbial vanillin production

The biotransformation of eugenol yields vanillin and its related metabolites, which have been found to have numerous applications and significant economic value (Ashengroph et al., 2011). Several research endeavors have been undertaken to explore the synthesis of vanillin from eugenol, utilizing both genetically modified and natural microorganisms (Harshvardhan et al., 2017). The degradation of eugenol has been observed in various bacteria and fungi, including but not limited to Corynebacterium, Pseudomonas, Byssochlamys, Penicillium, and Rhodococcus, which have displayed its potential to degrade eugenol (Xu et al., 2007).

A proposal suggests that the process of biotransformation of eugenol by Pseudomonas sp. commences with the epoxidation of eugenol, leading to the formation of eugenol oxide, which proceeds via intermediates of ferulic acid and vanillin (Unno et al., 2007). Moreover, the optimization of production parameters resulted in the production of 0.12 g/L of vanillin without any byproducts (Singh et al., 2019). Rhodococcus strains I24 and PD630 have effectively established a biosynthesis pathway for the production of vanillin by means of non-beta-oxidative coenzyme A-dependent conversion from eugenol precursor. (Plaggenborg et al., 2006). An additional instance entails achieving a substantial product yield from an initial concentration of 0.2 g/L of eugenol, utilizing microorganisms such as Serratia, Klebsiella, or Enterobacter, with the transformation process spanning 13 days (Sinha et al., 2012).

Pycnoporus cinnabarinus MUCL 39533 was supplemented with Amberlite XAD-2 resin to adsorb the produced vanillin and prevent the transformation of vanillic acid to methoxy hydroquinone. It appears that the production of vanillin increased upon the addition of resin at lower concentrations of Span 80 (Maskat et al., 2015). Upon incubated with ferulic acid, E.coli cells hosting the decarboxylase/oxygenase cascade efficiently produced vanillin (at a concentration of 8.0 mM or 1.2 g/L) through 4-vinylguaiacol in a single reaction vessel. Notably, this process yielded no discernible aromatic by-products (Furuya et al., 2017). A maximum vanillin concentration of 2.4 g/L was achieved with a molar yield of 52.5%, using wet weight cells from the MP24 strain cultivated with isoeugenol. These cells were harvested during the exponential growth phase at a concentration of 7.5 g/L (Ashengroph and Amini, 2017). Eugenol was utilized as the substrate in a biotransformation process with P. resinovorans SPR1, resulting in the production of 1.1 g/L of vanillic acid, achieving a molar yield of 44% (Ashengroph et al., 2011). Upon the optimization of the enzyme production by Pseudomonas nitroreducens Jin1, a noteworthy vanillin productivity of 115 g/L per day was achieved, with an impressive yield of 82.3% (Wang et al., 2021).

Enzymatic vanillin production

A viable approach for the production of natural vanillin entails biotransformation, whereby isolated enzymes are employed as biocatalysts (Nayak et al., 2022). The utilization of eugenol oxidase (EUGO) as an enzyme for vanillin production in an industrial setting shows great potential. The EUGO exhibits stability and activity across a broad pH spectrum; however, the optimal points for each parameter do not aligne (Guo et al., 2022). According to a study, increased activity reduced reaction time to 4 h when utilizing soluble EUGO. As a result, there was a 6.5-fold increase in STY, leading to the eugenol production for vanillin by 9.9 g prod L⁻¹ h⁻¹. Furthermore, the employment of immobilized EUGO enabled for the recycling of the biocatalyst across up to 18 reaction cycles, resulting in over a 12-fold increase in biocatalyst yield and consequently lowering the associated biocatalyst expenses (García-Bofill et al., 2021).

Immobilized system for vanillin production

The enzyme CSO₂ promotes the transformation of isoeugenol into vanillin without relying on coenzymes. The lack of dependence on coenzymes is a favorable characteristic for the synthesis of vanillin through the utilization of immobilized enzymes (Furuya et al., 2015). CSO₂ demonstrates significant efficacy within the pH range of 9.0–10.5 despite its isoelectric point being at pH 5.3. Based on the analysis, the substrate was efficiently converted to vanillin upon incubation with isoeugenol, facilitated by the immobilized CSO₂ on Sepabeads EC-EA (Martău et al., 2021). During the 24 h biotransformation process, the immobilized enzyme produced 4.6 mM of vanillin, while the cells produced 8.4 mM. Experimental confirmation shows that a portion of the substrate, isoeugenol, along with the product, vanillin, became attached to the carrier. In the second cycle, the immobilized enzyme helped produce 6.3 mM of vanillin.

A research study was conducted to examine the accumulation of vanilla flavor compounds, specifically vanillin, vanillic acid, and ferulic acid, in both suspended and immobilized cell cultures of Capsicum frutescens following isoeugenol feeding (Nedović et al., 2015). EUGO immobilized on epoxy agarose-UAB yielded similar results to its soluble form under identical conditions. The immobilized enzyme demonstrated the ability to be reused up to 5 and 18 cycles, resulting in 138 g of vanillin production and a 30.8 mg U^-1 biocatalyst yield. This marks a substantial 12.4-fold and 3.5-fold enhancement compared to using only the soluble enzyme or reusing the immobilized enzyme, respectively (García-Bofill et al., 2019, 2021). The method for the production of vanillin by eugenol was elaborated in Table 2.

Table 2.

Biological synthesis of vanillin using eugenol through microbial, enzymatic, immobilized system

System	Time (h)	pH	Initial concentration	Yield	References
Microbial
Bacillus fusiformis and resin HB-8	72	7.0	50 g/L	8.10 g/L	Zhao et al. (2006)
Pseudomonas resinovorans SPR1	30	7.0	2.5 g/L	240 mg/L	Ashengroph et al. (2011)
Candida galli strain PGO6	60	7.0	0.1% v/v	1.12 g/L	Ashengroph et al. (2011)
Serratia, Klebsiella or Enterobacter	312		0.2 g/l	18 mg/L	Sinha et al. (2012)
Psychrobacter sp. strain CSW	48	7.0	10 g/L	1.8 g/L	Ashengroph et al. (2012)
Pycnoporus cinnabarinus MUCL 39533 incorporated with amberlite XAD-2 resin	24		3.61 g/L	161 mg/L	Maskat et al. (2015)
Resting cells of Trichosporon asahii	32	5.8	5 g/L	4.2 g/L	Ashengroph and Amini (2017)
Bacillus safensis SMS1003	96	7.0	500 mg/L	120 mg/L	Singh et al. (2019)
Natural vanillin production from Isoeugenol using Pseudomanas putidia in bio-phasic	36	6.8	400 g/L	11.95 g/L	Karakaya and Yilmaztekin (2022)
Enzymatic
Eugenol oxidase from Rhodococcus jostii (EUGO)	4	9.5		9.9 g/L/h	García-Bofill et al. (2021)
Immobilized
CSO2 immobilized on Sepabeads EC-EA	24	9.0	10 mM	6.8 mg	Furuya et al. (2017)
Immobilized recombinant cells containing isoeugenol monooxygenase active aggregates	36	10.5	100 mmol/L	14.5 mmol/L	Zhao et al. (2019)
EUGO immobilized onto epoxy agarose-UAB	4	9.5		138 g	García-Bofill et al. (2021)
Other methods
Developing graphene oxide-supported transition metal catalysts for the conversion of isoeugenol into vanillin through oxidation	2	–	5 mmol	60%	Franco et al. (2017)
In vitro amplification of each gene in local bacterial isolates	24–48	2.3	1%	17.3 g/L	Mazhar et al. (2021)

Vanillic acid as substrate

Vanillic acid may be generated as a by-product during lignin breakdown processes or compete as a constituent in metabolic pathways, either through chemical or biological synthetic methods (Park et al., 2020). Ferulic acid and vanillic acid are prominent phenolic compounds obtained from lignocellulosic biomass and are widely recognized as crucial precursor for the production of biovanillin (Converti et al., 2010). Enhancing the efficiency of the reduction process for converting vanillic acid to vanillin can potentially increase the yield of vanillin production (Qu et al., 2018). Vanillic acid possesses the ability to undergo either oxidative decarboxylation, resulting in the formation of methoxy hydroquinone, or reduction, which yields vanillin and vanillyl alcohol (Zulkarnain et al., 2018). Vanillic acid represents a readily available and abundant substrate for the biocatalytic synthesis of vanillin (Gounaris, 2010; Li and Rosazza, 2000).

Microbial synthesis of vanillin

Vanillic acid is commonly recognized as an intermediate degradation product in a biotransformation process (Vaishnav and Variyar, 2020). In the two-step vanillin biosynthesis process, A. niger achieves an 88% molar yield in converting ferulic acid to vanillic acid, while Pycnoporus cinnabarinus achieves a 22% molar yield in conversion. The primary conversion of vanillic acid into methoxy hydroquinone by P. cinnabarinus reduces the vanillin concentration. Notably, supplementing the medium with enhances P. cinnabarinus's metabolic pathway, leading to a significant 51.7% increase yield of vanillin (Lesage-Meessen et al., 1997).

In another study, vanillic acid derived from A. niger fermentation was biotransformed into vanillin by P. cinnabarinus with XAD-2 resin. The utilization of Amberlite XAD-4 resin in P. cinnabarinus fermentation improved the conversion of vanillic acid to vanillin, effectively preventing its degradation into vanillyl alcohol and vanillic acid (Khai Lun et al., 2014). Adding sulfhydryl compounds like dithiothreitol and dithioerythritol to the bioconversion mixture enhanced vanillin accumulation during the microbial fermentation of ferulic acid. These sulfhydryl compounds serve as protective agents against oxidation for enzymes and vulnerable substances, thus boosting the metabolic pathway that yields produces 65 mg of vanillin from 16 g of vanillyl alcohol in specific microorganisms (Li and Rosazza, 2000).

The Amycolatopsis sp. ATCC 39116 strain achieved a maximum final yield of 9.18 g/L of vanillin, resulting in a corresponding molar yield of 96.1% with a minor accumulation of by-products (Ma and Daugulis, 2014). When S. sannanensis cultures were cultivated in a minimal medium where ferulic acid served as the sole carbon source, the predominant biotransformed product observed in the medium was vanillic acid. Additionally, a transient formation of vanillin was noted during this process. The highest recorded accumulation of vanillic acid reached 400 mg/L when the cultures were grown on a 5 mM ferulic acid substrate at a temperature of 28 °C (Ghosh et al., 2007).

Enzymatic synthesis of vanillin

The utilization of pure Nocardia carboxylic acid reductase led to the complete and quantitative reduction of vanillic acid, exclusively producing vanillin without any accompanying byproducts, through ATP- and NADPH-dependent mechanisms. The study investigates the potential of Nocardia sp. whole cells and enzyme preparations in synthesizing vanillin (Beld et al., 2013). The enzyme derived from Mycobacterium abscessus B1MLD7 gene exhibited superior performance among 14 carboxylic acid reductases tested in E. coli for vanillic acid reduction. Optimization of substrate and reducing power supply led to a successful production of 2.86 g/L vanillin with minimized by-products in whole-cell biocatalysis (Park et al., 2020).

C₆–C₃ phenylpropanoids as substrates

Phenylpropanoids constitute a diverse group of unique compounds pivotal in plant growth, development and the interactions between plants and their environment. These compounds are synthesized through the shikimic acid pathway, utilizing either phenylalanine or tyrosine, two aromatic amino acids found in specific plant species (Deng and Lu, 2017). The compounds that are derived from these amino acids share a common structure and are commonly referred to as phenylpropanoids (Barros and Dixon, 2020; Feduraev et al., 2020).

Enzymatic synthesis of vanillin

A biological technique entails utilizing the roots of Capsicum frutescens as a proficient source of caffeic acid, a precursor to vanillin (Upadhyay et al., 2023). In a research exploring the biosynthetic pathway of vanillin in Vanilla planifolia, the amino acids phenylalanine or tyrosine undergo deamination to produce a C6-C3 phenylpropanoid compound, a precursor for the vanillin production (Marchiosi et al., 2020). The introduction of novel genetic traits into cultivated vanilla has been shown to enhance its properties, particularly its resistance to Fusarium (Yuan et al., 2020). The present study transformed Vanilla planifolia using Agrobacterium tumefaciensand revealed that the conclusive enzymatic stage in the vanillin biosynthesis process involves the methylation of 3, 4-dihydroxybenzaldehyde (Yang et al., 2017).

Root cultures of Capsicum frutescens can biotransform externally provided precursors like caffeic acid, leading to the production of vanillin. The bioconversion of caffeic acid was observed by treating the cultures with a concentration of 10 µM methyl jasmonate (Vyas and Mukhopadhyay, 2018). Following this treatment, there was a notable rise in the enzymatic activity of caffeic acid O-methyl transferase. The root cultures subjected to methyl jasmonate treatment demonstrated a 1.93-fold increase in vanillin production on day 3 in comparison to the untreated cultures. After subjecting root cultures of C. frutescens to a 24-h treatment of methyl jasmonate, a noteworthy increase of 13.7-fold was observed (Suresh and Ravishankar, 2005).

Immobilized system for vanillin production

Vanillin production from C6–C3 phenylpropanoids was effectively facilitated by the immobilized system. The investigation demonstrated that the culture displayed a higher proficiency in biotransforming externally introduced protocatechuic aldehyde into vanillin rather than converting into capsaicin. In contrast, when the culture was exposed to caffeic acid, an inverse pattern was observed (Gupta et al., 2013). The highest vanillin accumulation occurred on the 6th day in immobilized C. frutescens treated with an intermediate, reaching 3.83 mg/L of capsaicin on the 15th day. When S-adenosyl-l-methionine was added to the immobilized cultures, vanillin production significantly increased on the 4th day, reaching 2.5 times more than cultures treated only with protocatechuic aldehyde (Kumar Basu and Krishna De, 2003).

Glucovanillin as substrate

Glucovanillin serves as a flavor precursor within the composition of vanilla beans, which undergo enzymatic hydrolysis during curing (Odoux et al., 2003).

Enzymatic synthesis of vanillin

Glucovanillin, sourced from green pods, holds the potential to undergo conversion into vanillin via cascade of enzymatic processes involving the degradation of cell walls and the hydrolysis of glucovanillin (Hartati et al., 2019). β-D-glucosidases are integral in the vanillin extraction process as they facilitate the breakdown of β-glucosidic linkages within cellulose. In the traditional curing process, only 2% of vanillin is obtained, primarily due to the substantial presence of 10–15% glucovanillin in uncured green beans (Karthika et al., 2021). Consequently, exogenous enzyme preparations comprising cellulase, pectinase, and β‐glucosidase enhanced the yield. However, the incorporation of exogenous enzymes on green pods resulted in producing a vanillin yield ranging from 4.25 to 7% (Perera and Owen, 2010). The extractive reaction exhibited a significantly higher efficiency, resulting in a vanillin extraction yield that was 3.13 times greater than the yield obtained using the Soxhlet method.

Other substrates

Several investigators have documented a range of substrates that can be utilized to produce vanillin. The aforementioned substances comprise cow dung, 4-hydroxybenzaldehyde, 3-bromo-4-hydroxybenzaldehyde, and 3-methoxy-4-hydroxybenzyl alcohol (Gu et al., 2010). A two-step method has been devised for synthesizing vanillin, involving electrophilic aromatic substitution followed by organometallic methoxylation, utilizing copper bromide and sodium methoxide (Saeed et al., 2022). The production of vanillin from various substrates were represented in Table 3.

Table 3.

Synthesis of vanillin by employing C6–C3 phenylpropanoids and other potential substrates

System	Time	pH	Initial concentration	Yield	References
Other C6-C3 phenylpropanoids
C. frutescens cell cultures that have been immobilized and treated with protocatechuic aldehyde	144 h	7.5	1.25 mM	5.63 mg/L	Rao and Ravishankar (2000)
Immobilized C. frutescens cell cultures treated with protocatechuic aldehyde and S-adenosyl-L-methionine (100 µM)	96 h	7.5		14.08 mg/L	Rao and Ravishankar (2000)
Immobilized C. frutescens cell cultures treated with caffeic acid	9 days	7.5	1.25 mM	2.68 mg/L	Rao and Ravishankar (2000)
Root cultures of Capsicum frutescens with caffeic acid treated with methyl jasmonate (10 µM)	72 h	8.0	10 mM	20.2 µM	Suresh and Ravishankar (2005)
Other substrates
Phanerochaete chrysosporium cultivated on green coconut agro-industrial husk through solid-state fermentation	24 h	4.5	1 g	52.5 µg	Barbosa et al. (2008)
Schizosaccharomyces pombe with glucose	48 h	8.3		65 mg/L	Hansen et al. (2009)
Saccharomyces cerevisiae with glucose	24 h	2.3		45 mg/L	Hansen et al. (2009)
Vanillin production using Brevibacillus agri 13	48 h	7.0	10%	1.7 g/L	Wangrangsimagul et al. (2012)
Vanillin production using rice bran oil by Enterobacter harmaechei	72 h	7.0		5.2 g/L	Mazhar et al. (2017)
Biovanillin production from lemongrass leaves hydrolysate using Phanerochaete chrysosporium ATCC 24725	72 h	6.0		93 mg/L	Galadima et al. (2020)
Molybdenum-catalyzed oxidative depolymerization of alkali lignin	3 h	> 12	12 wt%	9 wt %	Rawat et al. (2020)
Converting agricultural by-products into biovanillin via solid-state fermentation	48 h	7.5	100 g	0.029 g	Saeed et al. (2022)
Valorization of pomegranate peels into vanillin using Enterobacter harmaechei	24 h	6.5	1 g	0.462 mg	Mehmood et al. (2022)

Biological synthesis of vanillin

In the biotechnological extraction of vanillin, it is common to employ microbe-mediated fermentation procedures or bioconversions of natural precursors into vanillin through specialized microbial cells or enzymes. In recent decades, numerous researchers have documented the synthesis of vanillin using a variety of raw materials, including wood and agricultural residues (Chattopadhyay et al., 2018). A comprehensive investigation was conducted on a integrated reaction and separation process aimed at producing the vanillin from Kraft lignin. The oxidation of Kraft lignin in an alkaline environment was carried out in a batch reactor, with the primary objective of investigating the most favorable operational parameters for vanillin production (Silva et al., 2009). Another investigation was conducted on the continuous production of vanillin utilizing a structured bubble column reactor, with the primary objective of assessing its feasibility. The reaction stream, which contained degraded lignin and sodium vanillate, underwent an ultrafiltration process in order to retrieve the vanillate (Rodrigues Pinto et al., 2012). The recovery of vanillin can be achieved through an ion exchange process at pH 12–13. This process involves passing the vanillate solution through a column that contains an ion-exchange resin in the H⁺ form (Zabkova et al., 2007). Another study was conducted on specific components found in the waste material generated by the wood pulp industry to produce vanillin from lignin (Calvo-Flores and Dobado, 2010).

Enzymatic vanillin production

The Pseudomonas fluorescence E118 strain exhibits a broad range of substrate specificity, encompassing vanillyl alcohol into vanillin (Gallage and Møller, 2015). The enzyme preparations derived from Nocardia sp. were investigated for their potential in vanillin production and O-benzyl vanillic acid was utilized for its conversion into this product (Wohlgemuth, 2013). Carboxylic acid reductase in purified Nocardia cells fully converts vanillic acid to vanillin by utilizing ATP and NADPH. The study investigated creosol conversion with vanillyl alcohol oxidase (VAO), a two-step process where vanillyl alcohol initially forms and then turns into vanillin. Yet, limited catalytic activity is due to an unproductive complex between the enzyme's flavin and creosol. Additionally, vanillyl alcohol conversion is hindered during the second step because creosol competitively binds in the enzyme's active site (Basri et al., 2023).

The utilization of ligninolytic enzymes to facilitate the depolymerization of lignin into vanillin represents a highly favourable and promising method within the field of green chemistry (Basri et al., 2023). A robust biocatalytic platform was established by employing the immobilization of laccase and versatile peroxidase enzymes individually, and the co-immobilization of both enzymes onto magnetic silica microspheres, utilizing the Casuarina equisetifolia biomass as a key component. The depolymerization of lignin was conducted using both free and immobilized laccase. The results indicated that at pH 4.0 and a temperature of 30 °C, the vanillin yield achieved was 24.8% and 23%, respectively (Saikia et al., 2020).

Vanillin in the food sector

Vanillin, a versatile sweetening and flavor-enhancing agent, is widely utilized in an array of food and beverage applications, including confections, desserts, baked goods, and beverages. Typically incorporated post-baking to withstand high oven temperatures, thereby ensuring the preservation of its inherent properties. A related variant, known as vanillin sugar, contributes sweetness to baked goods and ice creams. Within the beverage sector, vanillin enhances the flavor profile of soft drinks, wines, and chocolate-infused beverages. Furthermore, beyond its role as a flavoring agent, vanillin has found application in food packaging and preservation owing to its antimicrobial properties.

Food preservative

Vanillin possesses the unique property of enhancing food preservation, making it a valuable ingredient in extending the shelf life of various food products. Vanillin is recognized as a safe and effective natural antimicrobial additive, showing substantial promise as a bio-preservative for food products (Table 4). A study assessed the antimicrobial properties of vanillin and its effectiveness in inhibiting yeast strains commonly associated with food spoilage: Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii. The results revealed minimum inhibitory concentration (MIC) values of 21 mM, 20 mM, and 13 mM for vanillin against Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii, respectively (Fitzgerald et al., 2003). In another study, Listeria monocytogenes and Escherichia coli were added to Granny Smith apple juice, with the pH adjusted to 4.0 and supplemented with 40 mm of vanillin. After 3 days at 4 or 15 °C, no bacteria were detected, showcasing vanillin's potential as a preservative for minimally processed apple products (Moon et al., 2006).

Table 4.

Application of vanillin in food sector towards food preservation and food packaging

Source	Concentration of vanillin	Food application	Remarks	References
Commercial	–	Utilized in the food packaging material with chitosan and poly(ethylene oxide)	• Antimicrobial activity against E. coli, S. aureus and C. albicans • Increases the shelf life to 7 days • Biodegradable	Andreica et al. (2023)
Commercial (plant derived)	0.1–2 mg/mL	Antibacterial activity against food-borne pathogens	• MIC value: 2 mg/mL • Decrease the ATP content and causes cell death	Chen et al. (2023)
Commercial	-	Food packaging material in the form of nano-fibres	• Enhanced the mechanical properties • Increased the elongation from 33.5–100% • Bacteriostatic agent	Liu et al. (2023b)
Natural vanilla extract	32–512 µg/mL	Flavoring agent and exhibit antifungal activity against Candida sp., Cryptococcus sp., Aspergillus sp.	• MIC value: 512 µg/mL • Active site binding energies: • − 9.1 to − 12.2 kcal/mol	Gazolla et al. (2023)
Commercial	0.74 g/cm3	Flavoring agent and food additive	• Enhances the anti-caking function • Exhibits 57.9% lower aroma release	Liu et al. (2023a)
Equisetum ramosissimum	–	Exhibits the antioxidant activity	• IC50 value: 0.064 ± 0.0004 mg/mL • Improves anti-inflammatory effects	Sissi et al. (2023)
Commercial	1 g	Food packaging by antimicrobial polymer and covalently linked vanillin motifs	• Enhance the shelf life by 50% • Inhibition rate: Escherichia coli—99.95%, Staphylococcus aureus—99.96% and Listeria monocytogenes—99.02%	González-Ceballos et al. (2022)
Commercial	3%	Utilized in emulsion formation with chitosan in food processing industry	• Mechanically stable • Shortening the substitute in cookies • Fat replacer	Brito et al. (2022)
Commercial	0.3–3 mM	Food additives and mediating the bitterness	• Improves sensory profiles • Flavoring agent	Morini et al. (2021)
Commercial	–	Utilized in food packaging and exhibit the antibacterial activity against L. monocytogenes and E. coli	• Exhibit the highest inhibitory effects with vanillin concentration of 1006 ppm • MIC value: 3002 ppm L. monocytogenes	Cava-Roda et al. (2021)
Commercial	10 mM	Utilized as food preservative	• Increase the stability of whey protein • Thermal stability: 80 °C	Boeve and Joye (2020)
Plant extract	0.2 mM	Antibacterial activity against E.coli	• Zone of inhibition: 1.42 cm • Increased the encapsulation efficiency	Marchianò et al. (2023)
Coffea canephora	–	Antibacterial and anti-adhesive activity against Staphylococcus aureus	• Antioxidant activity: 3.96 ± 0.08 μg/mL • MIC value: 40 µg/mL • Anti-adhesive activity: 70%	Aissaoui et al. (2020)
Commercial	0.1%	Antibacterial activity against L. monocytogenes and utilized in food packaging	• Increase the shelf life and tensile strength of material • Zone of inhibition: 25.24 mm	Lee et al. (2016)
Vanilla beans	–	Flavoring agent	• Helps in sensory evaluation • Recovery of vanillin by-product	Hadj Saadoun et al. (2022)

The preservative properties of vanillin were investigated in both apple juice and a peach-flavored soft drink. The efficacy of vanillin as a preservative was assessed concerning its impact on Saccharomyces cerevisiae and Candida parapsilosis. A biocidal impact on both yeast strains was noted within a span of 96 h with vanillin concentration of 20 mM. This finding suggests that vanillin displays potential as a preservative for fruit juices and low-lipid, low-protein soft drinks, effectively safeguarding them against S. cerevisiae and C. parapsilosis (Fitzgerald et al., 2004). In a study by Ngarsmak et al. (2006) vanillin demonstrated strong antimicrobial activity against various microorganisms, including four bacteria (Pantoea agglomerans, Aeromonas enteropelogenes, Micrococcus lylae, and Sphingobacterium spiritovorun), four fungi (Alternaria sp., Aspergillus sp., Penicillium sp., and Fusarium sp.), and three unidentified yeasts from spoiled fresh-cut mango slices. Vanillin's MICs were assessed in pH-adjusted (pH 5.0) laboratory media, indicating the microorganisms' sensitivity to vanillin, with MIC values ranging from 5.0 to 13.3 mM (Ngarmsak et al., 2006).

The combinations of vanillin with clove essential oil (EO) and vanillin with cinnamon bark EO demonstrated the most significant synergistic antimicrobial effects. In the case of L. monocytogenes, the most effective inhibition was observed with combinations of cinnamon bark EO (85 ppm) and vanillin (910 ppm), as well as clove EO (121 ppm) and vanillin (691 ppm). For E. coli, notable inhibitory effects were achieved using combinations of clove EO (104 ppm) and vanillin (1006 ppm), as well as cinnamon leaves EO (118 ppm) and vanillin (979 ppm) (Cava-Roda et al., 2021). Vanillin's ability to combat potential spoilage organisms, including Candida albicans, Lactobacillus casei, Penicillium expansum, and Saccharomyces cerevisiae, was investigated concerning contamination fresh-cut  products. The use of 12 mM vanillin resulted in a 37% reduction in total aerobic microbial growth in ‘Empire’ apples and an impressive 66% reduction in ‘Crispin’ apples during 19 days of storage at 4 °C (Rupasinghe et al., 2006).

Food packaging

Films composed of chitosan and poly(vinyl alcohol) were prepared by incorporating ethyl vanillin (CPEV) exhibited robust antibacterial properties against both Escherichia coli and Staphylococcus aureus. Consequently, it is evident that CPEV blends possess substantial potential as the source for active films, particularly for applications in food packaging (Narasagoudr et al., 2020). In a research study on food packaging, the use of vanillin at concentrations of 1 or 2 g/L, in combination with cinnamic acid at concentrations of 0.15 and 0.3 g/L, was explored to maintain the quality of fresh-cut Cantaloupe melon. The antimicrobial treatments exhibited notable effectiveness, especially in combating mesophilic bacteria, leading to a reduction of 1.5 log CFU/g. Additionally, these treatments proved to be highly effective in reducing Enterobacteriaceae by 2.2 log CFU/g (Silveira et al., 2015). In another study, a chitosan-vanillin film, developed for its antimicrobial characteristics, was employed in the packaging of butter cake. Over an 8-day storage duration, this film demonstrated an effective inhibition of mold growth, surpassing the performance of commercial stretch film. Remarkably, the quality of the cake remained unaltered throughout this period (Sangsuwan et al., 2015).

In their study, Das et al. (2021) endeavored to formulate an ALG-VAN film tailored for lettuce packaging, with the dual objectives of managing foodborne pathogens (Escherichia coli, Bacillus cereus, Shigella flexneri, Staphylococcus aureus, and Salmonella typhi) and extending the vegetable's freshness. This innovative film with light density, when employed for lettuce preservation, exhibits a substantial capacity to curtail bacterial counts, outperforming the conventional light density polyethylene film. Consequently, it emerges as a promising solution for upholding the quality of lettuce and mitigating the risk of foodborne bacterial contamination (Das et al., 2021). In another study, the impact of starfish gelatin films infused with 0.05% vanillin on the population of L. monocytogenes during a 9-day storage period at 4 °C on crab sticks. After 9 days, L. monocytogenes population on the control sample reached 8.91 log CFU/g, whereas the vanillin-containing starfish gelatin film-wrapped crab sticks showed a population of 7.43 log CFU/g, indicating a reduction of 1.48 log CFU/g. This suggests that vanillin has antimicrobial properties beneficial for crab stick preservation (Lee et al., 2016). In another research, chitosan, vanillin, and polyvinyl alcohol were employed as base materials to produce a composite electrospun nanofiber film. This resulting membrane demonstrated a robust ability to combat Shewanella putrefaciens and effectively prolonged the shelf life of turbot when stored at 4 °C (Mei et al., 2021).

In a different investigation, the inhibitory properties of polyhydroxybutyrate films infused with vanillin were tested against both bacterial (Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Staphylococcus aureus) and fungal (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus parasiticus, Aspergillus ochraceus, Penicillium clavigerum, and Penicillium viridicatum) food contaminants. It was found that the MIC against bacteria was equal to or greater than 80 μg/g of polyhydroxybutyrate, while for fungi, it was equal to or greater than 50 μg/g of polyhydroxybutyrate (Xavier et al., 2015). The Zn²⁺@VA/CMCS film exhibited biodegradability (degrading in 35 days in soil) and renewability, making it a promising option for the advancement of eco-friendly food packaging solutions (Lin et al., 2023). The study by Muppalla and Chawla, (2018) was to enhance the shelf life of ground chicken meat in chilled storage using active packaging incorporating Zanthoxylum rhetsa seed cover extract, particularly enriched with vanillin. Implementing these active films significantly extended the shelf life of ground chicken meat, reducing lipid peroxidation, and preserving freshness for up to 12 days under chilled conditions, in contrast to the less than 6 days achievable with standard packaging.

Future prospective and conclusion

Annually, roughly 16,000 metric tons of vanillin, valued at about $650 million, are produced, with less than 1% of this total stemming from natural vanilla pods. The consistent high demand for nature-identical vanillin within the food and other industries underscores its significance as a sought-after flavoring agent. Vanillin serves as a prevalent food flavoring agent while also playing crucial roles in both food preservation and various food packaging applications. Further exploration of its bioactive properties could pave the way for a more comprehensive assessment of its potential as a bioactive molecule with a wide range of applications. Adopting innovative biotechnology could enhance accessibility and cost effectiveness in producing nature-identical vanillin from natural sources, potentially replacing synthetic vanillin production. The comprehensive exploration of vanillin synthesis and its multifaceted applications opens up promising avenues for future research and innovation. Future prospects include the development of environmentally friendly, sustainable synthesis approaches that prioritize reducing the environmental footprint in vanillin production. Biotechnological advancements, such as genetic engineering and metabolic pathway optimization, offer opportunities to enhance production efficiency and cost-effectiveness (Fig. 3). Additionally, vanillin’s bioactive properties and potential health benefits suggest therapeutic applications that merit further investigation. In the food industry, tailored vanillin varieties catering to personalized and exotic flavors can be a focus for research, while its role as a natural preservative holds potential in an expanding global food market. Vanillin, recognized for its potent antibacterial properties, stands at the brink of revolutionizing food preservation, ensuring the integrity and extended shelf life of diverse food products. As a natural and efficient substitute for conventional synthetic preservatives, vanillin holds the promise of spearheading a fresh era in eco-friendly food packaging solutions. It has the potential to enhance sustainability and extend the shelf life of packaged products within the domain of food packaging. To fully unlock vanillin's potential, it is anticipated that interdisciplinary research collaboration among academics from diverse fields will be indispensable. This comprehensive review acts as a foundational resource, paving the way for future endeavors to unlock the vast possibilities of vanillin in both industry and academia.

Fig. 3.

Major challenges and future perspectives for bio vanillin production

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.