Microbial Production of Natural Flavours

Technology of Handling, Packaging, Processing, Preservation of Fruits and Vegetables: Theory and Practicals, pp 767-813 © 2019, Editor, V.K. Joshi New India Publishing Agency, New Delhi, India

35 Microbial Production of Natural Flavours RANJEETA BHARI AND R.S. SINGH* Department of Biotechnology, Punjabi University, Patiala 147 002 (Pb.) *Email: [email protected]

1. INTRODUCTION Flavour is a distinctive property of a substance affecting the gustatory sense. It may be defined as “The sum of those characteristics of any material taken in the mouth, perceived principally by the senses of taste and smell, and interpreted by the brain.” Flavour perception is a complex process which requires the particles to be mixed with saliva and salivary enzymes to promote the release of flavour. Flavour molecules are very diverse in nature. More than 5000 compounds in food have been identified as flavour components which can be detected at very low concentration and are thus, used in products at very low levels. Hexenols and hexenals, characteristic of green taste of fresh fruits and vegetables, can be perceived at a concentration of about 1 part per million (ppm). Naturally occuring flavour compounds are present at a very low concentration ranging from parts per trillions (ppt) to parts per millions (ppm) level. Majority of these compounds are volatile, with boiling points in the range of 20-300 °C. Most of these compounds are lipophilic and possess a great diversity of chemical group and structure with molecular weight ranging between 50-250 g/mol. Flavour and fragrance compounds are a part of expanding industry with an estimated cost of nearly $27 billion. These comprise of diverse molecules with unique structures and functional groups that contribute to varied aroma and flavour, ranging from floral to fruity notes. The processing operations modify the equilibrium between the different components in raw food that might weaken the original flavour, which require subsequent supplementation. These flavour supplements can be natural or chemically synthesized. Of all the available flavour compounds, around 80% are produced by chemical synthesis whose use is limited due to formation of racemic mixtures and lack of substrate specificity.

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Technology of Handling, Packaging, Processing Preservation

The growing awareness of consumers towards chemicals supplemented to food has led to the development of the flavouring compounds of biological origin referred to as natural flavours or bioflavours. The first major source of flavour compounds was primarily from plants. Identification of microbes producing vital food flavourants paved the way for alternative low cost, efficient and flexible method for the commercial production of flavour compounds. Flavours have been broadly categorized as natural or nature-identical. According to EC Flavour Directive (88/388/ EEC), natural flavours are defined as the substances or preparations which are obtained by appropriate physical, enzymatic or microbiological processes from material of plant or animal origin. It states that natural flavours include products obtained through microbial or enzymatic processes as long as the precursor/raw material are natural and obtained through physical or biological processes, and that the precursor and product can be found in nature or are a part of traditional food(s). Natural flavours are extracted from plants or prepared either by de novo synthesis in microbes or plants or through single-step biotransformation of natural substrates by microbes or their enzymes or plant cells. In de novo synthesis, microbes transform carbon or nitrogen compounds into flavour molecules with the help of enzymes that have found immense industrial potential for the biotransformation of cheap substrates to natural food flavourants. ‘Nature-identical’ flavours are synthesized chemically or by conversions of natural substrates, and include some aroma compounds like esters (ethyl and butyl acetates, ethyl butyrate, caproate, isobutyrate, isovalerate, 2-methyl butyrate, menthyl acetate), aldehydes and ketones (acetaldehyde, diacetyl), acids (acetic, butyric, caproic, caprylic, isobutyric, isovaleric, 2-methyl butyric) and lactones (gamma-decalactone) which can either be produced by microbial fermentation or by using the enzymes. Various classes of compounds responsible for particular aroma notes are listed in Table 1. Amoore has given a stereochemical theory based on the lock and key hypothesis to explain characteristic flavours exhibited by different substances. While thousands of molecules are known to have interesting aromas or tastes, only a few hundred are used regularly in flavours and still fewer are manufactured at large scale. The key compounds contributing to flavour in some of the foods are given in Table 2.

Microbial Production of Natural Flavours

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Table 1. Specific aroma notes and the possible class of flavorants. Aroma notes

Possible class of compounds

Fruity Floral Green Soily Almond

Aliphatic short chain aldehydes and esters Aromatic alcohol and esters Alcoholics, aldehydes, ketones, terpenes Terpenes Aromatic aldehydes, nitriles

Woody, musty Sugary Sweet Spicy Camphoraceous

Aliphatic alcohols Furan derivatives Alcohols, Thiols Terpenes Camphor

Sulphurous

Sulphur compounds

Source:

Flavours are bioactive compounds and their chirality influences the odor perception, thus strengthening the potential of biocatalysts in flavour production (Fig. 1). Contemporary microbiological techniques, including genetic engineering, are now being increasingly employed to enhance the efficiency of biocatalysts. Advantages of biotechnological approach for production of flavour compounds include independence of manufacturing process from climatic conditions, disease, application of fertilizers or pesticides and shortage due to local conditions of production (climate, disease, pesticides, etc.), easy scale-up and easy product recovery. Not only this, such processes involve conditions which are less damaging to the environment than chemical processes, and yield desirable enantiomeric flavour compounds. Raspberry ketone (threshold: 1-10 parts per billion) is a characteristic flavour compound of raspberries that can be produced chemically via condensation of p-benzaldehyde with acetone (Fig. 2a) or extracted from raspberries. The low yield of the ketone (3.7 mg/kg berries) poses economic restraints on commercialization of chemical synthesis due to high cost involved in the extraction procedure. However, hydrolysis of betuloside from Rhododendron using commercial -glucosidase from A. niger yields betuligenol, which could further be converted into the ketone via secondary dehydrogenase of Candida boidinii (Fig. 2b). In the present chapter, review of important flavouring compounds and their possible microbial synthesis has been described.

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Table 2. Key flavorants in some food.

contd...


Grapes

Isoamylacetate

Cinnamic aldchydc

Ethyl-(E,Z)-2,4decadienoate

771

772


Fig. 1. Effect of chirality on odor perception of nootkatone.

(a)

Fig. 2. Production of raspberry ketone by (a) Chemical synthesis; (b) Biotransformation approach.


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2. SOME IMPORTANT CLASSES OF FLAVOUR COMPOUNDS 2.1 Lactones These are cyclic esters of - and -hydroxy acids formed internally and are in equilibrium with the corresponding alcohols, and have a high flavour profile and contribute chiefly to fruity, coconut, buttery, creamy, sweet or nutty flavours in dairy products. Free lactones and their precursors are present in fresh butter imparting fruity, sweet flavour. -lactones are also present in animal fats and auto-oxidized vegetable oils. These impart pleasant flavour to candies and confectionaries. -Hexalactone or 5-methylpentanolide is a weak flavour, present in butter, coconut, raspberry, strawberry and tea. Some of the other important flavour -lactones and their properties are described in Table 3. Table 3. -Lactones and their properties.

contd...

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-Decalactone, also called as decan-4-olide, is an important lactone for flavour industry and is used as a fruit and dairy flavour. It has an extraordinarily tenacious odour and very powerful, creamy taste at a concentration below 5 parts per million. It is found in peach and used in peach, apricot, pear, maple, coconut, butterscotch and date flavours. It can be produced commercially by transformation of ricinoleic acid present in castor oil, the reaction being catalyzed by Yarrowia lipolytica or lipase from Candida guillermondii. Aspergillus niger, Pichia etchelisii and Cladosporium suaveolens have been reported to produce -decalactone from castor oil. Relatively lower product yields have been reported using other organisms such as Monilia fructicola, Sporobolomyces odorus and Rhodotorula glutinis. In early 1980s, -decalactone was extremely expensive, priced at more than US $ 10,000/kg. Optimization of microbial processes has resulted in a price drop to US $ 300/kg. 6-Pentyl-2-pyrone (6-PP), found as a major volatile constituent in Trichoderma viride cultures, exhibits a coconut aroma. Ricinoleic acid and coriolic acid can be transformed to -decalactone and -decalactone. Octalactones have been formed by fermentation of n-octanoic acid (caprylic acid) present in coconut oil by Mortinella species. -decalactone can be produced from 11-hydroxy palmitic acid present in sweet potatoes by yeast species. The various routes to lactone formation are illustrated in Fig. 3.


(a)

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(b)

Fig. 3. Biotransformation approaches for the production of flavour lactones.

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2.2 Pyrazines These are heterocyclic, nitrogen-containing compounds contributing to nutty and roasted flavours. Methoxy pyrazines are grape derived flavours, which give characteristic herbaceous, bell pepper or earthy aroma to grape wines. 3-Isobutyl-2-methoxy pyrazine and 3-isopropyl2-methoxy pyrazine are important determinants of green flavours in sauvignon blanc wines. 2,3,5-Trimethyl pyrazine is used as a chocolate flavour enhancer, while 2-methyl-3-methoxy pyrazine enhances the flavour of nuts. 2-Methyl-3-isobutyl pyrazine enhances the flavour of bell pepper while 2-acetyl-3-methoxy pyrazine is used as toasted corn flavour enhancer. These are formed through Maillard reaction during roasting of food but are also synthesized by some microbes, such as bacteria Corynebacterium glutamicum, that produces tetramethyl pyrazine from amino acids.

2.3 Esters Esters are the class of naturally occurring organic compounds in plants and animals and are common flavouring agents known for their fruity aroma. These are used in beverages, jams, jellies, candies, baked products, wine and dairy products like yoghurt, sour cream and cheese. Ethyl, methyl, propyl, butyl, isobutyl, amyl and isoamyl esters are commonly used flavours in food industry. Flavour of golden apples is attributed to hexyl-2-methylbutyrate while ethyl butyrate is known for its pineapple flavour. Methyl and ethyl cinnamates provide sweet, honey notes and are associated with strawberry flavour. Ethyl esters are formed by esterification or inter-esterification reactions catalyzed by lipases (Fig. 4). In plants, esters are formed from amino acids or fatty acids by the action of lipases and esterases. Acetate esters like isoamyl acetate and 2phenyl ethyl acetate produced respectively, by yeasts Hanseniaspora guillermondii and Pichia anomala are chief flavourants in grape-derived alcoholic beverages. E. coli has been successfully engineered to produce a variety of esters through the condensation of various alcohols and activated CoA species catalyzed by O-acyltransferases. Acetyl CoA is an abundant central metabolite. The co-expression of O-acyltransferase from S. cerevisiae and a 2-keto acid-derived alcohol production pathway has been reported to facilitate the conversion of 50 g/L glucose into 15–20 mg/L of each ester, ethyl acetate, propyl acetate, isobutyl acetate (IBA), 2-methyl-butyl acetate, 3-methyl-butyl acetate and 2-phenylethyl acetate. Butyl CoA, derived from 2-ketovalerate, can be condensed with butanol for the production of butyl butyrate (up to 14.9 mg/L) by feeding 3 g/L of both the substrates.


R1-COOH + R2-OH R1-CO-O-R2 + R3-OH

Esterification Transesterification

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R1-CO-O-R2 + H2O R1-CO-O-R3 + R2-OH

Fig. 4. Esterification-transesterification reactions for the production of esters.

2.4 Terpenes Terpenes have got their names from terpentine, fluid obtained by distillation of resins from certain trees. These are large and varied class of hydrocarbons, having general formula (C 5 H 8) n , where n=2 for monoterpenes, n=3 for sequiterpenes, n=4 for diterpenes, n=6 for triterpenes and n=8 for tetraterpenes. These are produced by a wide variety of plants, particularly conifers, though also secreted from osmeterium of some insects like swallowtail butterflies. These are the primary constituents of essential oils in plants and are widely used as natural flavour additives for food in traditional and alternative medicines. These can be cyclic, open-chained, saturated or unsaturated. Nerol, geraniol, citronellol and linalool are the most active bioflavourants that ensure proper taste and aroma of wines, especially Muscat and Cabernet Sauvignon. Terpenes are commonly produced by fungi belonging to genus Ceratocystis. Menthol, a monoterpene is a major constituent of peppermint or other mint oils and is one of the chief flavourants used extensively as a food additive, in pharmaceuticals, cosmetics, toothpastes, chewing gums and candies. Out of the seven possible isomers of menthol, (-)-(1R,3R,4S) isomer exhibits mint taste and can be obtained by crystallization of Mentha arvensis oil upon freezing or can also be produced using enantio-selective enzymes. The reaction is highlighted in Fig. 5.

Microbial lipase

Fig. 5. Stereoselective formation of L-menthol.

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Linear monoterpenes can also be synthesized from the mevalonate pathway. Pseudomonas putida displays high solvent tolerance, and has been utilized as a host for geranic acid production from glycerol that has a sweet, woody or leafy flavour with hints of citrus. Incorporation of mevalonate pathway as well as geraniol synthase in P. putida in a fedbatch system could support 193 mg/L geranic acid from 4.6 g/L glycerol over 2 days. Myrcene is a monoterpene often used as a scaffold for complex compounds such as geraniol and menthol. Heterologous expression of the mevalonate pathway along with a truncated specific geranyl diphosphate synthase from Abies grandis and a myrcene synthase from Quercus ilex L. has been reported for myrcene production in E. coli DH1. Production of 58 mg/L myrcene in 72 hours by optimizing gene expression levels, carbon source, and media along with a dodecane bilayer system has been documented. Limonene, another important terpene known for its unique citrus scent, is incorporated into many cleaning products, cosmetics, and perfumes. Expression of the eukaryotic mevalonate isoprenoid pathway in E. coli allows for conversion of acetyl CoA to the activated isoprene units, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which can be combined and cyclized to form limonene and its derivatives. Production of 435 mg/L limonene from 1% glucose take place. Addition of a P450 enzyme system has enabled production of perillyl alcohol from limonene at 105 mg/L from 1% glucose while limonene production (2.7 g/L) using glycerol as a carbon source has also been carried out.

2.5 Ketones Ketones are the molecules with carbonyl group (=C=O) and are responsible for many natural flavours and odors. Though a large number of saturated and unsaturated aliphatic, aromatic and cyclic ketones have been isolated from cheese, odd numbered (C 5-C11) 2-alkanones, along with free fatty acids and secondary alcohols give Penicillium-ripened cheese their distinctive flavour. A strong blue cheese flavour to dairy products is provided by the compound non-8-en-2-one while Oct-1-en3-one is responsible for metallic flavour in oxidized butter. Pineapple ketone (2,5-dimethyl-4-hydroxy-3(2H)-furanone) is found in pineapples, strawberries, raspberries and other food. It is found in cooked, roasted and fermented food such as coffee, roasted almond and soy sauce. It is formed by non-enzymatic browning during roasting and cooking operations. It has cotton-candy, caramelized-sugar flavour and is extensively used in food industry, particularly chewing gums. Methyl ketones are the primary components of cooked flavour associated with baked food, containing butter. The precursors to methyl


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ketones exist in fresh butter as alkanoic acids, which do not have flavouring characteristics. However upon heating, they are converted to methyl ketones which are the chief flavourants in heated and cooked food containing butter. Amongst this class of flavouring compounds, 2heptanone, 2-nonanone and 2-decanone contribute to flavour in blue cheese and fruits. These contribute to stale flavour of UHT milk. Coconut oil, rich in medium chain fatty acids, is a commercial raw material for the production of methyl ketones. These are produced by Aspergillus bisporus, A. niger, Trichoderma viride and Penicillium roquefortii. Methyl ketones can be readily produced in microbes by fatty acid -oxidation pathway. Their yield has been improved by overexpressing a few genes in E. coli. Acetoin production from glucose has been reported by installing acetolactate synthase and acetolactate decarboxylase genes from E. coli and Lactococcus lactis, respectively in E. coli. A strain of Candida glabrata optimized for pyruvate production, has been engineered for acetoin production by installing pyruvate decarboxylase from Saccharomyces cerevisiae. Diacetyl (2,3-butanedione) is a diketone flavourant, providing a rich buttery note and is used as an imitant of butter in dairy industry. It is a volatile yellow liquid ketone with a cheese-like smell with a characteristic flavour of buttermilk and sour cream. It imparts a yellow colour to dairy products and a buttery flavour to popcorns, chips, candies and pasteries. Chardonnay wines contain a high concentration of diacetyl from 0.005-1.7 mg/L. These are produced by bacteria such as Lactococcus lactis, Lactobacillus sp., Streptococcus thermophilus and Leuconostoc mesenteroides. Enzyme acetoin dehydrogenase present in lactic acid bacteria catalyzes dehydrogenation reactions in dough products forming diacetyl. Acetoin and diacetyl pathways utilized in E. coli have been transformed into cyanobacterium Synechococcus elongatus sp. PCC 7942, thereby, coupling CO2 fixation with acetoin (203 mg/L) and diacetyl production (2.4 g/L).

2.6 Aldehydes and Alcohols Aldehydes are chemical compounds containing –CHO group and have a low flavour threshold and produce desirable creamy, buttery flavours at low concentration while at high concentrations, they lead to oxidized off-flavours. Vanillin, benzaldehyde, acetaldehyde, anisaldehyde, phenylacetaldehyde and heliotropic are some of the examples. Odor of citrus is due to aliphatic aldehydes like decanal and oxygenated terpenes such as terpineol and citral. Cis and trans deca-2,4dienals impart a deep fat flavour to a variety of food as cottonseed oil, soybean oil, milk fat, beef fat, cooked chicken and rye crisp bread. Benzaldehyde, major component of cherry and almond flavour, has been

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identified as secondary metabolite of microorganisms. Alcohol oxidase from Pichia pastoris oxidizes benzyl alcohol to benzaldehyde. Streptococcus thermophilus and Lactobacillus bulgaricus possess enzyme threonine aldolase that catalyses the conversion of threonine to acetaldehyde and glycine. The most relevant compounds to obtain the “green” organoleptic characteristic are C6 aldehydes and their corresponding alcohols. Hexen1-ol, also called as leaf alcohol, has a strong odor of freshly cut grass and is an important flavourant for natural green notes. Bioproduction of green notes from linoleic acid and linolenic acid by enzymes lipooxygenases, hydroperoxide lyase and alcohol dehydrogenase is outlined in Fig. 7, in which the lyase reaction is rate-limiting. A novel strategy has been designed in which S. cerevisiae has been genetically modified to co-express the hydroperoxide lyase gene from banana and the lipoxygenase gene. Introduction of soybean Lox2 and watermelon Hpl into Saccharomyces cerevisiae has been reported for biotransformation of linolenic acid in resting cell catalysts, yielding mainly 3(Z)-hexenal (60 mg/L, 58% of theoretical yield) and a small amount of 2(E)-hexenal.

Fig. 6. Bioproduction of acetaldehyde by threonine aldolase from S. thermophilus and L. bulgaricus.

Fig. 7. Biocatalytic routes for the formation of green notes from linoleic and linolenic acid.


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Vanillin (4-hydroxy-3-methoxy benzaldehyde), obtained from a climbing orchid, Vanilla fragrans is the most widely used aromatic flavour compound in food, beverages, perfumes and pharmaceuticals. It develops in pods by the breakbown of glucosides in the curing process and contributes to about 2% (w/w) of the dry weight. Natural vanillin is highly priced due to limited availability of vanilla pods and labour intensive cultivation, pollination, harvesting and curing of vanilla pods. It is also a metabolic intermediate in the biodegradation of natural products such as phenolic stilbenes, eugenol, ferulic acid and lignin that provides an effective alternative for its commercial production. It can also be produced by fed batch fermentation employing Amycolatopsis sp. HR167 and 12g of vanillin has been produced per litre of the medium. Benzaldehyde, second most important flavouring compound next to vanillin, is the simplest aromatic aldehyde with a pleasant almondlike odor. It is the primary component of bitter almond oil extract and is present in cherry and other natural fruit flavours. It can be extracted from apricot, laurel leaves and peach seeds. Alternatively, it can be produced by biotransformation of phenylalanine using bacterium Pseudomonas putida and white rot fungi Trametes suaveolens, Polyporus tuberaster and Phanerochaete chrysosporium. It can also be synthesized from glycoside, amygdalin present in fruit kernels and nuts. 2-Phenyl ethanol is an aromatic alcohol with a rose-like odor and is present in many essential oils. It is used as a flavourant in soft drinks, candies and cookies. Rose petals contain about 60% of this alcohol, but the natural extraction from rose petals is economically not feasible. In 2002, it was marketed at around US $ 1,000/kg. Although, certain species of Corynebacterium have been found to produce 2-phenyl ethanol, it is being commercially produced using yeast strains of Hansenula anomala, Kluyveromyces marxianus and Saccharomyces cerevisiae. Yeasts are known to produce this alcohol by bioconversion of L-phenyl alanine through Ehrlich pathway (Fig. 8). Upon identification and deletion of uncharacterized aldehyde reductase encoding genes in E. coli, a variety of aldehydes have been reported from 2-keto acids. Vanillin (119 mg/L) has been produced from glucose (1.2%, w/v) in alcohol reductase knock out mutants of E. coli by assembly of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase from E. coli, 3-dehydroshikimate dehydrogenase from Bacillus thuringiensis, O-methyltransferase from Homo sapiens and carboxylic acid reductase from Nocardia iowensis. The conversion of benzoate to benzaldehyde has also been achieved in E. coli using a recombinant carboxylic acid reductase. Aldehyde reductase knockout strains fed with 606 mg/L benzoate were

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able to produce 350 mg/L benzaldehyde. Pichia pastoris has been utilized as a biocatalyst for the oxidative biosynthesis of benzaldehyde from benzyl alcohol. The presence of methanol induces expression of the native alcohol oxidases, which are responsible for oxidation of alcohol to aldehyde, as well as catalase, which aids in the dissimilation pathway for mineralized methanol. Benzaldehyde is known to exhibit a strong inhibitory effect on the oxidation of benzyl alcohol; sequestering benzaldehyde by an adsorptive polymer that alleviates this effect, and prevents product loss due to volatilization. In a 3 L culture, two-phase system has been reported to convert 70.7 g of fed benzyl alcohol to 14.4 g of benzaldehyde, with a productivity of 97 mg/L/h.

Fig. 8. Ehrlich pathway in yeasts for the formation of higher alcohols.

2.7 Fatty Acids Fatty acids are carboxylic acids with long unbranched saturated or unsaturated aliphatic tail having sour taste and intense smell. These are inexpensive natural flavours produced by oxidation of corresponding alcohols. Upon heating of animal and vegetable oils in the presence of air, alkenoic acids are formed which impart flavour to the food. Of the various fatty acids, C 1-C 10 are important food flavourants. It may be added that as the molecular weight increases, flavour becomes more rancid, buttery and cheesy, and then, becomes fatty. Common acids


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include acetic, propionic, butyric, isobutyric, valeric, isovaleric and lactic acids. 2-Methyl butyric acid is an important aroma constituent of cranberry while methylpentenoic acid imparts a sweet, green, sharp, strawberry character to the food. Propionic acid is an important flavour component of Swiss blue cheese and Propionibacterium is known to synthesize it de novo. A number of biotechnological processes have been developed for the production of fatty acids, such as hex-2-enoic acid and hex-3-enoic acid which are important constituents of raspberry flavour. Pseudomonas shermanii when co-cultured with Lactobacillus casei in whey could produce 30 g/L propionic acid, 10 g/L acetic acid, 8 g/L lactic acid after 90 h of fermentation. Clostridium sp. and Peptostreptococcus anaerobius convert leucin to isovaleric and isocaproic acids.

3. BIOPRODUCTION OF MICROBIAL FLAVOURS The natural flavours can either be directly extracted from plant material or can be produced biotechnologically. The extraction of natural flavours directly from plants however, is less promising due to low recovery of the desired compound, dependence on weather conditions and limitations imposed by spread of disease that makes the whole process expensive. Biotechnological approaches outlined in Fig. 9 include production of flavour compounds by plant cell cultures, microbiological methods and enzymatic techniques. Microbial production of flavour compounds using the microorganisms either for de novo synthesis of flavours i.e., production of flavour compounds by metabolizing cells or by biocatalysis i.e., the use of microbial cells to carry out specific chemical reactions have been described here (Fig. 9).

Fig. 9. Biotechnological production of flavour compounds.

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3.1 De novo Synthesis of Flavour Compounds by Microorganisms Microorganisms produce a wide range of secondary metabolites of which flavour compounds constitute an important class. The typical flavour characteristics of most of the fermented food and beverages are attributed to the presence of a large number of microorganisms as in Belgian gueze beer, where flavour characteristics depend upon mixed cultures. Pure microbial cultures can also generate specific and complex aroma profile as in beers, where Saccharomyces cerevisiae produces the final complex aroma. Various microorganisms are capable of producing specific flavours de novo, when grown in a specific medium.

3.1.1 Fungi Several fungal species known to produce complex flavours are enlisted in Table 4. Amongst the filamentous fungi, white-rot basidiomycetes, which live on dead or living timber, have ability to completely degrade lignin, a polymer of substituted p-hydroxy-cinnamyl alcohols, and to metabolize the resulting phenolic monomers (such as pconiferylic and p-sinapylic alcohols) into compounds of aromatic interest. Compared to other microorganisms, however, the ‘volatile spectrum’ of basidiomycetes is closest to those of plants and contains many different compounds such as highly potent aliphatic compounds such as 1-octen-3ol; aromatics including vanillin, benzaldehyde, phenylacetaldehyde, 1phenylethanone and methyl benzoate; and terpenoids such as citronellol and linalool (Table 4). Several species of Penicillium, Mucor and Oidium produce fruity odors. Ceratocystis moniliformis produces ethyl, propyl, isobutyl, isoamyl acetates, citronellol and gerianol. The strain also produces ô--decalactones having fruity banana, peach and citrus aroma. Genus Phellinus produces ethyl and methyl benzoates and methyl salicylate. Mucorales (M. racemosus, M. piriformis) impart a bitter almond flavour to butter. Ceratocystis species produce a variety of terpenes with floral/fruity odors. Benzyl alcohol and 2-phenethyl alcohol metabolites are produced in the fermentation medium of Trichothecium roseum, Phellinus ignarius, P. laevigatus and P. tremulae. Mushroom flavours have been found in cultures of Aspergillus and Penicillium species. Penicillium caseiocolum and P. camberti produce flavouring alcohols like 1-octen-3-ol, oct-1,5-dien-3-ol and octan-3-ol. Tyromyces sambuceus and Cladosporium suaveolens generate coconutflavoured -decalactone and -dodecalactone from ricinoleic acid and linoleic acid, respectively. Methyl ketones can be produced by Agaricus bisporus, Aspergillus niger, Penicillium roquefortii and Trichoderma viride using coconut fat as a substrate. -decalactones have been produced from ethyl decanoate by Mucor circillenoides. Trichoderma viride and T. harzianum


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produce coconut-flavoured lactone, 6-pentyl--pyrone and Marasmius alliaceus produce sulphur-containing volatile compounds containing the sulphurous flavours. Nidula niveo-tomentosa produces raspberry ketone in a culture medium supplemented with phenylalanine. Table 4. Flavour compounds in fungal cultures.

Phanerochaete chrysosporium, Pycnoporous cinnabarinus

sp., sp. contd...

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sp., sp., sp.,


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3.1.2 Yeast Pellicle forming yeast have been found to play an important role in the generation of aroma in various products. Food flavourants produced by yeast strains are enlisted in Table 5. Sporobolomyces odorus has been reported to produce peach-smelling -lactones. -decalactones have been formed by Candida sp., Yarrowia lipolytica, Monilia fructicola, Sporobolomyces odorus and Rhodotorula glutinis using castor oil as substrate (Table 5). Kluyveromyces lactis produces citranellol, linalool and geraniol. Geotrichum klebahnii produces pleasant fruity flavoured ethyl esters of branched carboxylic acids. Geotrichum fragrans forms ethyl isovalerates in cultures supplemented with ethanol (Fig. 10). Hanseniaspora guillermondii and Pichia anomala are potent producers of acetate esters while K. marxianus also produces 2-phenylethanol, Saccharomyces cerevisiae synthesizes ethyl acetate and Candida utilis produces acetaldehyde when grown on ethanol. Table 5. Flavour compounds produced by yeast cells.

contd...

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Saccharomyces cerevisiae

Fig. 10. Formation of ethy lisovalerate from L-leucine.

3.1.3 Bacteria The major food flavourants produced by bacteria are summarized in Table 6. Acetobacter species produces methyl-butyric acid, which is a precursor to common flavour esters, acetic acid bacteria form esters of ethyl acetate while, Lactobacillus lactis-acidi and Streptococcus hollandicus impart aromatic properties to milk and milk products (Table 6). Another group of butyric acid bacteria, such as Clostridium pasteurianum when grown in presence of oxygen produce a complex ester aroma resembling the apple odor while Corynebacterium glutamicum produces pyrazines in cultures. High levels of propionic acids are produced by Propionibacterium sp., while butyric acid is primarily produced by obligate anaerobes of genus Clostridium, Butyrivibrio, Eubacterium and Fusarium. Clostridium and Peptostreptococcus anaerobius when co-cultured produce high levels of isovaleric acid. High levels of butanol and isopropanol have been reported from batch fermentation of Clostridium beijerinckii, while mutants of Zymomonas mobilis produce acetaldehyde. Bacillus subtilis produces pyrazine when grown on soybean substrate, Streptococcus species produce various diketone flavours. Interstingly, Pseudomonas sp. HR 199 has been engineered for bioconversion of eugenol to vanillin. Cell immobilization has been employed successfully such as benzaldehyde can be produced from benzoyl formate by whole cells of Pseudomonas putida encapsulated in calcium alginate. Lactobacillus acidophilus and Pediococcus pentosaceus produce dairy flavours when grown on maizebased semi-solid medium.


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Table 6. Flavour compounds detected in some bacterial cultures.

sp.,

contd...

790


3.2 Flavour Production by Biotransformation Another approach for the production of food flavourants is by biotransformation. The specific substrates when added to the fermentation medium can be transformed into flavour compounds by microbial cells. Lactones, vanillin and various esters have been produced successfully by the bioconversion approach. Few important flavours produced by this technique are described here.


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3.2.1 Vanillin Various biotransformation approaches to vanillin synthesis have been reviewed earlier and different biotransformation routes to its synthesis are depicted in Fig. 11. It can also be synthesized from phenolic stilbenes like isorhapontin, commonly found in spruce bark and isoeugenol by enzyme dioxygenase present in Pseudomonas species. Another approach for vanillin synthesis is deamination of m-methoxytyrosine using Proteus vulgaris and then-, aldehyde group formation by treatment with mild base. Clove oil contains eugenol, which can also be converted to ferulic acid and then, to vanillin by Corynebacterium, Pseudomonas and Arthrobacter strains. Besides, biotransformation of eugenol to vanillin can also be achieved by Fusarium solani. However, a much cost effective method for vanillin synthesis employs maize and wheat-based agricultural wastes (beet pulp and cereal bran), which can be converted to ferulic acid-, using polysaccharide-degrading enzymes and feruloyl esterases, and further ferulic acid can be converted to vanillin by the action of Pycnosporus cinnabarius or via a two step fungal process in which A. niger transforms ferulic acid into vanillic acid, which is further converted to vanillin by basidiomycete, Pycnoporus cinnabarinus or Phanerochaete chrysosorium. Biotransformation of caffeic acid and veratraldehyde to vanillin has also been carried out successfully.

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Pycnosporus cinnabarius Streptomyces setonii

Corynebacterium& Pseudomonas

Eugenol

Ferulic acid Aspergillus niger Pseudomonas sp.

Pycnosporus cinnabarius, Phanerochaete chrysosporium

Vanillic acid (c)

©

Arthrobotrys globiformis

Eugenol

Ferulic acid

Coniferyl aldehyde

Feruloyl CoA

Vanilloyl CoA


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Vanillylamine

Capsaicin

Amine oxidase

(e)

Fig. 11. Production of vanillin by biotransformation.

3.2.2 Higher terpenes and terpenoids Carotenoids, when acted upon by free radicals, undergo oxidation reactions and form volatile compounds important for the flavour industry. Ionones present in blackberry, peach and apricot are one of them. Fungi such as Gyromitra esculenta, Clitocybe geotrapa and Fibroporia vaillantes possess enzymes such as polyphenol oxidases and lipoxygenases which carry out transformation of -carotenoids into ionones by cleavage at C13. In the culture supernatant of Ischnoderma benzoinum, Marasmus scorodonius and Trametes versicolor, -ionone is produced as the main metabolite.

3.2.3 Flavour lactones Ricinoleic acid present in the castor oil has been transformed into decalactone (Fig. 3) by partial -oxidation catalyzed by yeast strains of Sporidiobolus salmonicolor, Monilia fructicola, Rhodotorula glutinis, S. ruineii, S. johnsonii, S. odorus, S. pararoseus and Yarrowia lipolytica. Similarly, decalactone can also be synthesized by bioconversion of unsaturated fatty acids such as 11-hydroxypalmitic acid, corriollic acid and 2-decen5-olide using various yeast and fungal strains such as Saccharomyces cerevisiae and Cladosporium suaveolens. Dodecalactone with whisky flavour is formed by lactic acid bacteria and yeast from unsaturated fatty acids. Another flavouring compound, -decalactone in cultures of C. guillermondii growing in a medium rich in castor oil has been successfully demonstrated. Other known to be involved in formation of lactones involve P. chrysosporium, P. etchellsii, A. niger, Mucor species and Candida petrohillium and the various pathways to -decalactone synthesis are given in Fig. 12.

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Fig. 12. Various routes for production of 4-decanolide.

3.2.4 Methyl ketones 2-Heptanone, 2-nonanone and 2-undecanone can be produced by biotransformation of coconut oil, which is rich in medium chain fatty acids. Biotransformation of methyl ketones from fatty acids by Penicillium roquefortii is shown in Fig. 13. Similarly, octanoic acid can be converted to 2-heptanone by Penicillium roquefortii, Aspergillus niger and Trichoderma koningii (Fig. 14). Aureobasidium pullulans is known to produce 2-pentanone, 2-heptanone and 2-nonanone from octanoic and decanoic acids.

3.2.5 Other flavour compounds Gluconobacter sub-oxydans transform glucose/gluconic acid into 5ketogluconic acid, which is a good precursor for the chemical synthesis of savory flavour, monomethyl furanone while benzaldehyde, an important flavour after vanillin, is produced biocatalytically by microbial degradation of L-phenylalanine by a basidiomycete, Ischnoderma benzoinum (Fig. 15). Zygosaccharomyces rouxii has been shown to form 2,5-dimethyl-


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4-hydroxy-3-dihydroxyfuranone (DMHF) when supplemented with fructose-1,6-bisphosphate and glucose. Amino acid phenylalanine is transformed into raspberry ketone by cultures of fungus Nidula-niveo tomentosa. In the same way, raspberry ketone can be produced from 4hydroxy benzalacetone in cultures of Saccharomyces cerevisiae.

Fig. 13. Synthesis of methyl ketone by Penicillium roquefortii.

Octanoic acid

3-Ketooctanoic acid

2-Heptanone

Fig. 14. Biotransformation of fatty acids to methyl ketones.

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Fig. 15. Formation of benzaldehyde by Ischnoderma benzoinum.

3.3 Use of Enzymes for Production of Flavour Compounds Enzymes isolated from microbes or other natural sources have been widely used in food industry for cheese manufacturing, baking and brewing. Lipases, proteases, esterases and nucleases aid in flavour extraction. Enzymes used for production of important flavours are enlisted in Table 7. Enzyme catalysis offers several advantages such as flavour development in a single step, easy recovery and high process efficiency. Proteases, peptidases, lipases and esterases have been reviewed to produce enzyme modified flavour enhanced cheese. However, a few limitations of enzyme catalyzed reactions include substrate specificity, regioselectivity, enantioselectivity, use under mild reaction conditions and high cost of the biocatalyst. Some of the enzymes that play major roles in flavour production are discussed in this section.

Microbial Production of Natural Flavours Table 7. Some important enzymes in flavor industry.

lyase

alcohol

797

798


3.3.1 Lipases Use of lipases for production of chiral isomers in enantiomeric mixtures is a convenient method which requires only the identification of suitable alcohol or ester substrates for the transesterification reaction. Lipase from Candida rugosa has been found to catalyse L-methanol esterification with long chain unsaturated fatty acids to moderate its strong flavour. Immobilized lipase of Mucor meihei and Candida cylindracea have been employed to catalyze transesterification reaction in organic solvents for synthesis of geraniol. Bacterial lipases catalyze the enantioselective conversion of L-menthyl esters in the racemic mixture to L-menthol, a major mint flavourant (Fig. 5). Lipases from Rhizomucor meihei, Aspergillus niger and Aspergillus javanicus have catalysed the enantioselective synthesis of (S)-2-methylbutanoic acid methyl ester, a chief flavour component of apple and strawberry in presence of iso-octane. Staphylococcus epidermidis lipase could successfully catalyze the synthesis of flavour esters in aqueous media. Ethyl valerate (green apple flavour) and hexyl acetate (pear flavour) have also been produced by crude lipases immobilized on different supports.

3.3.2 Proteases Free amino acids and relatively short oligopeptides play a significant role in flavour industry. Different sequences of amino acids can be used to recreate the basic taste sensations of sweet, bitter, sour and salty. Proteases catalyze the hydrolysis of vegetable proteins producing a savory flavour, mainly due to formation of pyrazines and alcohols, earlier formed by heating the protein source at acidic pH. Serine and cysteine endopeptidases have been found to be particularly important for synthesis of peptides used as food additives. Proteases act on cray fish processing by-products and form benzaldehyde and pyrazines. Tetrapeptide AspAsp-Asp-Asp isolated from beer yeast seasoning, is known to mask the bitter taste of beer and can be synthesized by condensation of L-aspartic acid -dipropyl ester hydrochloride, the reaction being catalyzed by -chymotrypsin.

3.3.3 Glucosidases -Glucosidases from Vitis, Saccharomyces, Oenococcus, Aspergillus and Candida have been found to enhance wine aroma by hydrolysis of glycosidic-bound precursors like terpenols resulting in release of terpenes and other flavours. Commercial -glucosidase from Aspergillus niger converts betuloside (found in birch, maple and rhododendron) to betuligenol, which can then be transformed to raspberry ketone (Fig. 2)


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by Candida boidinii alcohol dehydrogenase. -glucosidases and alcohol oxidases of Candida methanolevescens have been found to transform glycoside salicin to almond flavoured salicyl aldehyde. The biotransformation reaction is illustrated in Fig. 16. β-glucosidase

Alcohol oxidase

Salicyl alcohol

Salicin

Salicylaldehyde

Fig. 16. Biotransformation of salicin to salicyl aldehyde.

3.3.4 Other enzymes Benzaldehyde can be produced from cyanogenic glycoside, amygdalin present in cherry stones, by the successive action of enzymes -glucosidase and mendelonitrile lyase (Fig. 17) present in crude almond meal. Rhamnosidases present in commercial Aspergillus pectinase preparations act on naringin and hesperidin, forming 6-deoxysugar rhamnose, which is a precursor to 2,5-dimethyl-4-hydroxy-2,3dihydrofuran-3-one (furaneol®) that exhibits strawberry flavour in dilute solutions and caramel-like flavour in concentrated form (Fig. 18). Alcoholic dehydrogenases from Lactobacillus kefir immobilized on polyvinyl alcoholic gel beads synthesize (R)-phenylethanol from acetophenone in presence of hexane. (+)-Germacrene A hydroxylase produces nootkatone from sequiterpenes. Vanillylamine from capsaicin (natural ingredient of pepper and capsicum) can be converted to vanillin by enzyme amine oxidase from Aspergillus niger. L-Glutamic acid, chief flavour ingredient of fermented soy sauce can be produced by action of microbial glutaminases. Agricultural wastes like beet pulp and cereal bran when treated with polysaccharide-degrading enzymes and esterases form ferulic acid that can be readily transformed to vanillin.

800


Fig. 17. Enzymatic synthesis of benzaldehyde.


Fig. 18. Conversion of citrus waste to furaneol.

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4. MICROBIAL PRODUCTION OF AROMA COMPOUNDS THROUGH SOLID STATE FERMENTATION Solid state fermentation (SSF) uses the growth of microbes on a nonsoluble material that acts both as physical support and source of nutrients in the absence of free flowing liquid. Solid state fermentation has several advantages over submerged fermentation in offering higher yields, better product characteristics, much lower costs, ease of operation and simple equipment. Coffee-derived residues have offered potential opportunities in flavour industry. Various studies have focused on production of aroma compounds by solid state fermentation using microorganisms such as Neurospora sp., Zygosaccharomyces rouxii and Aspergillus sp., on a variety of solid substrates like pre-gelatinized rice, miso and cellulose fibres, respectively. First flavour compound to be commercially produced by solid state fermentation was methyl ketones with a conversion yield of about 40%. Ceratocystis fimbriata has been reported to produce a strong pineapple aroma on coffee husk. Major flavour compounds produced on agro-waste residues have been identified as acetaldehyde, ethanol, ethyl acetate, ethyl isobutyrate and isoamyl acetate. The fruity aroma production on sugarcane bagasse using C. fimbriata cultures has been attempted. A strong banana aroma has been reported on agro-industrial residues supplemented with leucine or valine. Rhizopus oryzae has also been reported to substantiate volatile compounds on cassava bagasse, soybean meal and amaranth grain. The production of 6-pentyl--pyrone (6-PP), an unsaturated lactone with a strong coconut-like aroma could be produced using both liquid and solid substrates in SSF. Sugarcane bagasse has been used successfully as a substrate for growth and aroma production. Semisolid maize-based culture can be used to produce dairy flavour compounds like diacetyl, butyric acid and lactic acid by using mixed cultures of Lactobacillus acidophilus and Pediococcus pentosaceus. Similarly, Kluyveromyces marxianus could produce alcohols, aldehydes and esters on cassava bagasse and giant palm grain as substrate.

5. CONCLUSIONS Due to consumer preference for natural products and better economic benefits, biotechnological production of flavours has gained more importance. Biotechnological advancements have opened up new ways for the production of natural flavours. Low cost natural precursors can be transformed to expensive flavour compounds by microorganisms and their enzymes, and microbial production processes offer advantage over the traditional methodologies. However, a need for better understanding


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of metabolic mechanism arises to improve the processes significantly and to decrease the cost of production. Furthermore, identification and characterization of novel strains producing flavour compounds need to be optimized. Identification of alternative genes better suited for genetic engineering of existing strains could be pioneered. Enzymes that could be exploited in future are cyclases, which are involved in terpene synthesis in plants. Genetic engineering methods are being looked upon for development of strains with ability to produce flavours or incorporating genes in microorganisms to bring down the multistep reactions to a single step or development of significantly improved biocatalysts. Leuconostoc strain has been developed by protein engineering techniques, which is capable of lactose fermentation as well as diacetyl production. In beer production, yeast supplemented with gene encoding -acetolactate decarboxylase would result in removal of diacetyl and thus, the buttery off-flavour and hence, eliminating the need for lagering. Supplementing wine yeast with malolactic enzyme has resulted in improved composition of volatiles. A multifunctional O-methyl transferase catalyzing the formation of a compound with strawberry like flavour, 2,5-dimethyl-4methoxy-3(2H)furanone (DMMF) has been cloned and expressed in E. coli. Expression of flavour generating plant enzymes in microbes offers an opportunity for development of high level expression system. A pathway in the total synthesis of terpenoids involving 8 genes from S. cerevisiae has been engineered in E. coli. Further progress would essentially depend on better understanding of microbial metabolism. Genetic engineering techniques have helped a long way to elucidate biochemical basis of flavour production. However, to characterize the flavour molecule metabolism, identification of more genes and metabolic pathways is required. Many metabolic pathways have been engineered towards the accumulation of specific flavour compounds. Bacterial metabolisms can provide various biocatalytic tools for producing the value-added compounds of natural flavours and fragrances, and chemical synthasysis from inexpensive plant-derived biomass. However, many problems, such as toxicity of the parent compounds and products, efficient expression of the targeted genes, and physiological stability of the recombinant bacterial strains, should be overcome in order to produce expensive “natural” flavours and fragrances by engineered microorganisms. Further, fed batch fermentation, immobilization and in situ flavour extraction technique can circumvent the conditions where flavour compounds and their precursors are inhibitory or toxic to the producer strains. Accumulation of undesirable by-products and cost of raw

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materials pose economic constraints to the bioflavour production. For this, the undesirable intermediates could be metabolized to desirable products by other microbes eg., when ricinoleic acid is converted to decalactone, 3-hydroxy--decalactone is formed as a by-product which can be converted to 3,4-unsaturated -decalactone and then, reduced to -decalactone by S. cerevisiae. Biotechnological methods need to overcome several drawbacks like low water solubility and toxicity of precursors and products, and unwanted by-product accumulation. The industrial application of novel biocatalysis depends on scientific and technological feasibility, and also the public perception on the use of genetic engineering techniques for improved food quality and more environmental friendly production processes. A better understanding of the mode of action of enzymes as well as the mechanism of development of flavour compounds will further enhance the use of biotechnological methods to develop specific and desired flavours in food.

FOR FURTHER READING Aaslyng, M.D., Elmore, J.S. and Mottram, D.S., 1998. Comparison of the aroma characteristics of acid-hydrolyzed and enzyme-hydrolyzed vegetable protein produced from soy. J Agric Food Chem, 46: 5225-5231. Achterholt, S., Priefert, H. and Steinbüchel, A., 2000. Identification of Amycolatopsis sp. strain HR167 genes, involved in the bioconversion of ferulic acid to vanillin. Appl Microbiol Biotechnol, 54: 799-807. Aguedo, M., Ly, M.H., Belo, I., Teixeira, J.A., Belin, J.M. and Waché, Y., 2004. The use of enzymes and microorganisms for the production of aroma compounds from lipids. Food Technol Biotechnol, 42: 327-336. Allegrone, G., Barbeni, M., Cardillo, R., Fuganti, C., Grasselli, P., Miele, A. and Pisciotta, A., 1991. On the steric course of the microbial generation of (Z6)gamma-dodecenolactone from (10R,S) 10-hydroxyoctadeca-(E8,Z12)dienoic acid. Biotechnol Lett, 13: 765-768. Alonso-Gutierrez, J., Chan, R., Batth, T.S., Adams, P.D., Keasling, J.D., Petzold, C.J. and Lee, T.S., 2013. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab Eng, 19: 33-41. Amoore, J., 1971. Stereochemical and vibrational theories of odour. Nature, 233: 270-271. Aryan, A.P., Wilson, B., Strauss, C.R. and Williams, P.J., 1987. The properties of glycosidases of Vitis vinifera and a comparison of their -glucosidase activity with that of exogenous enzymes. An assessment of possible applications in enology. Am J Enol Vitic, 38: 182-188.


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Aungpraphapornchai, P., Griffin, H.G. and Gasson, M.J., 1999. Cloning, DNA sequence analysis, and deletion of a gene encoding diacetyl-acetoin reductase from Lactococcus lactis. DNA Sequence, 10: 163-172. Ayseli, M.T. and Ayseli, Y.I., 2015. Flavours of the future: Health benefits of flavour precursors and volatile compounds in plant foods. trends Food Sci Technol, 48: 69-77. Badings, H.T., Van der Pol, J.J.G. and Neeter, R., 1981. Aroma compounds which contribute to the difference in flavour between pasteurized milk and UHT milk. In: Flavour, Vol 81, Schreier P (ed), Walter de Gruyter, New York, USA, pp. 683-692. Baek, H.H. and Cadwallader, K.R., 1996. Volatile compounds in flavour concentrates produced from crayfish-processing by-products with and without protease treatment. J Agric Food Chem, 44: 3262-3267. Baqueiro-Pena, I. and Guerrero-Beltran, J.A., 2016. Vanilla (Vanilla plantifolia Andr.), its residues and other industrial by-products for recovering high value flavour molecules: A review. J Appl Res Med Aromatic Plants. doi: http://dx.doi.org/10.1016/j.jarmap.2016.10.003. Bauer, K., Garbe, D. and Surburg, H., 2001. Common Fragrance and Flavour Materials, 4th edn, Wiley-VCH, Weinheim, Germany. Belitz, H.D. and Grosch, W., 1999. Food Chemistry, 2nd Engl edn, Springer, Berlin, Germany. Berger, R.G., 1995. Application of genetic methods to the generation of volatile flavours. In: Food Biotechnology, Hui HY, Katchatourians G (eds), VCH, Weinheim, Germany, pp. 281-295. Besson, I., Ceruly, C., Gros, J.B. and Larroche, C., 1997. Pyrazine production by Bacillis subtilis in solid state fermentation on soybeans. Appl Microbiol Biotechnol, 47: 489-495. Bodie, E.A., Goodman, N. and Schwartz, R.D., 1987. Production of propionic acid by mixed cultures of Propionibacterium shermanii and Lactobacillus casei in autoclave-sterilized whey. J Ind Microbiol Biotechnol, 6: 349-353. Bonnin, E., Brunel, M., Gouy, Y., Lesage-Meesen, M., Asther, M. and Thiobault, J.F., 2001. Aspergillus niger I-1472 and Pycnoporus cinnabarius MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases. Enzyme Microb Technol, 28: 70-80. Boog, L.G.M., Peters, A.L.J. and Roos, R., 1991. Process for producing deltalactones. EP Patent CA2021270 BO.36025. Bramorski, A., Christen, P., Ramirez, M., Soccol, C.R. and Revah, S., 1998 Production of volatile compounds by the edible fungus Rhizopus oryzae

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Microbial Production of Natural Flavours

Microbial Production of Natural Flavours

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