FLAVOURS PRODUcTION IN FERMENTED FOODS

Introduction Flavour is defined as the sum of

characteristics of any material taken in the mouth, perceived principally by the sense of taste and smell and also the general, pain and tactile receptors in the mouth, as received and interpreted by the brain (European Council).

The word fermentation (from the Latin word fermentare meaning to cause to rise) describes enzyme-catalyzed reactions in which compounds such as carbohydrates are broken down anaerobically by bacterial or yeast enzymes, leading to the formation of pyruvic acid and other end products).

Campbell-Platt (1987) has defined fermented foods as those foods which have been subjected to the action of micro-organisms or enzymes so that desirable biochemical changes cause significant modification to the food..

Types of Fermented Foods

Fermented foods

Alcoholic Beverages

Diary Product

s

Fruits and

Vegetables

Cereals and

Legumes

Meat and

Meat Product

s

Fish and Fish

Products

Miscellaneous

Types of Fermentation Processes 1. Liquid Fermentation

Most industrial fermentations are carried out in liquid media, also called submerged fermentation, in which microorganisms are dispersed into a nutrient liquid using a bioreactor. The reactor, a stirred or non-stirred tank, could run as batch or continuous.

Examples of flavour production in submerged fermentation:

The production of (R)-d-dodecanolide, a lactone applied as a butter flavor in margarine.

The bio-conversion of 5-ketododecanoic acid which takes place on a 30,000 L fermentor using baker’s yeasts (Janssens et al., 1992).

The production of peach aroma (4-decalactone) which involves the conversion of ricinoleic acid, oil obtained from the seeds of castor oil.

The production of vanillin, which is widely used as a flavoring agent in a wide range of foods and fragrances.

Contd………. 2. Solid-State Fermentation. Solid-state fermentation (SSF) can be defined as a method to cultivate microbial

cells in which organisms are grown on solid substrates or support in the absence of free water (Pandey, 2003).

Due to a low level of water activity, SSF processes have advantages in comparison with submerged liquid fermentation.

These include high volumetric productivity, low capital investment and energy requirements, the possibility of using alternative substrates (e.g. solid residues) without pre-treatments, less wastewater output, and easier product recovery.

However, there are some disadvantages, such as the difficulty in removing excessive heat generated, problems in fermentation control, sterilization, and contamination (Feron et al., 1996).

Flavors from Fermented Foods

Pathways for generation of microbial flavours

The organoleptic properties of fermented foods usually differ from those of the unfermented substrate and are dependent upon the biochemical

activities of the associated microorganisms.

Metabolic Pathways for formation of flavours in fermented foods

Common flavours produced during fermentation

The flavor compounds of traditionally

fermented foods originate from a

complex microflora that acts in the

chemical precursors of a food matrix.

Fermented Food Products and their flavours 1. Beer Many of the flavour chemicals characteristics of beer are produced by yeast enzymes acting

on amino acids present in the malt. For example, leucine can undergo transamination and decarboxylation to produce 3-methylbutanal.

The individual yeast strains used to brew some specialty beers produce different flavour compounds. For instance, concentrations of vinylguaiacol of over 3 mg/L are found in German top-fermented wheat beer. In the case of other beers it is generally an off-flavour originating from wild yeasts.

One important change that occurs in the maturation of lagers is a reduction in the concentration of diacetyl, which gives an undesirable buttery flavour note. Diacetyl is metabolized very slowly by yeast during the long lautering period, but now the enzyme diacetyl reductase can be added to accelerate this process and reduce maturation costs.

Beer ……..Contd

Hops are a good example of a multifunctional

ingredient. This is because, in addition to their

flavouring properties, hops act as a beer clarifier by

precipitating proteins, they have a preservative effect,

and the pectins present help to stabilize beer foam.

Wine The flavour chemicals responsible for the flavour and aroma of wines are

either derived from the original grapes, such as ethyl-3-thiol propionate, as metabolites of the yeast fermentation, or develop during the maturation of the wine.

This is mostly determined by the flavour molecules extracted from the grape, so that the amount of time that the grape skins remain during the fermentation is important, whereas less commonly the wine flavor is modified by the practice of leaving the wine in contact with the yeast lees for an extended period after fermentation has ceased.

Wine flavour is especially reliant on ethyl esters (ethyl acetate, propanoate, pentanoate, hexanoate, octanoate, decanoate) as well as the hexyl, 2-phenylethyl, 3-methylbutyl and ethyl acetates, plus a number of type-specific flavour chemicals.

Many of the flavour compounds present in wine occur as glycoside precursors in the grape juice and are released during the wine-making process.

Contd.. Muscat grapes have been shown to contain concentrations of free linalool, geraniol, nerol and terpineol of 100, 0.5, 0.5 and 0.5 g/kg fruit, respectively, so that only the linalool contributes to taste, as linalool has a flavor threshold of only 6 g/L.

Ethyl-3-thio-propanoate is also a characteristic flavour chemical of concord grapes, having a flavour threshold of 2 × 10-3 g/L. It has a flavour of fruit and grape at low concentrations.

Furthermore, 3-thiohexanol and its acetate occur both in Sauvignon wine and in passion fruit wine, explaining why some wine tasters describe Sauvignon wines, especially Sauvignon blanch, as having a passion fruit character.

Desirable improvements in quality are also common during the maturation of wines. For instance, during the maturation of sherry, the amounts of acetals, esters and sotolone increase relative to the concentrations of ethanol and volatile acids.

For instance, Jorgenson et al. (2000) analyzed elder flower wine by the GC-sniffing technique. They found that cis-rose oxide, nerol oxide, hotrienol and nonanal contributed to the special elderflower aroma, whereas linalool, terpineol, 4-methyl-3-penten-2-one and cis-ocimene contributed to floral notes of country wines.

Diary Products

In cheese and yoghurt, the rapid conversion of lactose, present in milk, into lactic acid is the most important feature of lactic acid bacteria. The resulting reduction of pH, removal of oxygen and the absence of lactose inhibits the growth of undesired bacteria.

The two main events during cheese maturation, largely following the fast fermentation of lactose, are proteolysis followed by amino acid degradation and conversion of fat. The activities of rennet enzymes and proteolytic enzymes from lactic acid bacteria (LAB) yield small peptides and free amino acids from casein. In yoghurt, no rennet is applied and the LAB (S. thermophilus and L. bulgaricus) here closely cooperate to liberate the amino acids necessary for growth from the proteins in their environment.

The most important flavour compounds originating from fat are free fatty acids produced by lipolysis. Lactic acid bacteria usually possess relatively weak lipolytic activities.

In cheese, free fatty acids are precursors of many important flavour compounds, such as methyl ketones, lactones, esters, aldehydes and secondary alcohols.

Contd….

Overview of general casein conversion pathways relevant for

flavour

Chocolate Some of the flavour compounds characteristic of chocolate and cocoa, especially savoury flavour

chemicals, are formed during the fermentation process that the beans undergo after harvesting.

During the fermentation, first sugars are metabolized by yeasts to produce ethanol, then lactic acid bacteria convert citric acid from the bean extracts into ethanol, and then acetic acid bacteria metabolize this ethanol into acetic acid.

Some other flavours are produced by the fermentation, such as ethyl-2-methylbutanoate, tetramethylpyrazine and some other pyrazines. The bitter taste notes are provided by theobromine and caffeine carried over from the original beans, together with diketopiperazines formed from the thermal decomposition of proteins during the roasting step.

Other amino acids released during the fermentation are the precursors for other flavor chemicals such as 3-methylbutanol, phenylacetaldehyde, 2-methyl-3-(methyldithio)furan, 2-ethyl-3,5-dimethyl- and 2,3-diethyl-5-methylpyrazine.

2-Acetyl-1-pyrroline is formed by Bacillus cereus acting in the later stages of the fermentation, and also during the drying of the pulp, and so at least some of the acetyl-1-pyrroline of cocoa appears to be formed microbially, and not just by thermally induced reaction during the later stages of cocoa processing (roasting etc.).

Tea Tea flavour and aroma is complex and depend greatly on the particular type of tea leaf used and the processing conditions. For instance, fully fermented black tea has a very different character from that of partially fermented oolong tea.

Initially, the leaves of Camellia sinensis are allowed to wither, allowing a loss of water, and then the leaves are macerated so that phenolics and other phytochemicals especially flavanols, such as epigallocatechin gallate, are allowed to react with endogenous enzymes.

Then, the macerated leaves are allowed to ‘ferment’ at an elevated temperature so that extensive enzyme reactions take place, followed by ‘firing’ at higher temperatures, which cuts short the enzyme activity but allows chemical reactions, often involving the products of the enzyme reactions, to proceed at a high rate.

Flavours such as diacetyl, methylpropanal and 2- and 3-methylbutanals are produced during tea fermentation. Many of these chemicals are produced by degradation of carotenoids and unsaturated fatty acids originally present in the tea leaf by the action of endogenous enzymes.

Flavors Produced from Bread during Sourdough Fermentation

The microflora involved in fermentation of baladi bread are the LAB L. brevis and L. fermenti, and yeast.

In sourdough fermentation, organic acetic acid and volatile flavoring compounds produced are dependent on the microorganisms in the dough. The volatile compounds are produced both in lactic acid fermentation and in alcoholic fermentation, but the levels of these compounds are much higher in yeast fermentation (Meignen et al., 2001).

However, Schieberle, 1996 concluded that volatiles formed do not affect the final flavor of the bread. The compounds having a high flavor dilution factor would have a significant impact on the final odor.

In a French yeast sourdough, more than 40 flavoring components have been identified: 20 alcohols, seven esters, six lactones, six aldehydes, three alkanes, and a single sulfur compound (Frasse et al., 1993).

Contd…… There are two kinds of aromatic compounds that are produced during

fermentation of sourdough. Non-volatile compounds include organic acids produced by homo- and heterofermentative bacteria that acidify, decrease the pH, and produce aroma in the bread dough.

Volatile compounds of sourdough bread are composed of the alcohols, aldehydes, ketones, esters, and sulfur-containing compounds produced by biological and biochemical actions during fermentation and contribute to flavor.

Major Flavouring Compounds in

Fermented Foods

1. Lactones Lactones are associated with fruity, coconut, buttery, sweet, or nutty flavors. Trichoderma viridae,

a soil fungus, generates a characteristic coconut flavor due to the production of 6-pentyl-2-pyrone.

The main component of peach flavor, 4-decalactone, can be synthesized by Sporobolomyces odorus. Aspergillus niger can transform b-ionone into a complex mixture resembling tobacco flavor. Lactones make a significant contribution to the flavor of several fermented foods like dairy products and alcoholic beverages.

Some microorganisms such as Ceratocystic moniliformis, Trichoderma viride, Sporobolomyces odorus, and some species of Candida have been reported as lactone producers. However, the production is not very significant and has low yields (mg/L), except for the in situ production of lactones from dairy products.

Among lactones, 6-pentyl-a-pyrone (6-PP) presents the most interesting flavor properties. The production of 6-PP by Trichoderma harzanium with sugar cane bagasse by solid-state fermentation was studied by Sarhy-Bagnon (1999) as an alternative for the production by submerged fermentation, giving a six fold raise in concentration.

Contd…….

Esters

Of the fermented foods, esters are probably the most important class of flavor compounds in alcoholic beverages. Maarse and Visscher (1989) listed 94 different esters as being identified in beer. Most of the esters found in beer are formed via primary fermentation.

They have been linked to the metabolism of lipids by yeast. Lipid metabolism provides a large number of acids and alcohols that may undergo esterification to yield a variety of esters.

While pure chemical reactions can lead to ester formation, this reaction is too slow to account for the esters in most foods.

Candida utilis was found to ferment glucose to ethyl acetate when grown on a medium limited in iron. Ethanol could be converted into either ethyl acetate or acetaldehyde, depending on the initial concentration of alcohol.

Acids The acid of greatest importance to the flavor of fermented dairy products is lactic

acid. Lactic acid is an optically active acid existing as the D, L or optically inactive mixture depending upon the microorganism involved in its synthesis.

The organisms most commonly associated with lactic acid formation are classified as being either homofermentative (e.g., Lactobacillus bulgaricus, which produces primarily lactic acid, > 85 percent of metabolic end products) or heterofermentative (e.g., Leuconostoc sp., which produces lactic acid, acetic acid, ethanol, carbon dioxide, and other metabolites).

The homofermentative organisms employ the Embden-Meyerhof pathway for lactose fermentation, while the heterofermentation organisms metabolize lactose via the hexose-monophosphate pathway.

Lactic acid is also formed in wines during fermentation from L-malic acid. Malic acid is a very tart acid present in grapes that needs to be converted to lactic acid to "smooth out" the harsh acidity.

Carbonyls Carbonyl flavor compounds make a particularly significant contribution to the flavor of fermented dairy

products. Diacetyl is one of the most important carbonyls to the flavor of these products.

It has a buttery, nut-like flavor. Diacetyl is produced via the fermentation of citrate in dairy products. The most important citric acid fermenters are Leuconostoc citrovorum, L. creamoris, L. dextranicum, Streptococcus lactis subspecies diacetylactis, S. thermo philus and certain strains of Proprionibacterium shermani.

Kempler and McKay (1981) have presented a summary of the two best accepted pathways for the synthesis of diacetyls. Both pathways involve the degradation of citrate to acetate and oxaloacetate, which is then decarboxylated to form pyruvate.

A second dairy product where carbonyls are considered as flavor impact compounds is yogurt. Here, acetaldehyde is credited with providing the characteristic pungent green character.

Acetaldehyde is a metabolic end product of L. bulgaricus and/or S. thermophilus during the fermentation of milk to yogurt. Carbonyls are quite important to the flavor of cheeses. Maarse and Visscher (1989) noted that a total of 30 aldehydes and methyl ketones have been identified in the volatile fraction of Cheddar cheese.

Carbonyls Carbonyls (methyl ketones) may arise in

fermented products initially via lipase activity of the microorganism.

Methyl ketones and aldehydes may also be formed via microbially induced lipid oxidations.

A final mechanism for the formation of carbonyls via microorganisms involves the transamination and decarboxylation of free amino acids.

Aldehydes

Aldehydes are typically synthesized by microorganisms as intermediates in the formation of alcohols from keto acids.

While the aldehydes are normally reduced to alcohols, the use of fast-fermenting yeast strains, high fermentation temperatures, the presence of readily available nutrients and good aeration during fermentation promote the accumulation of aldehydes.

Even higher concentrations (35 g/1) of acetaldehyde have been produced using P. pastoris. The alcohol oxidase system of P. pastoris was found to have low substrate specificity and was active with the C5-C6 primary alcohols as well as benzyl alcohol.

Ketones

Ketones are important contributors to the flavor of cheeses, particularly the mold-ripened cheeses (e.g. blue and Camembert). Of the ketones, the C3 to C11 methyl ketones are of the greatest significance.

The methyl ketones are formed from the lipids of milk fat through hydrolysis (lipase), oxidation and addition of an acyl group followed by decarboxylation to yield a methyl ketone of one less carbon.

Blue cheese flavor concentrates have been manufactured using whey that has been supplemented with fats, oils or specific fatty acids and fermented with Penicillium roqueforti.

Alcohols Alcohols typically make a minor contribution to flavor unless present in relatively high

concentrations or they are unsaturated (e.g. 1-octen-3-o1).

Alcohols may arise via primary metabolic activity of a microorganism (e.g. ethanol) or by reduction of a carbonyl to the corresponding alcohol. The figure (Next slide) metabolic pathways leading to the major congeners during grain fermentation.

It shows that the fusel alcohols (alcohols larger in size than ethanol) can be formed from either carbohydrate or amino acid metabolism.

Alcohol production from amino acids may occur by transamination, decarboxylation and reduction or by oxidative deamination followed by decarboxylation and reduction.

In either pathway, the product is always the amino acid minus the amine group and one carbon atom. The alternate metabolic pathway yielding alcohols involves the reduction of the corresponding carbonyl.

Contd…..

Terpenes There are no fermented food products commercially available that derive their characteristic

flavor from terpenes. The interest in terpene synthesis by microorganisms is due to the potential for the commercial production of specific terpenes via fermentation.

Collins and Halim, identified geraniol, citronellol, linalool, neral, citral, geranyl acetate and citronellyl acetate as metabolic products of Ceratocystis variospora. Later, nerol, citronellol and geraniol were found to be produced by Trametes odorata.

The biosynthetic pathways leading to terpene formation in microorganisms is well characterized. It is assumed that a pathway similar to that found in plants operates in microbial syntheses.

That basically involves the conversion of mevalonate (MVA) into isopentyl pyrophosphate and 3,3-dimethyl allyl pyrophosphate, which condense to form geranyl pyrophosphate. This pathway occurs in the synthesis of geraniol in Ceratocystzs moniliformis.

Pyrazines Bacillus subtilis was the first organism found to produce a pyrazine. Since that time, several

pyrazines (2-acetyl, dimethyl, 2-methoxy-3-ethyl, 2,5 or 2,6-diethyl-3-methyl, trimethyl, tetramethyl and trimethylpyrazine) have been identified in aged cheese (Liardon et al., 1982).

It has been assumed that pyrazines are formed as a result of enzyme catalyzed reactions. Work by some studies has shown that pyrazines can be formed via non-enzymatic pathways at ambient conditions.

This raises the possibility that microorganisms are providing low molecular weight precursors of the pyrazines as metabolic end products, but pyrazine formation proceeds by purely chemical pathways.

Pyrazines have also been linked to the generation of off-flavors in foods due to microbial action. Pseudomonas taetrolens has been shown to produce a musty-potato aroma (2,5-dimethyl pyrazine and 2- methoxy-3-isopropyl pyrazine) in milk and other refrigerated foods

Formation of pyrazines by microorganisms.

Sulphur Compounds The origin of the majority of sulfur-containing aroma compounds formed by microorganisms is

sulfate, which is initially incorporated into the sulfur amino acids (L-methionine and L-cysteine) and the peptide, glutathione.

Cysteine may react with carbonyls to yield flavor compounds (e.g., trithiolanes) or be decarboxylated to give cysteamine, deaminated to provide α-keto-3-thiopropionic acid or degraded to free H2S.

H2S is a flavor compound in its own right and also is very reactive with carbonyls and free radicals to form very potent aroma compounds (e.g., ethyl sulfide, diethyl disulfide, amyl mercaptan, and 3-methyl-2-butenethiol).

Methionine may be degraded to yield numerous sulfur-containing aroma compounds, the most important of which is methional.

Methionine may also be degraded by microbial lyases to yield methanethiol. Methanethiol may make a contribution to flavor itself or through secondary reactions that yield various sulfides, disulfides, tetrasulfides, and thioesters .

Contd….

Conversion of methionine and Acyl-CoA to thioesters.

Factors affecting the production of flavours during fermentation

Biological Factors i.e. presence of desired microorganisms

pH

Temperature

Nutrients i.e. Nitrogen and Carbon sources e.t.c.

Water activity

Aeration

Conclusion

For thousands of years, products such as cheese, yogurt, sausage, bread, wine and beer have been produced, preserved and flavored by means of microorganisms.

These classical fermentation systems lead to the production of complex flavor compositions as a result of microbial metabolism. Certain of the lactic acid bacteria used for the production of fermented dairy products are capable of producing lactic acid and the volatile compounds, diacetyl and acetaldehyde.

These compounds are produced during the fermentation as a result of microbial metabolism and are responsible for the characteristic buttery flavor and aroma of certain fermented dairy products.

Flavor may be developed from the primary metabolism of the microorganism or from residual enzymatic activity once the microbial cell has lysed. For example, primary metabolism is responsible for much of the flavor of alcoholic beverages, while residual enzymatic activity is essential for the development of aged cheese flavor.

REFERENCES

Feron, G., Bonnarme, P and Durand, A. 1996. Prospects for the microbial production of food flavours. Trends Food Sci Technol, 7:285–93.

Janssens, L., De Poorter, H.L., Vandamme, E.J., Schamp, N.M. 1992. Production of flavours by microorganisms. Proc Biochem 27:195–215.

Marilley, L. and Casey, M.G. 2004. Flavours of cheese products: metabolic pathways, analytical tools and identification of producing strains. Int J Food Microbiol 90(2):139–59.

McSweeney, P.L.H. 2004. Biochemistry of cheese ripening. International Journal of Dairy Technology, 57(2/3): 127–144.

Pandey A. 2003. Solid-state fermentation. Biochem Eng J 13:81–4.

Soccol, C.R and Vandenberghe, L.P.S. 2003. Overview of applied solid-state fermentation in Brazil. Biochem Eng J 13:205–18.

Toelstede, S., Hofmann, T. 2008. Sensomics Mapping and Identification of the Key Bitter Metabolites in Gouda Cheese. Journal of Agricultural and Food Chemistry, 56:2795–2804.

Ziadi, M., Bergot, G., Courtin, P., Chambellon, E., Hamdi, M and Yvon, M. 2010. Amino acid catabolism by Lactococcus lactis during milk fermentation. International Dairy Journal, 20(1), 25-31