Kluyveromyces marxianus and Debaryomyces hansenii (Guneser et al., 2015). 2.3 Microbial and Enzymatic Bioconversion/Biotransformation. Bioconversion ...
CHAPTE R 2
Different Bioengineering Approaches on Production of Bioflavor Compounds Muge I. Hosoglu*, Onur Guneser**, Yonca K. Yuceer* *Canakkale Onsekiz Mart University, Canakkale, Turkey; **Usak University, Usak, Turkey
1 Introduction Flavors and fragrances affect consumer preference and acceptance of the products. They are commonly used additives especially in food, feed, cosmetic, and pharmaceutical industries. Flavor and fragrance compounds are strong-smelling organic compounds with pleasant odors. Still, they are similar to chemical structures, chemical messengers, and their receptors are olfactory cells in the nasal system, they are called perfume or flavor depending on their usage. The term fragrance is used for pleasant odors in cosmetics and consumer products, but flavor is associated only with food products. The worldwide market of flavors and fragrances is estimated at approximately 24.7 billion US dollars in 2015. A higher consumption of flavors and fragrances is observed in Asia-Pacific, North America, Western Europe and South America countries, respectively. Givaudan (Switzerland), Firmenich (Switzerland), IFF (United States), Symrise (Germany), Takasago (Japan), Mane SA (United States), Frutarom (Israel), Sensient Flavors (United States), Robertet SA (France), Huabao Intl. (China), and T. Hasegawa (Japan) have been noted as the leading companies of the flavor and fragrance markets in the world (Anonymous, 2016; Reineccius, 1994; Scragg, 2007; Surburg and Panten, 2006). Flavor is the most important factor for determining the sensory quality of foods. The flavor of food results from its natural raw material characteristics and formed by many chemical or biochemical reactions during food processing and storage. Moreover, it also originated from added other food ingredients (Cabaroglu and Yilmaztekin, 2010; Reineccius, 1994; Ziegler, 2007). The compounds responsible for flavor include categories such as aldehydes, alcohols, esters, ketones, lactones, short- to medium-chain free fatty acids, phenolic and sulfur compounds (Gupta et al., 2015; Longo and Sanroman, 2006). Nowadays more than 6500 flavor compounds are known in the food industries, but only approximately 300–400 of flavor compounds are widely used for foods especially beverages, dairy products, and sauces (Scragg, 2007; Ziegler, 2007).
Role of Materials Science in Food Bioengineering http://dx.doi.org/10.1016/B978-0-12-811448-3.00002-4
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Copyright © 2018 Elsevier Inc. All rights reserved.
38 Chapter 2 Food flavors are distinguished by three categories: natural, natural-identical, and artificial flavors. According to the European Council Directive (Directive 88/388/88 EEC), “Natural flavors” are defined as being obtained from plant or animal raw materials either by physical (extraction, distillation, concentration, crystallization process) or enzymatic and microbiological processes. “Natural-identical flavors” are produced by chemical synthesis or isolated by chemical processes. They are organoleptically and chemically identical to natural flavor compounds from plant or animal sources. “Artificial flavors” are produced by chemical synthesis and are not found in natural plant and animal sources. Although “artificial flavors” are not identified in a natural product, their sensory characteristics are the same as natural ones and they are chemically different (Anonymous, 1985, 1988). Most flavor compounds are produced by distillation/extraction process from animal and plant sources (natural flavor) or synthesized in chemical ways (artificial flavor). Several drawbacks such as the low quantities of flavor compounds in plant and animal source, the cost of distillation/ extraction process, and the effect of environmental factors on flavor compounds in natural sources have directed flavor and fragrances companies to produce flavors and fragrances by chemical synthesis. The production of vanillin and coumarin can be given as good examples for chemical synthesis. The chemical name of vanillin is 4-hydroxy-3-methoxybenzaldehyde and it is produced naturally from vanilla beans by an extraction process, but its synthetic form is produced from spent sulphite liquor or guaiacol and glyoxylic acid by several condensation, oxidation, and decarboxylation reactions. The synthetic vanillin costs about US$12/kg, whereas the natural form is relatively expensive and priced at US$4000/kg (Dignum et al., 2001; Lomascolo et al., 1999; Rao and Ravishankar, 2000). Production of flavor compounds by chemical synthesis is cheaper and higher in yield (Cabaroglu and Yilmaztekin, 2010), but there are some disadvantages of producing flavor compounds by a chemical process, which include the formation of undesirable racemic mixture compounds, environmentally unfriendly production steps, and complex reaction conditions in chemical synthesis. These problems have led to the emergence of new natural flavor production processes in the flavor and fragrance industries. Nowadays, scientists who work on flavors and fragrances search for alternative routes that differs from extractive and chemical synthesis processes for producing natural flavor compounds (Akacha and Gargouri, 2015; Longo and Sanroman, 2006; Reineccius, 1994). Biotechnology is featured as one of the emerging and the most attractive way for the production of natural flavors and fragrances. New biotechnological ways for flavor synthesis are based on de novo synthesis by plant or tissue cells, microbial metabolisms, and bioconversions of natural precursors using microbial cells or enzymes. Due to an increasing chemophobia on the part of consumers and consumers’ preferences for natural food additives and other compounds of biological origin because of health concerns, there has been an increasing trend toward the production of flavor compounds by biotechnological processes (bioflavor) during recent decades. The industrial application of these techniques has started to grow since the end of the 20th century. Biotechnological processes have produced more than 100 flavor compounds, and
Different Bioengineering Approaches on Production of Bioflavor Compounds 39 they are commercially available (Berger, 2015; Guneser et al., 2015; Mantzouridou and Paraskevopoulou, 2013; Medeiros et al., 2000). This chapter reviews the production of flavor compounds by biotechnological processes. The discussion of the current state of the art of developments in industrial and academic research particularly focuses on microbial production processes. Bioengineering provides promising technical options for increasing the productivity of bioflavors, such as different fermentation strategies (batch, fed-batch, and continuous fermentation); gas-phase or two-phase reactions; specific reactor constructions such as membrane, solid-state, or closed loop reactors; optimization and modeling approaches of the bioprocess; and in situ recovery of product. The art of bioprocess used to improve productivity of bioflavor compounds, with emphasis on using different bioengineering approaches are also discussed.
2 Biotechnological Processes for the Production of Bioflavor and Fragrance Compounds Biotechnological processes are alternative and innovative ways for producing natural flavors and fragrances. Compared to chemical synthesis, biotechnological processes are performed at mild conditions; specific enantiomers (chemo-, regio-, and stereo-selective) of desired compounds can be produced in these processes and possible shortages such as the effects of climatic conditions on raw material, trade restrictions, and socio-political instabilities are not concerns for biotechnological processes. Moreover, harmful or toxic wastes are not generated during biotechnological processes (Bicas et al., 2010; Krings and Berger, 1998). Biotechnological production of flavors and fragrances can be performed in three different ways: (1) plant cell or tissue cultures (PTCs), (2) microbial fermentation (de novo synthesis), and (3) microbial and enzymatic bioconversion/biotransformation (biocatalysts). Of these, microbial fermentation and biotransformation are the main techniques, which are widely investigated and applied to the production of flavors and fragrances (Bicas et al., 2010; Hrazdina, 2006; Schrader et al., 2004).
2.1 Production of Flavors and Fragrances by Plant Cell or Tissue Cultures Plants are good sources of flavors and fragrances. They are naturally produced from differentiated plant tissues such as fruit, flower, leaf, or root at certain stages of organ and tissue development in the plant. For instance, benzaldehyde (BA) is mainly produced in mature seeds and not by any other tissues of apricot. The production of flavor compounds from a plant is greatly limited by tissue specificities. Hence, climatic and cultivation conditions of plants affect the quality and quantity of flavor compounds. Owing to these limitations, the PTC technique was initially considered a potential biotechnological process for the production of flavor compounds, which allows the cultivation of single cells, tissues, and whole organs of selected plant with specific properties in a liquid or solid medium. PTC
40 Chapter 2 is a useful technique to directly produce flavor compounds from specified plant cells or tissue in bioreactors without growing plants in agricultural areas (Berger, 1995; Harlander, 1994; Havkin-Frenkel and Belanger, 2009; Medeiros et al., 2000). For the biosynthesis of flavor compounds by PTC, specified plant cells or tissues are first isolated from meristems, roots, leaves, or immature fruits and aseptically transferred to solid media containing plant growth factors (i.e., auxins) and nutrients (carbohydrates, mineral salts, vitamins). Then, the plant cells or tissues proliferate into undifferentiated cell masses known as callus. Callus can be transferred in culture media and cultured as suspension culture for the flavor production in fermenters, or multiple callus cultures can be propagated on agar disks to reconstitutite functional tissue of the whole plant (Reineccius, 1994; Tretzel and Marx, 2007) (Fig. 2.1). Higher amounts of flavor compounds can be achieved by PTC. However, there are still problems in the production of secondary metabolites of PTCs. Sensitivity to shear stress, the instability of cell lines, relatively long growth cycles, low yields, progressive loss of biosynthetic activity, and unusual production secretions are some of the problems and drawbacks that need special attention in PTCs. Therefore several strategies have been developed to overcome production problems and to stimulate cultured plant cell or tissue for biosynthesis of flavor compounds by manipulation of culture medium composition, manipulation of environmental factors, addition of exogenous elicitors, and induction of cell differentiation (Dicosmo and Misawa, 1995; Longo and Sanroman, 2006; Reineccius, 2006). Several flavor compounds including terpenoids, phenylpropanoids, aliphatic compounds, polyines, glycosides, and nitrogen-and sulfur-containing compounds have been produced in PTCs. However, their productions are not developed sufficiency for commercially applicability. Researchers have a great deal of interest in producing vanillin and strawberry-like flavor compounds by PTCs (Hrazdina, 2006). In a study by Sudhakar Johnson et al. (1996), ferulic acid and vanillylamine was biotransformed to capsaicin and vanillin by immobilized plant cell cultures of Capsicum frutescens Mill. Maximum concentrations of capsaicin and vanillin were observed as 190 and 315 µg/mL culture, respectively. It was also reported that isoeugenol can biotransformed to vanillin, vanillic acid, ferulic acid, and capsaicin by the same plant cell cultures (Rao and Ravishankar, 1999). The other flavor compound associated with fruity notes is p-hydroxyphenyl-2-butanone known as the raspberry ketone. It has the key metabolite characteristic of raspberry flavor in more than 230 identified compounds with raspberry aroma. It is commonly used for beverages, and dairy and confectionary products in the food industry (Hrazdina, 2006; Scragg, 2007). Pedapudi et al. (2000) indicated that the raspberry ketone is produced in cell suspension cultures of Rubus idaeus, and it can be rapidly increased 2- to 3-fold
Different Bioengineering Approaches on Production of Bioflavor Compounds 41
Figure 2.1: Production of Flavor Compounds by Plant Cell or Tissue Cultures (Tretzel and Marx, 2007). Reprinted with permission from John Wiley & Sons, Inc.
by elicitation with methyl jasmonate in the concentration range of 10–50 µM in a culture medium. The production of flavor compounds associated with fruity aroma was also investigated with strawberry cell suspension cultures (Hong et al., 1990). The researchers found that strawberry cell suspension cultures have converted α-ketovalerate to butanal and butanol and produced ethyl butyrate and butyl butyrate from sodium butyrate. Moreover, it was reported that α-terpineol and nerol can be produced by Camellia sinensis cell-suspension cultures in Murashige and Skoog synthetic media containing 5 mg/L of 2,4-dichlorophenoxyacetic and 1 mg/L of 6-benzyladenine (Grover et al., 2012).
42 Chapter 2
2.2 Microbial Fermentation (De novo Synthesis) The microbial fermentation process has been used for the production of fermented foods since ancient times. It provides several varieties of flavor compounds for fermented foods such as cheese, yogurt, kefir, beers, wines, soy sauce, sausages, sauerkraut, kimchi, and fermented fish products. During fermentation, carbohydrates, fats, and proteins are catabolized by microbial cells (bacteria, yeasts, and molds) and they further convert into the breakdown products to flavor molecules. The fermentation process is also called de novo synthesis. Alcohols, esters, aliphatic acids, aldehydes, lactones, ketones, sulfur compounds are major flavors produced by microorganisms as secondary metabolites in de novo synthesis (Fig. 2.2).
Figure 2.2: De Novo Synthesis and Biotransformation of Natural Flavor Compounds (Macedo Alves et al., 2010). Reprinted with permission from John Wiley & Sons, Inc.
Different Bioengineering Approaches on Production of Bioflavor Compounds 43 The production of these compounds by microorganisms is usually performed in batch process in which microorganisms are grown in bioreactors. In general, bioreactors are grouped as stirred-tank and solid-state bioreactors in two different fermentation types including submerged fermentation (SmF) and solid-state fermentations (SSF), respectively (Dastager, 2009; Lee, 2015; Reineccius, 2006). Both fermentation techniques and examples of productions will be discussed in detail in the following sections (Section 3.1). Fundamentally, flavor compounds were produced from simple molecules through complex metabolic pathways in microbial fermentation. The Ehrlich pathway and β-oxidation of fatty acids are the main biochemical pathways for the production of flavor compounds, which are used by microbial cells (Bicas et al., 2010; Krings and Berger, 1998; Lee, 2015; Macedo Alves et al., 2010; Reineccius, 2006). Production of esters and alcohols was achieved via the Ehrlich pathway especially in the metabolism of yeasts. The Ehrlich pathway covers sequential biochemical reactions such as transamination, decarboxylation, oxidation, and reduction of some branched chain amino acids (i.e., leucine), aromatic amino acids (i.e., phenylalanine) and sulfur-containing amino acids (i.e., methionine). Acetyl-coenzyme A/alcohol acetyl transferase reactions are also crucial for flavor production in yeast metabolisms (Hazelwood et al., 2008; Mason and Dufour, 2000) (Fig. 2.3).
Figure 2.3: Ehrlich Pathway for Production of Flavor Compounds in Yeasts (Mas et al., 2014).
44 Chapter 2 Fatty acids can be oxidized via α-, β-, w-oxidation pathways, and β- of fatty acids especially was used by yeast for the production of lactones. β-Oxidation is a cyclic oxidation system of fatty acids. Four main reactions occurred in the reaction; dehydrogenation, leading to trans2,3-enoyl-CoA; hydration of the unsaturation, leading to 3-hydroxyacyl-CoA; oxidation into 3-ketoacyl-CoA; and finally, the release of acetyl-CoA and acyl-CoA shortened by two carbons. In the case of a saturated fatty acid, this four-reaction cycle is repeated several times until the complete degradation of fatty acid into acetyl-CoA molecules. During this, β-oxidation enzymes, including acyl-CoA oxidase, enoyl-CoA hydratase, OH-acyl-CoA dehydrogenase, and ketaoacyl-CoA thiolase convert the intermediates of β-oxidation cycles to different lactones (Endrizzi et al., 1996; Vandamme and Soetaert, 2002; Wache et al., 2001) (Fig. 2.4). On the other hand, fungi also oxidize fatty acid for eight-carbon volatiles such as 1-octen3-ol, 1-octen-3-one (Vandamme and Soetaert, 2002). Production of eight-carbon volatiles, which is associated with mushroom aroma, is largely an unknown in fungal metabolism and is related to the glucose and fatty-acid metabolism of both fungi, because these flavor compounds are end products of fungal fatty-acid metabolism. Also, acetyl-CoA, which is a breakdown substance of glycolysis, can be used as a precursor for the production of these compounds. In the biosynthesis pathway, the oxidation step of polyunsaturated fatty acids was achieved by haem dioxygenase, which is different from lipoxygenase (LOX). The cleavage stage of intermediate hydroperoxide compounds by fungal-specific 10-hydroperoxide lyase results in the production of eight-carbon volatiles (Combet et al., 2006). Simple and inexpensive sources of carbon and nitrogen, and sometimes vitamins, minerals, or other micronutrients were used as culture media in microbial fermentation (Harlander, 1994). Nowadays, using agrowastes as culture media has been suggested in the biotechnological production of flavor and fragrance by several researchers due to the low costs and rich content for microbial growth. Cassava bagasse, tapioca bagasse, sugar beet, beet molasses, wheat bran, apple pomace, soy bean meal, rice bran, fruits and vegetables pomaces, and whey are the agrowastes that have the most potential for the production of natural flavors and fragrances by microbial fermentation (Dastager, 2009; Pandey et al., 2000). Because of their high fermentation capability with low-growth requirements and high-enzyme catalyzed systems (Gupta et al., 2015; Hausler and Münch, 1997), it was noted that metabolisms of yeasts and fungi are more suitable for fermentation of agrowastes than bacteria. In the literature, several high impact studies can be found on the production of flavor compounds by fermentation of certain yeast and fungus (Guneser et al., 2014, 2015; Lalou et al., 2013; Mantzouridou et al., 2015; Oliveira et al., 2015; Wilkowska et al., 2015). In a more recent study by Mantzouridou et al. (2015), it was reported that de novo biosynthesis of isoamyl acetate, ethyl dodecanoate, decanoate, octanoate, and phenyl ethyl acetate from orange peel can be achieved using S. cerevisiae. Similarly, in a study performed in our laboratory, some esters and alcohols including isoamyl alcohol, phenyl ethyl alcohol, isoamyl acetate,
Different Bioengineering Approaches on Production of Bioflavor Compounds 45
Figure 2.4: β-Oxidation of Cycle With Lactones Formation in the Yeasts (Świzdor et al., 2012).
and phenyl ethyl acetate can be synthesized from tomato and pepper pomaces by using Kluyveromyces marxianus and Debaryomyces hansenii (Guneser et al., 2015).
2.3 Microbial and Enzymatic Bioconversion/Biotransformation Bioconversion and biotransformation are other approaches for the production of flavor and fragrance compounds described as the use of microbial cells or enzymes to perform specific modifications or interconversions of precursor substances to a structurally similar
46 Chapter 2
Figure 2.5: Some Microbial Biconversion of the Certain Precursors (Longo and Sanroman, 2006).
flavor compound. Several precursors, which are inexpensive and easily available, can be converted to more valuable flavor compounds in these reactions (Fig. 2.5). Compared to de novo synthesis, bioconversion and biotransformation yield a higher level of desired flavor compounds than de novo synthesis. Therefore these approaches for production of flavor compounds offer a more attractive industrial process from an economic viewpoint (Amaral et al., 2010; Welsh et al., 1989). Different biochemical reactions such as oxidation, reduction, hydrolytic reactions, dehydration, and formation of new C–C bonds occurred in these processes. The biotransformation process comprises single-step reactions, whereas multistep reactions occur in bioconversion processes. Fatty acid, amino acid, and terpenes from essential oils are the main precursors and are commonly tested for flavor production by many researchers. The synthesis of γ-decalactone (GDL) from ricinoleic fatty acid in castor oil by yeasts and vanillin from ferulic acid in several cereal brans by fungus are particularly good examples of the use of biotransformation/bioconversion (Amaral et al., 2010; Cheetham, 1993; Krings and Berger, 1998). The yeasts, such as Candida tropicalis, Yarrowia lipolytica, Sporobolomyces odorus have the ability by biotransformation of ricinoleic acid to GDL. In the production of lactone by yeasts via biotransformation, ricinoleic acid first is metabolized to 4-hydroxy decanoic acid. This compound then is chemically lactonized to GDL and other lactones (Amaral et al., 2010; Waché et al., 2003). In case of vanillin production, one of the wellknown biotransformation routes is a two-stage process in which a strain of Aspergillus niger is first used to convert ferulic acid to vanillic acid, which was then reduced to vanillin by a laccase-deficient strain of Pycnoporus cinnabarinus (Walton et al., 2000). Other favorite precursors for bioconversion and biotransformation are terpenes and terpenoids, which are constituents of essential oils. α-Pinene, β-pinene, d-limonene, and citral are used in large quantities for bioconversion into more valuable flavor compounds. Nerol and
Different Bioengineering Approaches on Production of Bioflavor Compounds 47 geraniol are monoterpenes, which are associated sweet rose-like flavor, finding applications in flavors designed for teas and other beverages. Both geraniol and nerol can be prepared by biotransformation of citral with Saccharomyces cerevisiae. Hence, d-limonene from the citrus peel oils is one of the most studied monoterpene. It is used for the biotransformation to α-terpineol, perillyl alcohol, carveol, carvone, and menthol by Penicillum spp. and Pseudomonas spp. (Duetz et al., 2003; Labuda, 2009; Van Der Werf et al., 1997). Benzaldehyde (BA) is the second most important flavor compound after vanillin for the food and cosmetic industries. It is typically derived from a cyanogenic glycoside amygdalin found in fruit kernels and it is mostly produced from mature seed from apricots. However, the toxicities of hydrocyanic acid and hydrogen cyanide as by-products present a safety problem in the extraction process. Biotransformation of phenylalanine to BA is a natural alternative to the extraction process. Bacterium Pseudomonas putida, white rot fungus Phanerochaete chrysosporium, and basidiomycetes Ischnoderma benzoinum and Polyporus tuberaster have been studied for the production of this flavor. It was emphasized that biotransformation of phenylalanine was achieved through phenylpyruvate and phenylacetaldehyde to phenylacetate, which is converted into mandelate and benzoylformate. Both compounds then are converted into benzaldeyhde by dehydrogenase and decarboxylase enzyme of the microorganism (Feron et al., 1996; Labuda, 2009; Lomascolo et al., 1999).
3 Bioengineering Approaches Used for the Production of Bioflavor Compounds: Current State and Prospects The demand of using natural flavor compounds obtained through biotechnological means in several industries has led to strengthening the techniques used in the laboratory to a large scale. When developing an industrial fermentation technique, designing the medium composition and environmental factors are of critical importance because they significantly affect product concentration, volumetric productivity, and the cost of process. The development of suitable culture media formulations for microbial growth and metabolite production becomes a key factor for success of bioprocesses. Culture media used in fermentation processes can be synthetic substrates or by-products of food and agricultural industries to reduce the production cost. The bioreactor is the heart of any fermentation or biotransformation processes and creates a controlled environment for the needs of microorganisms to be carried out with the highest adherence to process parameters. Otherwise, uncontrolled conditions may result in the production of unwanted compounds instead of the bioactive compounds of interest. There are many experimental design approaches and technologies used for controlling and optimizing such complex systems. By supplementing the medium with precursor compounds, high product concentrations can be achieved. However, the desired product or by-products can inhibit reactions. Thus in situ product removal can be essential for such processes. Here, the authors’ intention is
48 Chapter 2 to give examples of bioengineering approaches currently used or under investigation for industrial-scale productions for some important bioflavor compounds (vanillin, aliphatic aldehydes, and alcohols (C6 and C9 compounds), esters, 2-phenylethanol, γ-lactones) rather than to present the great variety of them produced by biotechnological approaches.
3.1 Bioprocess Designs With Different Bioreactor Configurations and Fermentation Strategies for Production of Bioflavor Compounds Bioprocesses must be designed to provide a higher degree of control over process parameters by maintaining the desired biological activity and eliminating or minimizing undesired activities. The aim of applying different types of bioreactor design and fermentation strategies is to control and positively influence the biological reaction. The cylindrical tank, either stirred or unstirred, is the most common reactor type in bioprocessing. Bioreactors are generally classified as stirred-tank or solid-state bioreactors with mainly two different fermentation techniques, such as SmF and SSF, respectively. At the research level, both SmF and SSF have been used for bioflavor production processes. New types of reactors are constantly being developed for special purposes (Pescheck et al., 2009). Some designs yielded better results than others, which depend on the nature of reactants, catalyst, products, and also cost of the process (Akacha and Gargouri, 2015; Schrader et al., 2004). Here, the authors tried to discuss the engineering aspects and applications for a variety of bioreaction/ fermentation strategies for the aforementioned bioflavor compounds including the challenges of each and the advantages and disadvantages of the respective technologies. The majority of reactions are performed in a type of SmF in which microorganism and substrate are present in the liquid medium, and products produced by microorganisms are recovered from the liquid medium and purified. Gas/air mixture needed for fermentation is supplied to a culture medium in sterile conditions. Bioreactors are equipped with pH, dissolved oxygen, and foam sensors. Mechanical agitation achieves mixing and bubble dispersion. Microbial production of bioflavor compounds by SmF was studied by several researchers (Barghini et al., 2007; Löser et al., 2012; Medeiros et al., 2001; Tai et al., 2016; Urit et al., 2011). Vanillin is the world’s most preferred additive, which attracted the attention of biotechnologists. PTC techniques can also be used to produce vanilla-like flavor compounds. However, unlike microbial growth rates, plant cells grow more slowly and need long fermentation cycles, which increases contamination risks and cost for such systems. Also, low productivity values are obtained in most PTC systems compared to microbial fermentations. Hence, industrial applications of PTC-derived flavors have been limited (Rao and Ravishankar, 2000). Most efficient biotechnological approaches for the synthesis of natural vanillin are based on bioconversion of certain natural substances such as lignin, ferulic acid, eugenol, isoeugenol, and vanillic acid or on de novo biosynthesis using microorganisms such as bacteria, yeasts, and fungi for an industrially applicable process (Hua et al., 2007; Kaur and Chakraborty, 2013; Zheng et al., 2007). Using metabolically engineered strains
Different Bioengineering Approaches on Production of Bioflavor Compounds 49 carrying the genes encoding for the bioconversion of those precursors to vanillin could be an interesting alternative to natural strains for industrial productions (Barghini et al., 2007). Pseudomonas was used for the production of vanillin by making its recombinant strain using gene encoding feruloyl-CoA synthetase and feruloylhydratase/aldolase to convert ferulic acid into vanillin in a 3 L stirred tank reactor (STR) (Di Gioia et al., 2011). This strain produced up to 8.41 mM vanillin, the highest concentration of vanillin produced by a Pseudomonas strain reported. The same research opened new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes, which contain ferulic acid. Lactones are other industrially important flavor compounds associated with fruity, coconut-like, buttery, sweet or nutty impressions that are widely distributed in foods, fruits, and beverages. Among them, GDL is commonly used compound in the flavor industry (Alchihab et al., 2010; Romero-Guido et al., 2011). Demand for natural flavor compounds by consumers has encouraged food scientists to produce natural GDL through biotechnology and with a significant decrease in its price (around US$300 kg/L) (Antonio Rocha-Valadez et al., 2006; Fadel et al., 2015; Ramos et al., 2008). The use of castor oil, ricinoleic acid or its methyl ester has been extensively employed for the microbial production of GDL (Romero-Guido et al., 2011). Production of GDL from castor oil in batch cultures of Y. lipolytica W29 in stirred tanks and airlift bioreactors was compared (Braga et al., 2015). Airlift bioreactors have great potential for developing processes based on aerobic cultures due to the need for high oxygen transfer rates. A two-time increase in GDL concentration (around 3 g/L) was reported in the airlift compared to the STR. The highest product concentration of up to 11 g GDL/L was reported in 55 h without a genetically modified strain and raw castor oil as substrate (Schrader et al., 2004). Effects of physical (pH, temperature, aeration, and agitation rates) and nutritional factors (carbon and nitrogen sources, trace metals, etc.) on both the growth and production of flavor compounds in SmF were examined at both lab and industrial scale productions (Alchihab et al., 2009; Bicas et al., 2008; Romero-Guido et al., 2011; Yilmaztekin et al., 2013). Volatile esters constitute the most important class of flavor compounds providing “fruity” notes. They are produced naturally by yeast or other microbial species, and are responsible for the fruity and perfume-like aroma of fermented beverages such as wine, beer, and sake. The most common representatives are the acetate esters, such as isoamyl acetate (banana aroma), isobutyl acetate (fruity aroma), and phenylethyl acetate (rose-like, honey, fruity aroma) and the ethyl esters of medium-chain fatty acids, such as ethyl hexanoate (anise, apple-like, strawberry aroma), ethyl octanoate (sour apple, pineapple aroma) and ethyl decanoate (grape-like aroma) (Etschmann and Schrader, 2006; Longo and Sanroman, 2006; Park et al., 2009). Saerens et al. (2008) revealed that a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, whereas a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Another alternative for natural volatile ester production process is using enzymes and other biocatalysts that produce products with better odor and color (Cass et al., 2000;
50 Chapter 2 Schrader et al., 2004). Lipase-catalyzed esterification or interesterification reactions can be carried out for this purpose (Garlapati and Banerjee, 2013; Ju et al., 2009; Rajendran et al., 2009). Immobilization of enzymes results in several advantages reported by many researchers (Garlapati and Banerjee, 2013; Zhao et al., 2015). C. rugosa lipase and porcine pancreatic lipase were immobilized into calcium alginate (Ca-Alg) gel beads by means of entrapment and were used to produce three industrially important flavor esters—namely, isoamyl acetate, ethyl valerate, and butyl acetate (Ozyilmaz and Gezer, 2010). In industrial applications of immobilized enzymatic reactions for bioflavor synthesis, packed-bed bioreactors operated especially in continuous mode, and fluidized bed reactors are the most frequently used (Akacha and Gargouri, 2015; Ju et al., 2009). The medium used in SmF can be by-products of food and agricultural industry in order to reduce production costs (Etschmann et al., 2002; Izawa et al., 2015; Löser et al., 2012; Urit et al., 2011; Yilmaztekin et al., 2008). Zheng et al. (2007) treated waste residue of rice bran oil with water–ethanol to obtain the ferulic acid enriched fraction that was then used in transforming into vanillin by a combination of fungal strains A. niger CGMCC0774 and P. cinnabarinus CGMCC1115. The highest yield reached 2.2 g/L of vanillic acid by A. niger CGMCC0774 in a 25 L fermenter when concentration of ferulic acid was 4 g/L. Batch fermentations constitute 60% of the reported bioflavor production processes and the remainder were on fed-batch, although a few were repeated-batch ones (Van Hecke et al., 2014). Choosing the right operation strategy has a significant effect on substrate conversion, product concentration, and susceptibility to contamination. For example, fed-batch processes are characterized by the addition of one or more nutrients to the bioreactor during the operation, maintaining the products inside the bioreactor until the final fermentation. The fed-batch processes are employed to prevent the inhibition of cells by substrate or metabolic products. One of the most important rose-like aroma is 2-phenylethanol (2-PE). Biotransformation of l-phenylalanine (l-Phe) to 2-PE is considered to be the most promising strategy because of the advantages of high substrate selectivity and relatively mild reaction conditions (Mihaľ et al., 2013; Wang et al., 2011). Several organisms, including Brevibacterium linens, A. niger, K. marxianus, S. cerevisiae, Pichia fermentans and Hansenula anomala, have been reported to have the ability to synthesize 2-PE from l-Phe (Etschmann et al., 2002; Mihaľ et al., 2013; Stark et al., 2003). Eshkol et al. (2009) screened for a robust, stress-tolerant S. cerevisiae natural isolates for production of 2-PE from l-phenylalanine. One thermo-tolerant strain, Ye9-612, was the most efficient and under optimal conditions produced 4.5 g/L 2-PE in a batch-fed fermentation compared to 0.85 g/L in shake flasks. Stark et al. (2003) investigated inhibitory impacts of the bioconversion of l-phenylalanine (l-Phe) to 2-PE on the metabolism of S. cerevisiae in batch, fed-batch, and continuous cultures. The maximum production rate of 2-PE was obtained in the fed-batch cultures up to 0.42 g/L/h, compared to a value of 0.3 g/L/h in the batch culture and 0.33 g/L/h during the continuous cultures. The repeated-batch operations,
Different Bioengineering Approaches on Production of Bioflavor Compounds 51 which combine advantages of fed-batch and batch processes can be alternative process strategy for production of flavor compounds. The repeated-batch process involves repeated cycles of fermentation by reinoculating a part or all of the cells from one batch fermentation medium into the next batch medium. Thus this strategy mainly is possible to conduct during long periods and to improve the production compared to the batch process (Romero-Guido et al., 2011). Compared to the SmF process, microorganisms grow on nonsoluble material that acts both as physical support and source of nutrients in the absence of or near absence of free water in solid state fermentation. SSF technology has several processing advantages such as higher fermentation productivity, more concentrated form of products with higher stability, which are important properties for downstream processing. Also, this technology allows the use of various wastes from food and agricultural industries, as compared with SmF, which makes the process economically competitive (Fadel et al., 2015; Singhania et al., 2009). Using SSF for the production of flavor compounds by yeasts and fungi via de novo synthesis or transformation was studied (Akacha and Gargouri, 2015; Longo and Sanroman, 2006; Mantzouridou et al., 2015; Medeiros et al., 2000, 2003, 2006). De Aráujo et al. (2002) studied production of 6-pentyl-alpha-pyrone (6PP) using SSF higher than that was used for the liquid fermentation process. The amount of 6PP (3 mg/g) produced during solid-state-fermentation process is higher than that reported in literature for submerged process. Additionally, a mixed-culture of fungi and yeasts, which cannot be cultured in SmF, can produce, in a synergistic way, various aroma-active components in SSF (Hölker et al., 2004). Bacteria have been considered as unsuitable for solid-state fermentation because of their higher water-activity requirements. However, growing bacteria on solid or semisolid mediums for the production of flavor compounds has been reported (Akacha and Gargouri, 2015; Besson et al., 1997; Escamilla-Hurtado et al., 2005). Although, SSF is the oldest technology used since ancient times and has several biotechnological advantages, scaling up in this technology has been a limiting factor so far, mainly due to the heterogeneous nature of the substrate, mass, and heat transfer problems (Akacha and Gargouri, 2015; Hölker et al., 2004; Singhania et al., 2009). However, some digital imaging technologies have been developed for evaluating the growth kinetics in filamentous fungi in SSF, which can be further improved for application of mathematical modeling tools to describe the system for scale-up studies (Couri et al., 2006). Additionally, researches have been directed toward the development of new reactor designs such as a rotating drum bioreactor, an immersion bioreactor, and a mixed solid-state bioreactor, which would overcome the difficulties for large-scale productions (Longo and Sanroman, 2006; Medeiros et al., 2006). The recent demand for natural flavor compounds has forced the discovery of alternative fermentation techniques. However, due to the variations among different fermentation techniques, a lot of work still needs to be done in terms of comparison of these techniques
52 Chapter 2 and identifying sustainable substrates and processes to maintain high productivity and quality. Those can help in increasing the production and reducing the cost of bioflavor compounds.
3.2 Bioengineering Approaches Used for Scaling Up and Downstream Processes of Bioflavor Compounds Research has been conducted in the last two decades to accelerate the bioflavor production process. During the process scale-up of bioflavor compound production from laboratory scale to pilot or industrial scale, a number of physiological changes were observed (i.e., microbial growth, production rates, and sporulation), which were triggered as result of differences in the production systems. Additionally, certain constraints must be taken into account when choosing the process to recover aroma compounds from liquid effluents owing to the chemistry of both substrate and products. However, it is necessary to assure the molecule integrity (according to the low thermostability of flavor compounds), and it is important to have an efficient process that is easy to install and maintain. Both substrate and products are often not very water soluble, easily get lost through the exhaust gas stream of the reactor, and characterized by low productivity because of their toxicity against the producing cells. Hence, the product stream is generally dilute; this leads to high costs in the subsequent isolation and purification steps (Souchon et al., 2004; Stark and Von Stockar, 2003). One additional tool that could often yield significant increases in the productivity of many processes is the use of in situ product recovery (ISPR) techniques (Hua et al., 2007, 2010; Rito-Palomares et al., 2001; Stark and Von Stockar, 2003). Several ISPR configurations, with their advantages and limitations, have been applied through the process lines. The total contribution of flavor/fragrance category in ISPR reports during the period of 2003–13 was 46%, which was examined by Van Hecke et al. (2014) in whole-cell biotechnology. Different configurations of ISPR are possible depending on the separation unit, that is, separation can be done inside the bioreactor where the reaction takes place (internal) or in an external integrated unit (external). Alternatively, configuration was done directly via the whole fermentation medium (direct) or rather from the supernatant obtained from cell separation (indirect) outside the reactor. Van Hecke et al. (2014) reported a vast majority of researches (60%) applied the internal-direct configuration, as compared to 42% of the external type (both direct and indirect). When the presence of microorganisms adversely affects the performance of the ISPR technology, an indirect configuration is preferred. The technique used is dependent on the properties of the product to be separated (e.g., volatility, molecular weight, size, charge, solubility, and hydrophobicity; Heerema et al., 2011). For all designs, integration of the downstream process step with bioreactor ensures the direct removal of product during growth and progression of reactions, potentially increasing the productivity of the biocatalyst. There are many advantages of ISPR, which allow decreasing wastewater volume, fermenter volume, and the stress on microorganisms resulting from oxygen limitation and shear stress caused by the cycling of the fermentation
Different Bioengineering Approaches on Production of Bioflavor Compounds 53 broth. Additionally, there is facilitation further downstream from processing the product and a decrease in the total process costs and processing time (Bluemke and Schrader, 2001). A product can be removed from the fermentation medium by evaporation, which is the most commonly used method for concentration of liquids; however, it will be lacking in aroma compounds and coupled with water vapor. Yanniotis et al. (2007) presented a new system in which the vapor generated in an evaporator passes through an absorber where a hygroscopic solution partially absorbs water vapor. In this way, the relative concentration of the aroma compounds was 1.8–2.8 times higher than in the condensate obtained from comparative simple distillation experiments. The use of nonconventional media such as ionic liquids (ILs), supercritical fluids (sc-fluid), and solid–gas interphase reactions has definitely opened new perspectives in recent years. ILs have been considered the green solvents and could substitute for the toxic and flammable organic solvents (Cantone et al., 2007; Schlosser et al., 2005). Different biocompatible ILs were tested for 2-PE recovery capability by determining their distribution coefficients. A 3- to 5-fold increase in the total 2-PE production was obtained with 1-methyl-propylpiperidinium bis(trifluoromethylsulfonyl)imide (MPPyr[Tf2N]), methyltrioctylammonium bis(trifluoromethylsulfonyl)imide (OMA[Tf2N]), and 1-butyl2-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMIM[Tf2N]) (Sendovski et al., 2010). The biosynthesis of GDL in IL containing cosolvent system by immobilized whole cells of Y. lipolytica has been reported recently (Zhao et al., 2015). Under the optimized conditions, the GDL yield was up to 8.05 g/L. After 10 times of reuse, the GDL yield was 7.51 g/L, corresponding to 93.3% of that obtained in the first batch, suggesting a good reusability and potential for industrial applications (Zhao et al., 2015). Supercritical fluid extraction (SFE) uses fluids at supercritical conditions to selectively extract substances from solid or liquid mixtures. The most used sc-fluid for SFE is CO2. The extraction of aromas and flavors from herbs, spices, and fermented foods by means of supercritical CO2 is an attractive alternative to conventional aroma recovery processes such as distillation, adsorption, and solvent extraction (Brunner, 2005). The comprehensive review can be examined for the detailed information about ILs and sc-fluids, biotransformation performed in them and their combining applications, which are promising methods for bioflavor industry (Brunner, 2005). Lu et al. (2011) used SFE method for the first time to recover aroma compounds from Zhenjiang aromatic vinegar, which is produced from sticky rice through solid-state fermentation. Fabre et al. (1999) dealed with the feasibility and optimization of the process of achieving an extractive fermentation of 2-PE coupling fermentation with K. marxianus and SFE. Their first results revealed that direct SFE was not possible, due to a drastic CO2 effect on cell viability. Therefore they performed cell separation prior to the extraction. The most investigated ISPR techniques in research and scale-up applications of several bioflavor processes were two-phase systems (Etschmann and Schrader, 2006; Morrish et al., 2008; Wang et al., 2011). Aqueous two-phase system (ATPS) has been suggested to use as an alternative system, and it is usually composed of a mix of two or more incompatible
54 Chapter 2 polymers or a polymer-salt system in aqueous environment (Akacha et al., 2007; Lu and Zhang, 2008). Polyethylene glycol (PEG)-potassium phosphate and PEG-magnesium sulfate are among the most frequently used polymer-salt systems (Porto et al., 2008). The technique has several advantages including ease of scale-up, biocompatibility, low cost. Partition behavior of 6PP and Trichoderma harzianum mycelium in PEG-salt and PEG-dextran two-phase systems were determined (Rito-Palomares et al., 2001). PEG-dextran systems were unsuitable for in situ recovery of 6PP because both 6PP and biomass partitioned to the same phase or a large extraction phase was required for this process. However, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and more suitable in the potential application for in situ recovery of 6PP. The highest 2-PE concentration (26.5 g/L) and 6.1 g/L 2-PEAc in polypropylene glycol were obtained in an optimized batch-fed procedure using K. marxianus CBS 6001200 reported by Etschmann and Schrader (2006). However, emulsion formation upon stirring caused problems in the product recovery process reported by the same researchers. Macroporous adsorbent resins have been developed and evaluated for in situ removal of bioflavor compounds during the production with direct or external contact configurations. Especially, adsorption on activated carbon and cross-linked polystyrene resins are suitable methods to extract and concentrate volatile compounds from aqueous medium (Fernandes et al., 2003). The successful application of adsorbent resins, with wide variations in their functionality, porosity, and surface area, in the bioproduction of flavor/fragrance compounds, such as 2-PE (Ang et al., 2009; Gao and Daugulis, 2009; Hua et al., 2010), fruity esters (Medeiros et al., 2006), and vanillin (Hua et al., 2007; Ma and Daugulis, 2014). Ma and Daugulis (2014) showed that polymers have advantages in ISPR applications by maintaining sensory quality of the desirable product. Such improvements in the performance of the bioprocess can result in the desired final product concentration significant for developing an industrial-scale biotransformation process (Ma and Daugulis, 2014). Because of the low adaptability of standard bioreactors to work with solids resins, the use of external units are more applicable where adsorption, desorption, and regeneration occur. A highly efficient continuous bioprocess with two resin columns, which can be run on line and regenerated alternatively by switching the lines, offering excellent industrial production of the natural 2-PE has been described by Wang et al. (2011). A few attempts have also been made to use different resins as a product-removal technique to increase the productivity in vanillin production. During the two-step bioconversion process of vanillin from vanillic acid, some filamentous fungi strains subsequently further transform vanillin into vanillyl alcohol (Ma and Daugulis, 2014). High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1 (Hua et al., 2007). Due to the toxicity of vanillin and the product inhibition, a batch-fed biotransformation strategy using adsorbent resin was also investigated. Several macroporous adsorbent resins (CAD40, CD180, DM11, DM130, HZ803, and HZ816) were tried to adsorb vanillin in situ during the biotransformation. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g/L
Different Bioengineering Approaches on Production of Bioflavor Compounds 55 ferulic acid could be added continually and 19.2 g/L vanillin was obtained within 55 h (Hua et al., 2007). Medeiros et al. (2006) used activated carbon, Tenax-TA, and Amberlite XAD2 adsorbent columns connected to the column-type bioreactor (lab scale) for recovering fruity characteristics produced by Ceratocystis fimbriata. They reported that all compounds present in the headspace of the reactor were adsorbed in Amberlite XAD-2. Acetaldehyde was adsorbed in higher concentrations with Tenax-TA. However, the recovery found by using the activated carbon was very low. During the last decade, membrane processes for liquid separation such as nanofiltration and reverse osmosis, pervaporation, and membrane-based solvent extraction (perstractive systems) have been investigated to extract aroma compounds from aqueous (Adler et al., 2011; Bocquet et al., 2006; Heerema et al., 2011; Stefer et al., 2003) solutions. There are polymeric or zeolite membranes usually serve as a barrier for such processes. When a membrane is in contact with a liquid mixture, one of the components can be preferentially removed from the mixture due to its higher affinity with, and/or quicker diffusivity in, the membrane. In pervaporation, a partial vaporization of the liquid solution occurs through a dense membrane. Solutes are dissolved in the polymer material at the upstream face of the membrane and then diffuse into the polymeric network and are finally evaporated in the downstream gas phase (Lamer et al., 1996; Stefer et al., 2003). Controlling step of mass transfer depends on the properties of flavor compounds and on the stripping phase (Fernandes et al., 2003; Schlosser et al., 2005). The production of de novo synthesized natural aroma compounds (ethyl acetate, propyl acetate, isobutyl acetate, isoamyl acetate, citronellol, and geraniol) by Ceratocystis moniliformis with an integrated bioprocess was implemented by means of interlinking a pervaporation membrane module with a producing bioreactor (Bluemke and Schrader, 2001). Authors stated that by circumventing inhibiting product concentrations and thus intensifying aroma production, the total yield of aroma compounds produced was higher in an integrated bioprocess compared with batch cultivation. In addition, permeates obtained from pervaporation consisted of highly enriched mixtures of produced flavors and fragrances (Bluemke and Schrader, 2001). In applying a suitable membrane configuration, such as a hollow fiber, rotating annular, and so forth, fluids should flow on opposite sides of the membrane and the fluid/fluid interface forms at the mouth of each membrane pore (Gabelman and Hwang, 1999; Schlosser et al., 2005). The membrane acts as a physical barrier between the aqueous feed and the stripping phase and imparts no selectivity to the separation in hollow-fiber membrane contactors (HFMCs). Liquid-liquid extraction of flavor compounds with hollow-fiber contactors has been examined in a few studies (Schlosser et al., 2005; Souchon et al., 2004). Unlike columns and other traditional contactors, HFMCs offer flexibility in the choice of operating conditions. Hence, there are no loading, flooding, or emulsification problems; except for the pumping of the flowing phases, no agitation or moving part is needed (Gabelman and Hwang, 1999; Schlosser et al., 2005). Definitely, the use of HFMCs also brings some disadvantages. The membrane induces another resistance to mass transfer, which results in a loss in efficiency. Additionally, membranes are subject
56 Chapter 2 to fouling and have a finite life, so that the cost of periodic membrane replacement should be considered (Gabelman and Hwang, 1999). Nevertheless, HFMCs seem to be interesting devices to carry out aroma compound extractions as shown by different works in this area (Bocquet et al., 2006; Mihaľ et al., 2013; Souchon et al., 2004). Hollow-fiber modules applied for industrial applications are available from a variety of sources. They can be designed for pressure-driven filtration processes or concentration-driven mass transfer (Gabelman and Hwang, 1999). Bocquet et al. (2006) investigated the extraction of PE, BA, ethyl butyrate, and dimethyltrisulfide by hexane in the most well-known two Liquicel cross-flow hollow-fiber modules (hollow-fibers X-30 and X-40 module) made of polypropylene offered by Celgard LLC (Charlotte, NC; formerly Hoechst Celanese). Modules are designed for concentrationdriven mass transfer explained in detail by Gabelman and Hwang (1999). Researchers studied the parameters of the process, which were the position of the feed: inside or outside the fibers and the nature of the commercial module X-30 or X-40 module. Simulations and experiments showed that the best configuration for the extraction is when the feed flows inside the shell with the X-30 module. Mihaľ et al. (2013) presented a work to investigate the possible yield increase of 2-PE production in a hybrid system consisting of membrane extraction performed by a hollow-fiber membrane module immersed in the down flow of an airlift reactor (ALR). The proposed hybrid system by researchers consisting of an ALR and a membrane module for membrane extraction immersed in the down flow of the ALR seemed to be a very suitable device for in situ 2-PE removal from the fermentation medium. A handmade hollow-fiber membrane module was stable during the extraction experiments without any leakage of the organic solvent to the water phase or vice versa. Direct contact of the biomass with the membrane module during the extraction experiments did not cause any clogging of the fibers and the biomass had no influence on the PEA extraction kinetics or on the PEA partition coefficient. The membrane extraction in the hybrid system could be also satisfactorily predicted using mathematical modeling comprising correlations taken from literature. One of the disadvantages of this hybrid system reported by the researchers was the loss of the used organic solvents during the membrane extraction process caused by the weak continuous dissolution of the solvent in the water phase, followed by its stripping with air out of the ALR. The solution proposed by the same researchers to avoid this problem was to use solvents that were less soluble or insoluble in water (Mihaľ et al., 2013). Some strategies have also been developed in order to reduce the toxicity of lactone toward organisms. In a study, three different techniques (in situ trapping in oily phases, in porous hydrophobic sorbents and in β-cyclodextrins) were used to overcome the toxicity of GDL during a bioconversion process using ricinoleic acid as a precursor. Oily phases added to the media (olive, Miglyol, tributyrin, and paraffin) had a protective effect on Sporidiobolus salmonicolor, and they improved the lactone production (Dufossé et al., 1999). Alchihab et al. (2010) studied the production of GDL and 4-hydroxydecanoic acid by the psychrophilic yeast Rhodotorula aurantiaca. Productions were performed in 20 L and 100 L bioreactors with the addition of gum tragacanth to the medium at concentrations of 3 and 4 g/L. They stated that
Different Bioengineering Approaches on Production of Bioflavor Compounds 57 it could be an adequate strategy to enhance GDL production and to reduce its toxicity toward the cell by adding the gum. Six carbon-aldehydes, (2E)-hexenal and (3Z)-hexen-1-ol, with “green characters” widely used flavor compounds to give both the green note and the impression of freshness in food and fragrance applications. Although the traditional source of such compounds are plants, they cannot be obtained in sufficient amounts, which motivated researchers finding an alternative way (Akacha and Gargouri, 2009; Cass et al., 2000; Schade et al., 2003). In plants, C6-aldehydes are formed through the LOX pathway, in which unsaturated fatty acids are converted in a sequential action involving the enzymes LOX and hydroperoxide lyase (HPL) into hexanal. It is possible to reuse the biocatalyst by keeping its operational stability for a long time by using immobilized enzymes in such bioprocesses. Enzyme templates, responsible for the synthesis of hexanal from linoleic acid (18:2) isolated from naturally enriched tissues of carnation petals, strawberry and tomato leaves were immobilized in an alginate matrix and used as a biocatalyst in a packed-bed bioreactor (Schade et al., 2003). In addition, continuous product recovery was achieved by using a hollow-fiber ultrafiltration unit coupled to the packed-bed bioreactor to have one-step purification for hexanal production. Under optimized conditions, hexanal production 112-fold higher than endogenous steady-state levels in a proper amount of plant tissue could be obtained over a 30-minute period. An extractive fermentation process for 6PP production by T. harzianum was scaled-up using criteria volumetric power drawn (P/V) from 500 mL shake flasks to 10 L stirred tank bioreactors. Two general effects of P/V over 6PP production were reported by authors: at P/V values under 0.4 kW/m3, a gradual increase in P/V improved 6PP production; meanwhile, at P/V values above 0.6 kW/m3, 6PP concentration was significantly reduced due to higher hydrodynamic stress. Most of the reported ISPR techniques have been attempted at the laboratory scale and in some cases at the pilot plant scale. Many of the process configurations proposed at the pilot-scale are difficult to apply or at least not economically profitable in the large scale. However, an efficient integrated-process modeling has been done to develop the process from the engineering point of view by Adler et al. (2011). The authors developed an unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-PE and 2-PEAc by K. marxianus. They obtained the notably improved agreement model between model and experimental data by using results from the conventional and the ISPR process. Therefore the offered model could be a useful tool for the development and optimization of an efficient integrated bioprocess.
3.3 Statistical Optimization and Mathematical Modeling Studies Used in Bioflavor Production Processes It is important to define an efficient procedure for monitoring the effect of the process variables (factors) on one or more response of interest (dependent variable). The goals in the design of experiment are to collect information about the process and to state the factors
58 Chapter 2 and their levels affecting the quality and characteristics of the process. Statistical design of experiments is a very useful methodology by which selected factors are intentionally varied in a controlled manner to evaluate their effects on a response and followed by the analysis of the findings. After the introduction of process analytical technology (PAT) by the United States Food and Drug Administration, biotechnology industry has been trying to integrate those emerging regulatory demands not only in the pharmaceutical field but also in diagnostics, foodstuffs, commodities, or fine chemicals (Mandenius and Brundin, 2008). Therefore the use of experimental design and mathematical modeling approaches to make production process much safer and reproducible is becoming increasingly important. Experimental design approaches are also of great importance to bioflavor production processes; because the process is very complex and influenced by many factors such as flavor producing strains, organic or inorganic nutrients and environmental factors of the process. Hence, an appropriate experimental design can be used to work on effects of various factors on the process for making it better understood and optimized for improving its performance. This chapter summarizes experimental design methods that have been used to investigate various factors relating to bioflavor production processes. There are many different experimental design approaches used in natural flavor productions like full factorial design, fractional factorial design [i.e., Plackett–Burman design, Taguchi design, Box–Behnken, and central composite design (CCD)], and artificial neural networks (ANNs). Those design methods were briefly introduced, and then their application through bioflavor production processes were analyzed. The design of experiments can be grouped into two groups: one-factor-at-a-time design (single-factor design) and factorial design (multiple-factor design). The level of the factor investigated in single-factor design is changed while the other factors are kept constant (Kennedy and Krouse, 1999). Because, this design is easy to use and analyse, it has been widely used to study the effects of various factors on bioflavor production processes as seen in Table 2.1. For example, Fonseca et al. (2013) investigated the effect of different sugars on the growth, substrate consumption, metabolite formation, and respiratory parameters of K. marxianus CBS 6556 at two different incubation temperatures. All sugars tested one by one at the predefined concentrations for defined responses while keeping the levels of other factors constant. They explained the results with several plotted graphs to show the effects of each carbon source on growth, substrate consumption, metabolite formation, and respiratory parameters. Additionally, dual effects of sugars were investigated with another experimental design. Single-factor design has some disadvantages. It does not take into consideration the interaction effects between factors. These lead to wrong decision about the system especially when the interaction between factors is significant. Another one is that it is laborious and time consuming to carry out the experiments because of relatively large number of experiments, specifically when the number of factors is large (Mandenius and Brundin, 2008). Factorial design can be generally divided into a full factorial design and a fractional factorial design. Full factorial design is a design that consists of two or more factors at
Different Bioengineering Approaches on Production of Bioflavor Compounds 59 Table 2.1: One-factor-at-a time design approaches in bioflavor production processes. Biocatalysts
Flavor Compounds
Factors Studied
References
Trichoderma viride EMCC-107 Seven strains of Wickerhamomyces pijperi
6-Pentyl-α-pyrone (6-PP) Alcohols, aldehydes, acetate esters, ethyl esters α-Terpineol
Sugarcane bagasse concentration, time Effects of composition of culture media
Fadel et al. (2015)
(R)-(+)-limonene concentration
Tai et al. (2016)
Penicillium digitatum DSM 62840 Kluyveromyces marxianus Saccharomyces cerevisiae var. cerevisiae Williopsis saturnus var. saturnus K. marxianus Candida antarctica lipase B displaying Pichia pastoris
Fusel alcohols, esters Carbon and nitrogen source Esters Aeration conditions
Isoamyl acetate
Temperature and aeration
Ethyl acetate Esters
Fe content Solvent, temperature, substrate concentration, ratio of acid to alcohol, and amount of whole-cell biocatalyst Ferulic acid concentration in the bioconversion buffer, pH of the bioconversion buffer and cell concentration of the suspension Temperature, pH, concentration of adsorbent resin Different substrates and gums Temperature, pH, detergents, metal ions and inhibitors, and salt concentrations Enzyme and substrate concentrations Polymer bead concentration
Vanillin
S. cerevisiae P-3
2-PE
Rhodotorula aurantiaca Bacillus licheniformis S-86
Hydroperoxide-lyase (HPLS) K. marxianus
γ-Decalactone Type II esterase activity and isoamyl acetate C6-aldehydes and alcohols 2-PE and 2-PEA
Rh. aurantiaca A19
γ-Decalactone
S. cerevisiae
2-PE
Streptomyces sp. strain V-1 Vanillin
Izawa et al. (2015)
Gethins et al. (2015) Mantzouridou and Paraskevopoulou (2013) Yilmaztekin et al. (2013) Löser et al. (2012) Jin et al. (2012)
Di Gioia et al. (2011)
Hua et al. (2010) Alchihab et al. (2010) Torres et al. (2009)
Akacha and Gargouri (2009) Gao and Daugulis (2009) Alchihab et al. (2009)
Temperature, initial pH, and castor oil concentration Glucose and l-phenylalanine Eshkol et al. (2009) concentrations Concentration of adsorbent resin Hua et al. (2007)
different combination of levels in which the interaction effect of factors are also tested (Montgomery, 2005). Typical important factors in a bioprocess can be strain, medium components, temperature, initial pH, aeration, and inoculation rate (Keskin Gündog˘du et al., 2016; Mandenius and Brundin, 2008). Full factorial design is convenient to evaluate the effects of the factors providing the reduced cost of a large number of experiments. Two-level full factorial designs are some of the most widely used experimental designs and are represented as 2k designs. k indicates the number of factors included in the
60 Chapter 2 model. For example, if k = 4 then the design would be a 24 full factorial consisting of 16 runs (Briggs, 2011). Full factorial design is not preferred when the number of factors is large in the design, since, the number of runs needed for full factorial designs increase exponentially. Using a fractional factorial design is the most widely used option in this situation. Researchers conducted only a fraction of the runs in full factorial designs (Briggs, 2011). Some of the main effects and two-way interactions can be complex and cannot be separated from the effects of other higher-order interactions in the fractional factorial design. Therefore it is more practical than full factorial design (Keskin Gündog˘du et al., 2016; Montgomery, 2005). The application of fractional factorial design methods such as the Taguchi method allows a more flexible way of studying several parameters using a small number of experimental trials achieved by the design of an orthogonal array (OA). Meanwhile, the effects of many factors on a response are studied at two or more levels. Instead of testing all combinations like full factorial design, Taguchi method uses pairs of combinations. The Taguchi method can be effectively used when the number of variables is between 3 and 50 (Keskin Gündog˘du et al., 2016). Among the reviewed studies that were published in recent years on the production of flavor compounds, Koh et al. (2013) performed the fermentation process by K. lactis KL71 for bioproduction of methionol using Taguchi design (L27 OA). Among these studied factors, shaking speed was found to be the most significant factor that affected the bioproduction of methionol, followed by incubation time, pH level, and l-methionine concentration. The average yield of methionol obtained under the optimum fermentation conditions was 990.1 ± 49.7 µg/mL. Plackett–Burman design (PBD), a two-level fractional factorial design has been widely used to define the most important factors from many candidates with a significant effect on the response. The number of runs for a PBD is set to multiples of four (e.g., 12, 16, 20, or 24) (Briggs, 2011). Plackett–Burman design has also been widely used to determine effective factors in the production of flavor compounds (Bicas et al., 2008; Kaur and Chakraborty, 2013; Ramos et al., 2008). Xiao et al. (2007) performed a study in which the nutritional requirements for acetoin production by Bacillus subtilis CICC 10025 were optimized statistically in shake flasks using Plackett–Burman design. The medium components considered for initial screening comprised a-molasses (molasses submitted to acidification pretreatment), soybean meal hydrolysate (SMH), KH2PO4·3H2O, sodium acetate, MgSO4·7H2O, FeCl2, and MnCl2, in which the first two were identified as significantly (at the 99% significant level) influencing acetoin production. Factors screened by the Plackett–Burman and Taguchi designs can be further investigated using response surface methodology (RSM) (Bicas et al., 2008; Wang and Wan, 2009). RSM is used for developing, improving, and optimizing processes based on various statistical and mathematical techniques. Central composite design (CCD) combines a two-level full or fractional factorial design, developed by Box and Wilson, with external points and at least one point at the center of experimental region, which is necessary to obtain several properties,
Different Bioengineering Approaches on Production of Bioflavor Compounds 61 such as rotatability or orthogonality, to fit the quadratic polynomials (Keskin Gündog˘du et al., 2016; Wang and Wan, 2009). Box–Behnken design is a three-level fractional factorial design and developed by Box and Behnken, widely used experimental designs for surface RSM to estimate a second-order polynomial approximation to a response in the region (Keskin Gündog˘du et al., 2016; Montgomery, 2005). For RSM, a second-order polynomial model [Eq. (2.1)] is usually proposed to describe the effects of various factors on a response based on experimental results from a CCD or Box–Behnken design;
k
k
i =1
i =1
y = β 0 + ∑ β i xi + ∑ β i i xi2 + ∑ β i j xi x j
(2.1)
i< j
where y is the response, β0 is the constant and βi is the linear coefficient, βii is quadratic coefficient, βij is the interactive coeeficient, and xi is the coded factor level. A second-order polynomial model can also be displayed as a surface plot and a contour plot, by varying only two factor levels, while keeping other factor levels constant (Montgomery, 2005; Wang and Wan, 2009). For example, Barghini et al. (2007) investigated the effects of substrate concentration and biomass on the vanillin production by RSM using a CCD. A second-order polynomial model and surface plots were used to show the effects of factors on the production rate of vanillin. Based on the analysis of variance for the estimated model, the RSM was an effective way to optimize the process. The highest vanillin molar yield (about 75%) was found at low ferulic acid concentrations (0.5 mM) using 4.5 g (wet weight)/L of biomass. As shown in Table 2.2, among the reviewed studies, RSM has been widely used for optimization of different flavor compounds. However, the application of screening methodologies like Plackett–Burman and Taguchi designs for identification and a better selection of medium factors, followed by execution of RSM approaches according to the screening results is not very common in bioflavor production processes (Medeiros et al., 2000). Such approaches are mostly used for optimizing microbial growth. For example, Li et al. (2009) applied RSM to optimize culture conditions for the growth of C. utilis with bamboo wastewater. A significant influence of initial pH, fermentation time and yeast extract on biomass of C. utilis was evaluated by Plackett–Burman design. These factors were further optimized using a CCD. A combination of initial pH 6.1, fermentation time 69 h and yeast extract 1.17 g/L was determined as the optimum for maximum biomass of C. utilis. The ANN and the genetic algorithm (GA) have been used as two of the most efficient methods for empirical modeling and optimization of bioprocesses, specifically for nonlinear systems in the past two decades (Desai et al., 2008; Franco-Lara et al., 2006; Nagata and Chu, 2003). ANN is a nonlinear computational model based on biological neural networks. It simulates the human brain learning process by mathematically modeling the network structure of interconnected node cells. ANN has been utilized with high success for system design, modeling, optimization, and control, mainly due to their capacity to learn, filter noisy
62 Chapter 2 Table 2.2: Factorial design applications for bioflavor production processes. Biocatalysts
Flavor Compounds Designs
Vanillin Pediococcus acidilactici BD16 MTCC 10973 Lipase Nonyl caprylate
Factors Studied
Central Substrate, temperature, composite design time, biomass
Central Reaction time, reaction composite design temperature, amount of enzyme and shaking speed Lipozyme IM77 C6-aldehydes Box–Behnken Temperature, mixture design flow rate, and lauric acid concentration Plackett–Burman Medium composition, the Fusarium R-(+)-α-terpineol presence of a cosubstrate, screening, oxysporum the cultivation conditions optimization (temperature, agitation), using a central composite design the substrate concentration, and the inoculum/culture medium ratio, temperature, agitation rate, substrate (%), cosubstrate Recombinant Vanillin Central Biomass and substrate Escherichia coli composite design concentration Enzymes 2E-Hexenal A 23 factorial Nitrogen flow, reactor design temperature, and trap temperature Immobilized Hexanal A 22 factorial Substrate concentration and enzyme design catalyst loading Alcohols, esters and A 25 factorial Initial pH, addition of K. marxianus aldehydes design, a 22 glucose, cultivation factorial design temperature, initial substrate moisture and inoculum size, pH and glucose
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signals, and generalize information through a training procedure (Franco-Lara et al., 2006). When a generalized ANN model has been developed, its input space is optimized using GA. This combined methodology is referred to as ANN-GA. The GA resembles the principles of biological evolution, namely, “survival of the fittest” and “random exchange of data during propagation,” followed by biologically evolving species. GA has been considered as an ideal technique to solve optimization problems in biochemical engineering (Desai et al., 2008; Franco-Lara et al., 2006). The usage of ANN and GA models for optimizing a bioflavor production process is very limited, but both models are highly recommended by some researchers. For example, Etschmann et al. (2004) used a GA to improve the bioconversion of l-phenylalanine (l-phe) to 2 phenylethanol (2-PE) with K. marxianus CBS 600. Within four generations plus an additional temperature screening, corresponding to 98 parallel experiments altogether, a maximum 2-PE concentration of 5.6 g/L was obtained, equivalent to an increase of 87% compared to the nonoptimized medium.
Different Bioengineering Approaches on Production of Bioflavor Compounds 63
3.4 Requirements and Perspectives Nowadays, most of the flavor compounds used commercially are produced by chemical synthesis. However, the current and future prominence of production of natural flavor compounds by biotechnological routes is obvious from the vast amount of published scientific reports about this topic and the number of patent applications and grants. Although, plants are the source of a variety of natural volatile compounds, the yields of those compounds are too low to be commercially competitive. To overcome this problem, researchers have started to develop new technologies such as PTCs through biotechnological approaches. However, the yield is still not sufficient for commercialization. Microorganisms produce a vast variety of flavor compounds during fermentation. At this point, it should be emphasized that there is a need to exploit extended use of already approved natural GRAS (generally regarded as safe by FDA) microorganisms for the production of flavor compounds. Some of the recent developments of commercialized processes are based on biotransformation, which leads to natural precursors into valuable flavor and fragrance compounds through microbial metabolic pathways. With the increasing knowledge of those metabolic pathways, it is possible to know the enzymes and genes involved through the process. For this reason, new opportunities are being revealed with industrially applicable engineered microorganisms for the production of flavors. It is possible to perform such biotransformation through immobilized microbial cells. Optimization of these microbial production systems with respect to nutrient sources, and environmental and bioprocess conditions is necessary to meet the increasing demand for bioflavor compounds. High production costs involved in such a process are one of the major bottlenecks for its application to the industrial scale. Much attention has been given to cost and yield optimization by searching for cheaper substrates (i.e., industrial residues) as a culture medium for microorganisms with suitable bioreactor types, which are critical factors affecting the commercial viability of bioflavor processes. If future studies focus on the control of stability and the microenvironmental changes around the immobilized cell, as well as on the development of simple bioprocess designs, biotransformation studies done with immobilized cells are very promising and present great potential for producing the flavor compounds at an industrial level. Also, during a process scale-up (from flask to bioreactor), a number of physiological changes can be observed in both free and immobilized cells, which could not assure similar production rates or yields in two systems. It is important to select a suitable scale-up criterion depending on the transport property of the system for defining the overall economy of a bioprocess. The alternative, enzymatic synthesis of flavor compounds is advantageous to obtain one specific flavor compound with high specificity and productivity values. However, the process is limited only to a laboratory scale. The majority of these enzymatic synthesis reactions are carried out using lipases. The searches for alternative enzymes from solvent-tolerant microbial cells, because of their stability in organic solvent, are also required for the flavor industry. In addition, a design of continuous enzymatic reactors where biosynthesis of flavor compounds was coupled to their extraction provides a satisfactory process for the production of bioflavor compounds on an industrial scale.
64 Chapter 2 The fundamental advantages of application ISPR techniques to bioflavor processes lie in avoiding or reducing inhibition or toxicity caused by substrates or products, stabilization of products, and facilitation of further downstream processing. Among them, in situ product adsorption applying macroporous adsorbent resins, an ATPS, and membrane-based solvent extraction have been widely used in the production of different natural flavors. It is important to consider adsorbent resins with high specific surface area that are odorless, thermotolerant, biocompatible, and cheap, while using a solid-liquid two-phase partition bioreactor system. Regarding membrane-based technologies, finding the best configuration of the membrane modules according to the flavor compounds properties is important. Generally, further progress could also be established with the development of new extractants or membrane phases. Development of effective and cheaper contactors, preferably hollow-fiber ones, would help in the application of these processes on an industrial scale. Additionally, ILs have definitely opened new perspectives in extractive separations of flavor compounds. Significant developments take place in online monitoring and process modeling, which are useful supporting tools to develop the control scheme for the overall integrated bioprocess. Implementation of such process modeling and simulation programs for bioflavor processes would definitely facilitate control of the process toward the desired outcome. The primary economic driving force for the synthesis/extraction of bioflavor compounds is the desire to establish reliable and economically profitable production systems that are environmentally benign in comparison with the classic production approaches based on largescale chemical synthesis.
4 Conclusions The production of natural flavor compounds through microbial cultures, their enzyme preparations, or plant tissue cultures generates great interest in both industry and the general public for development of alternative and attractive bioengineering approaches. Industrial application of bioengineered microorganism (metabolically and genetically modified forms) for de novo synthesis enhances the production of desired metabolite. Although microbial biotransformation has been recognized as having a significant economic potential, technical problems have been encountered and prevent the commercialization of the process. Enzyme catalyzed reactions and enzymatic reactors for production of flavor compounds have undergone a rapid development with lots of advantages over de novo synthesis. Much attention should be given to productivity and cost optimization of the process by seeking cheaper nutrient sources (i.e., industrial wastes). Once the association of substrate and biocatalyst is established, the accurate choice of bioreactor type and the optimization of operation conditions are also of great interest. Bioflavor production processes have been limited also by product inhibition, unstable characteristics of products or formation of toxic by-products. A feasible solution to overcome such drawbacks is in situ removal of products
Different Bioengineering Approaches on Production of Bioflavor Compounds 65 as they are formed. Although highly efficient continuous bioprocesses with different in situ product removal techniques for the production of various types of bioflavor compound have been described in the literature, final product concentrations are still far below what is required for commercially competitive processes.
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