Production of Food Additives

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Production of Food Additives 1071

34 Production of Food Additives Pierre Fontanille and Christian Larroche

1. INTRODUCTION Many people enjoy making bread, cakes, wine, beer and ice cream at home. However, most of today’s food is bought from shops and supermarkets and has to stay in top condition over a much longer period of time than home-cooked food. That’s why food additives have become a necessity of all types of food products and food industry. Right from the aroma of the beverage, the texture of the food and its visual appeal, have to be enriched to make it acceptable. Additives also improve the nutritional value of some foods and can make them more appealing by improving their taste, texture, consistency or color. Because many chemical additives are banned by food legislation and by customers, the need for natural alternatives has increased. That’s why more and more additives are produced by fermentation or will be produced in future. This chapter first summarizes the main legislations on food additives in the world. Then, the authors’ aim is to show examples of molecules belonging to the main categories of food additives produced by fermentation for industrial application. Bioprocesses currently used in industry or under investigation are also considered. The choice of these products is based on global food additives market (Fig. 1). Future prospects and new products likely to become new food additives are more detailed in other specific chapters of this book. 2. DEFINITIONS 2.1. The Codex alimentarius definition of food additives The internationally recognized standards, codes of practice, guidelines and other recommendations relating to food, food additives, food production and food safety are given by the Codex Alimentarius (Latin for “food code” or “food book”). The Codex Alimentarius Commission, where

1072 Food Fermentation Biotechnology 7,000

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Fig. 1: Global food additives market by category, 2007(Value in USDm) excluding sales of vitamins, minerals, fat replacers and functional ingredients (http://www.leatherheadfood.com/food-additivesmarket—global-trends-and-developments-4th-edition, 2008).

sit the representatives of about 200 countries, was created in 1963 by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) to try and achieve harmonization in the use of food additives through-out the world by developing food standards, guidelines and related texts such as codes of practice under the Joint FAO/WHO Food Standards Programme. Food additives are clearly defined in the codex general standard for food additives (CODEX STAN 192-1995) as: “Any substance not normally consumed as a food by itself and not normally used as a typical ingredient of the food, whether or not it has nutritive value, the intentional addition of which to food for a technological (including organoleptic) purpose in the manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food results, or may be reasonably expected to result (directly or indirectly), in it or its byproducts becoming a component of or otherwise affecting the characteristics of such foods. The term does not include contaminants or substances added to food for maintaining or improving nutritional qualities”. Even though the Codex alimentarius is recognized as international standard, a food additive definition is included in the European Council Directive 89/107/EEC. This definition is, however, quite similar to the Codex one. In the USA, the Food Additives Amendment 1958 to the Federal Food, Drug and Cosmetics Act contains a more complex definition.


The term food additive means any substance the intended use of which results or may reasonably be expected to result, directly or indirectly, in its becoming a component or otherwise affecting the characteristics of any food (including any substance intended for use in producing, manufacturing, packing, processing, preparing, treating, packaging, transporting, or holding food; and including any source of radiation intended for any such use), if such substance is not generally recognized, among experts qualified by scientific training and experience to evaluate its safety, as having been adequately shown through scientific procedures (or, in the case of a substance used in food prior to January 1, 1958, through either scientific procedures or experience based on common use in food) to be safe under the conditions of its intended use; except that such term does not include: (1) a pesticide chemical residue in or on a raw agricultural commodity or processed food; or (2) a pesticide chemical; or (3) a color additive; or (4) any substance used in accordance with a sanction or approval granted prior to the enactment of this paragraph 4 pursuant to this Act [enacted Sept. 6, 1958], the Poultry Products Inspection Act (21 U.S.C. 451 and the following) or the Meat Inspection Act of March 4, 1907 (34 Stat. 1260), as amended and extended (21 U.S.C. 71 and the following); (5) a new animal drug; or (6) an ingredient described in paragraph (ff) in, or intended for use in, a dietary supplement. This amendment categorized food chemicals as: (1) those Generally Recognized As Safe (GRAS); (2) those with prior sanction; and food additives. Pesticides on raw agricultural products and food color additives were excluded from the legal definition as they were covered by other legislation. 2.2. How are additives approved for use in foods? New additives and new process production of existing additives are evaluated by the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) which normally meets twice a year. It is an international expert scientific committee that is administered jointly by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). It has been meeting since 1956, initially to evaluate the safety of food additives. To date, JECFA has evaluated more than 1500 food additives. The Committee has also developed principles for the safety assessment of chemicals in food that are consistent with current thinking on risk assessment and take into account recent developments in toxicology and other relevant scientific areas such as microbiology, biotechnology, exposure assessment, food chemistry including analytical chemistry and assessment of maximum residue limits for veterinary drugs. The “Codex General Standard for Food Additives” (GSFA, Codex STAN 192-1995) lists and sets

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forth the conditions under which permitted food additives may be used in all foods. To search for the provisions of a food additive, GSFA Database is available at: http://www.codexalimentarius.net/ gsfaonline/additives/search.html?lang=en. In the United States, food and color additives are strictly studied, regulated and monitored by the FDA which has the primary legal responsibility for determining their safe use. To market a new food or color additive (or before using an additive already approved for one use in another manner not yet approved), a manufacturer or other sponsor must first petition FDA for its approval. These petitions must provide evidence that the substance is safe for the ways in which it will be used. As a result of recent legislation, since 1999, indirect additives have been approved via a premarket notification process requiring the same data as was previously required by petition. Under the Food Additives Amendment, two groups of ingredients were exempted from the regulation process. GROUP I - Prior-sanctioned substances - are substances that FDA or USDA had determined safe for use in food prior to the 1958 amendment. Examples are sodium nitrite and potassium nitrite used to preserve luncheon meats. GROUP II - GRAS (generally recognized as safe) ingredients - are those that are generally recognized by experts as safe, based on their history of extensive use in food before 1958 or based on published scientific evidence. Among the several hundred GRAS substances one finds salt, sugar, spices, vitamins and monosodium glutamate (MSG). Manufacturers may also request that FDA review the industry’s determination of GRAS Status. The Food and Drug Administration (FDA) maintains a list of over 3000 ingredients in its data base “Everything Added to Food in the United States” available at: http://www.fda.gov/Food/ FoodIngredientsPackaging/FoodAdditives/FoodAdditiveListings/ucm091048.htm 2.3. Why are Additives Used in Foods? Additives perform a variety of useful functions in foods that are often taken for granted. Most people today have come to rely on the many technological, aesthetic and convenience benefits that additives provide in food. Additives are used in foods for five main reasons: • To maintain product consistency. Emulsifiers give to products a consistent texture and prevent them from separating. Stabilizers and thickeners give smooth uniform texture. Anticaking agents help substances such as salt to flow freely. • To improve or maintain nutritional value. Vitamins and minerals are added to many common foods such as milk, flour, cereal and margarine to make up for those likely to be lacking in a person’s diet or lost in processing.


• To maintain palatability and wholesomeness. Preservatives retard product spoilage caused by mold, air, bacteria, fungi or yeast. Bacterial contamination can cause foodborne illness, including life-threatening botulism. Antioxidants are preservatives that prevent fats and oils in baked goods and other foods from becoming rancid or developing an off-flavor. They also prevent cut fresh fruits such as apples from turning brown when exposed to air. • To provide leavening or control acidity/alkalinity. Leavening agents that release acids when heated can react with baking soda to help cakes, biscuits and other baked goods to rise during baking. Other additives help modify the acidity and alkalinity of foods for proper flavor, taste and color. • To enhance flavor or impart desired color. Many spices, natural and synthetic flavors enhance the taste of foods. Colors, likewise, enhance the appearance of certain foods to meet consumer expectations. 2.4. Classification of additives Food additives can perform a number of technological functions during food processing and storage and in a few cases the same substance may have more than one function. A comprehensive, but not exhaustive, list of additive functions is given in the Codex Standard on Food Labeling (Table 1). The food additive functional classes are based on the Codex Class Names and the International Numbering System (INS) for Food Additives (CAC/GL 36-1989). This list illustrates the diversity of functions covered by additives and in most countries it forms the basis for the classification of approved additives. 3. FLAVOURING AGENTS AS FOOD ADDITIVES Flavouring agents are one of the largest single groups of food additives. Food and beverage applications of flavours include dairy, fruit, nut, seafood, spice blends, vegetables and wine flavouring agents. They may complement, magnify, or modify the taste and aroma of the foods. They represent a worldwide market of about US$ five billion and constitute over a quarter of the global market of food additives. Indeed, olfaction has a dramatic impact on our food perception because it can elicit strong emotions and lasting memories (Shepherd, 2006). The subdivision of volatile flavors into natural, natural identical (EC only) and artificial is based on legal definitions: the EC Guidelines 88/388/EEC, 91/71/EEC, 91/72/EEC and the US code of Federal Regulation. The term “natural”, considered as consumer friendly, is defined in Europe as “flavoring substances or preparations which are obtained by appropriate physical processes or enzymatic or microbiological processes from material of vegetal or animal origin” (EC Flavor Directive, 88/388/EEC). In the USA, the term natural flavor means “the essential oil, oleoresin, essence or extractive, protein hydrolysate, distillate of any product of roasting, heating or

1076 Food Fermentation Biotechnology Table 1 : Food additives functional classes according to codex alimentarius Technological purpose

Food additives functional class

Definitions

Acidity regulator

Acidity regulator, acid, acidifier, alkali, Alters or controls the acidity or alkalinity of a base, buffer, buffering agent, pH food. adjusting agent

Anticaking agent

Anticaking agent, antistick agent, Reduces the tendency of particles of food to drying agent, dusting powder, release adhere to one another. agent

Antifoaming agent

Antifoaming agent

Antioxidant

Antioxidant, antioxidant synergist, Prolongs the shelf-life of foods by protecting against deterioration caused by oxidation, such as sequestrant fat rancidity and colour changes

Bulking agent

Bulking agent, filler

A substance, other than air or water, which contributes to the bulk of a food without contributing significantly to its available energy value.

Colors

Color

Adds or restores color in a food.

Color retention agent Color fixative, color stabilizer

Prevents or reduces foaming.

Stabilizes, retains or intensifies the color of a food.

Emulsifier

Clouding agent, dispersing agent, Forms or maintains a uniform mixture of two or emulsifier, plasticizer, surface active more immiscible phases such surface as oil and water in a food. agent, surfactant, wetting agent

Emulsifying salt

Melding salt, sequestrant

Rearranges cheese proteins in the manufacture of processed cheese, in order to prevent fat separation.

Firming agent

Firming agent

Makes or keeps tissues of fruit or vegetables firm and crisp, or interacts with gelling agents to produce or strengthen a gel.

Flavor enhancer

Flavor enhancer, flavor modifier, Enhances the existing taste and/or odor of a food. tenderizer

Flour treatment agent Bleaching agent, dough improver, A substance added to flour to improve its baking quality or color. flour improver

Production of Food Additives 1077 Food additives functional class

Technological purpose

Definitions

Foaming agent

Aerating agent, whipping agent

Makes it possible to form or maintain a uniform dispersion of a gaseous phase in a liquid or solid food.

Gelling agent

Gelling agent

Gives a food texture through formation of a gel.

Glazing agent

Coating, polish, sealing agent

A substance which, when applied to the external surface of a food, imparts a shiny appearance or provides a protective coating.

Humectant

Moisture/water retention agent, Prevents food from drying out by counteracting the effect of an wetting agent atmosphere having wetting agent a low degree of humidity.

Preservative

Antimicrobial preservative, anti- Prolongs the shelf-life of a food by protecting mycotic agent, bacteriophage control against deterioration caused by microorganisms. agent, chemosterilant, disinfection agent, wine maturing agent

Propellant

Propellant

Raising agent

Binder, leavening agent, raising agent A substance or combination of substances which liberate gas and thereby increase the volume of a dough.

Stabilizer

Colloidal stabilizer, firming agent, Makes it possible to maintain a uniform dispersion foam stabilizer, moisture/water of two or more immiscible substances in a food. retention agent

Sweetener

Artificial sweetener, sweetener, sweetener

Thickener

Bodying agent, texturizer, thickening Increases the viscosity of a food. agent

A gas, other than air, which expels a food from a container.

nutritive A non-sugar substance which imparts a sweet taste to a food.

enzymolysis, which contains the flavoring constituents derived from a spice, fruit juice, vegetable or vegetable juice, edible yeast, herb, bud, bark, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products or fermentation products thereof, whose significant function in food is imparting flavoring rather than nutrition” (Code of Federal Regulations, 1990). Both definitions state that the precursor or product must be present in nature or be part of traditional


foods and those natural flavors must be obtained via physical or bio-processes. Microbial flavors can occur along de novo microbial syntheses (fermentation), or in bioconversions upon adding a suitable precursor compound to the micro-organisms or enzymes (biocatalysis). Physical processes include extraction, distillation, concentration, crystallisation etc. The authors’ intention is to illustrate examples of compounds currently used or under investigation in industry rather than to present the large variety of flavor compounds generally made available by biotechnology. More detailed review on this topic is given elsewhere in the book. There are over 1200 different flavoring agents used in foods to create flavor or replenish flavors lost or diminished in processing, and hundreds of chemicals may be used to simulate nature flavours. Alcohols, esters, aldehydes, ketones, pyrazines or terpenes are examples of flavouring agents. 3.1. Vanillin, benzaldehyde and aromatics Vanillin, the main flavor compound of the bean of Vanilla planifolia, is by far the most important flavor chemical in terms of amount and value. Annually more than 10 000 tons are produced and less than 1% is extracted from the vanilla bean. Whereas chemically produced vanillin is a cheap flavor compound (US$ 11 kg–1), natural vanillin derived from microbial processes currently costs up to US$ 1000 kg–1 and yields a market volume of several tons per annum (Schrader et al., 2004). Limited supply and the high price of the natural vanillin extracted from the botanical source (up to US$ 4000) stimulated research for a biotechnological substitution. Different biotechnological production processes are based on bioconversion of ferulic acid, phenolic stilbenes, isoeugenol, or eugenol and on de novo biosynthesis, applying bacteria, fungi, plant cells or genetically engineered microorganisms (Priefert et al., 2001). Two commercial processes are well known: pressure hydrolysis of curcumin from curcuma roots and degradation of natural feluric acid, frequently occurring in plants and mainly isolated from rice bran, by Pseudonocardia. Benzaldehyde, with an annual global consumption of 7,000 tons, is the second most important aroma after vanillin. It has a cherry or almond taste and can be extracted from fruit kernels. Only a few organisms have been reported to produce benzaldehyde in high amounts, mainly because it is toxic towards microbial metabolism (Desmet et al., 2008). Cinnamic acid esters (exotic fruit), cinnamaldehyde (cinnamon), eugenol (clove), methyl benzoate (dry fruit) and benzyl acetate (jasmine) were among the volatiles found in submerged cultures of basidiomycetes. 3.2. γ-Decalactone γ-decalactone is the most important lactone for flavor application. It has an oily-peachy aroma, extraordinarily tenacious odour and a very powerful, creamy-fruity, peach-like taste at concentrations below 5 mg L–1. In the early eighties natural γ-decalactone was an extremely expensive, rare natural


flavor (>US$ 10,000 kg–1). The subsequent introduction and optimization of microbial processes has resulted in a price decrease to approx. US$ 300 kg–1 and a market volume of several tons per year (Schrader et al., 2004). Most of the commercial processes for the formation of γ-decalactone use ricinoleic acid, the main fatty acid of castor oil or esters thereof. The formation of γ-decalactone, with the same enantiomeric configuration as the lactone naturally found in peaches and other fruits, was first observed by Okui et al. (1963) in the catabolism of ricinoleic acid by yeasts of the genus Candida. The processes with the highest product concentrations use Yarrowia lipolytica strains. Using a genetically engineered multiple auxotrophic mutant, designated PO1D, Nicaud et al. (1996) obtained high yields of γ-decalactone from ricinoleic acid methyl ester. After 75 h the culture broth yielded 9.5 g γ-decalactone L–1 (Pagot et al., 1997). In a production process established by Haarmann & Reimer GmbH (H&R), Germany, up to 11 g γ-decalactone L–1 was obtained in 55 h without a genetically modified production strain and with raw castor oil as substrate (Rabenhorst & Gatfield, 2000). Kümin & Münch (1997) at Givaudan, Switzerland, used Mucor circillenoides on ethyl decanoate as substrate for the production of γ-decalactone. They obtained 10.5 g γ-decalactone L–1 after 60 h. 3.3. Esters Aliphatic and terpenic esters can be found in almost every food. Those with a relatively low molecular weight are usually fruity in character. The word ‘estery’ is an established term to describe this sensory impression. Ethyl esters belong to the most important esters that are relevant for flavors since ethanol is widely present in plant metabolism. Methyl, propyl, butyl, isobutyl, amyl and isoamyl esters are also of specific interest for flavors in the food industry. They are generally produced by many microorganisms by oxidative shortening of fatty acids and partial reduction of the degradation products, by degradation of free amino acids, or by conversion of terpenoid precursors (Berger, 2000). A great advantage of reactions involving lipases or esterases is that they can be carried out in organic solvents without the need for any cofactor. This is of special interest for ester formation since the water formed can be removed simply by distillation or adsorption. 3.4 Green aldehydes and alcohols C6 aldehydes and their corresponding alcohols are the industrially most relevant compounds to obtain the ‘green’ organoleptic characteristic. For example, 3(Z)-hexen-1-ol (‘leaf alcohol’) has a powerful odour of freshly cut grass and is an important flavor and fragrance material used for natural green top notes. The market of natural green notes is estimated at 5–10 t year–1 and US$ 3000 kg–1 (Muller et al., 1995a). Since the traditional preparation of natural green notes by distilling plant oils, e.g. mint terpene fractions, is costly and cannot meet the increasing market demand for natural flavors, different biocatalytic syntheses have been developed, all containing


at least one enzymatic reaction catalyzed by crude plant material (Goers et al., 1989, Brunerie & Koziet, 1997, Belin et al., 1998, Holtz et al., 2001). In comparison with these patents, one filed by Firmenich (Muller et al., 1995b) reported the highest yields, e.g. 4.2 g 3(Z)-hexen-1ol kg–1 and 1.5 g 2(E)-hexenal kg–1 were produced (Schrader et al., 2004). 3.5. Ketones The most important ketone aroma is 2,3-butanedione (diacetyl), which is related to the flavor of butter (Bartowsky & Henschke, 2004). It is produced by lactic acid bacteria and other microorganisms present in food, and is, therefore readily available. Methyl ketones, such as 2-heptanone, are the largest contributors to stale flavors in UHT milk, and are used for the flavoring of blue cheese. A interesting method to produced 2-heptanone has been developed by Larroche and coworkers (1992) with spores of Penicillium roquefortii in a water-organic solvent two-phase system. 3.6. Terpenes and terpenoids Terpenes and terpenoids are molecules composed of isoprene units and can be found in the essential oil of plants (Loza-Tavera, 1999). The terpenes with the highest flavor intensity are geraniol, citronellol, nerol and linalool. Terpenoid flavor compounds represent an area of high commercial interest to the industry due to the large number of key aroma compounds which are in principle accessible by biotransformation of abundant natural terpene hydrocarbons (Schrader & Berger, 2001). For this class of molecule, the feasibility of novel biocatalytic flavor syntheses will much depend on process engineering features. This is especially obvious in the case of biotransformation which have to overcome several drawbacks, such as the low water-solubility of the precursors, toxicity of precursors and products, and metabolic diversity which leads to unwanted by-products or further degradation of the target molecules. One example showing that extremely high yields can nevertheless be obtained has been given by Fontanille & Larroche (2003). Up to 400 g cis2-methyl-5-isopropyl-2,5-hexadienal (isonovalal) l–1, an artificial fragrance compound for potential use in perfume formulations, has been reported to be produced from α-pinene oxide within 2.5 h using permeabilized Pseudomonas rhodesiae CIP 107491. The bioprocess was performed with in situ product recovery using hexadecane in a biphasic medium and by sequential feeding of biomass and precursor to compensate the irreversible biocatalyst inactivation by the product. 3.7. Pyrazine Pyrazines are heterocyclic, nitrogen-containing compounds which have been shown to contribute significantly to the unique taste and aroma of roasted or toasted foods since the mid-1960s (Seitz, 1994). Thermally treated foods accumulate these heterocycles from aminoketone precursors through the Maillard reaction. The demand for bacterial pyrazines as food additives was initiated by the development of modern cooking processes (i.e. micro-waving) which do not result in the formation


of pyrazines. The GRAS list of substituted pyrazines shows that 34 such compounds are approved for use in flavoring. They can be produced in foods by various microorganisms and in broth systems by actinomycetes, Gram negative, Gram positive and filamentous fungi. Fermentation or biotransformation processes with submerged techniques or solid substrate fermentations are claimed to be an useful tool for the production of this kind of natural components (Larroche et al., 1999). 4. HYDROCOLLOIDS AS FOOD ADDITIVES Hydrocolloids are thickening, gelling and stabilizing agents, which play a major role in numerous food and beverage products. Recently, Morris (2006) has described the wide potential application and the latest research in the hydrocolloid field of bacterial polysaccharides in food industry. They are essentially produced for their applications as good substitutes of gums from plants and marine algae. The water-binding properties of hydrocolloids are used for viscosifying and gelling purposes in food. Several of the products also interact with proteins, allowing for protein stabilization and protection applications. To a large extent, the use of hydrocolloids also influences the texture and consequently the mouth-feel and eating experience of foods. These last years a lot of authors have tried to provide a comprehensive overview of novel aspects of the main classes of polysaccharides employed in food products underscoring their nutritional contribution and all their biotechnological properties including organoleptic properties, health effect, role in emulsions and foams formation, interaction with particles, flavour encapsulation, food texturation, etc (Alistair, 2006; Williams & Philipps, 2007, Delattre et al., 2008). Only main industrial microbial polysaccharides productions are presented here. More details can be found elsewhere in the book. Since many years, microbial exopolysaccharides such as xanthan and dextran have been commercial products. For example, approximately 40 000-50 000 tones of xanthan per year are produced due to its success based on its important rheological properties at low concentrations (Demain, 2000). Others exopolysaccharides produced by fermentation such as gellan or curdlan have worthwhile industrial food applications. 4.1. Xanthan Xanthan gum is the most versatile microbial exopolysaccharide. It is produced by the phytopathogen bacteria Xanthomonas campestris pv campestris. It is largely used in food industry (cheese, beer, sauces, juices, ice cream, salad…) alone or in association with others polysaccharides as galactomannans or glucomannans (Sutherland, 1998) reflecting its physico-chemical properties, as a gelling, emulsifying, thickening, and stabilizing agent (Delattre et al., 2008). Xanthan gum is a ramified heteropolysaccharide made up of a principal cellulosic backbone substituted on every


second unit with a trisaccharide side chain linked on C-3 position of the glucosyl residue (Jansson et al., 1983). The xanthan gum is produced in batch fermentation. Components of the media used in industry are mainly inexpensive and complex, being natural raw materials. Successful fermentation requires a clear understanding of the microbial environment with particular attention to the high viscosity of the fermentation broth. The kinetics are influenced significantly by spatial variations in the concentrations of substrate, biomass and the polymer itself. Final concentration can reach 15-30 kg m–3 after 96h of fermentation. 4.2. Gellan Another interesting exopolysaccharide formally called gellan gum (E418) is prepared by aerobic submerged fermentation process from Sphingomonas paucimobilis (Pollock, 1993). It is used in food and pharmaceutical industry for its wide various texture properties. Gellan is a gelling agent obtained by mild alcalin deacetylation and is commercially called under the trade name Kelcogel® and Gelrite® (Banik et al., 2000). Other bacterial species from the bacterial genus Sphingomonas have been described as producer of gellan like polysaccharides called sphingans. New polysaccharides belonging to this new family (gellan, welan and rhamsan) have been produced and sold for applications in food hydrocolloids market. Gellan has been approved in USA and EU for food uses as suspending, stabilizing, thickening, binding and gelling agent, either alone or in combination with other hydrocolloids (Sutherland, 1998; Sa-Correia et al., 2002; Sanderson & Clark, 1983). 4.3. Curdlan Curdlan (E424) is a bacterial neutral linear and no branching b-D-glucan polysaccharide consisting of β-1,3-linked glucose units produced by Agrobacterium biovar.1 (Alcaligenes faecalis var. myxogenes strain 10C3), few Rhizobium and Gram-negative Cellulomonas, including C. flavigena KU (Lee, 2002; Kenyon & Buller, 2002; McIntosh et al., 2005). At ambient temperature, curdlan is totally insoluble in water and in most organic solvents but can form two types of heat-induced gels termed “high-set gels” and “low-set gels” via the heating of aqueous solution (Harada et al., 1996; Lee, 2002). Curdlan gels have properties similar to the gelatine elasticity and the agar fragility. These specific properties are particularly appreciated by food industry in Korea, Taiwan, and Japan (Spicer et al., 1999), since it can be applied as texturant processing aid, stabilizer and thickener or texture modifier in a wide range of food preparations including processed meat, surimi products, sauces, vegetarian foods, or other functional foods (Harada & Sato, 1978). 5. AMINO ACIDS AS FOOD ADDITIVES For almost 50 years now, biotechnological production processes have been used for industrial


production of amino acids. In terms of market volume, development over the last 20 years has been tremendously bullish in the so-called feed amino acids L-lysine, DL-methionine, L-threonine, and Ltryptophan, which constitute the largest share (56%) of the total amino acid market, estimated in 2004 at approximately US $4.5 billion (Leuchtenberger et al., 2005). The food sector represents a small part of the market, and is determined essentially by three amino acids: L-glutamic acid in the form of the flavor enhancer monosodium glutamate (MSG) and the amino acids L-aspartic acid and L-phenylalanine, both of which are starting materials for the peptide sweetener L-aspartyl Lphenylalanyl methyl ester (Aspartame), used, for example, in “lite” colas. Monosodium Glutamate (MSG), the sodium salt of glutamic acid, is the most closely studied taste enhancer. MSG enhances the savory flavors imparted by glutamic acid, which occurs naturally in proteinaceous foods (eg, meats, seafood, stews, soups, sauces). MSG is on the list of GRAS ingredients, but the US FDA requires manufacturers to list MSG and related compounds (monopotassium glutamate, monoammonium glutamate) on the food label. Indeed, MSG was banned from the production of infant foods because of the occurrence of irreversible retinal lesions in neonatal rodents. 5.1. Monosodium glutamate The rapid development of the amino acid market since the 1980s is due for a large part to major successes in cost effective production and isolation of amino acid products. Among the four production methods for amino acids-extraction, synthesis, fermentation, and enzymatic catalysisit is particularly the last two biotechnological processes, with their economic and ecological advantages, that are responsible for this spectacular growth. The extraction method for obtaining L-glutamate was superseded nearly 50 years ago by fermentation, following a sharp increase in demand for the flavor-enhancer MSG. The discovery of the soil bacterium, Corynebacterium glutamicum, which is capable of producing L-glutamic acid with high productivity from sugar, paved the way for the success of the fermentation technique in amino acid production (Kinoshita et al., 1957). It was advantageous here that the wild strain could be used on an industrial scale under optimized fermentation conditions for mass production of glutamate. Glutamate biosynthesis and methods for improving production strains have been investigated in depth (Kimura, 2003). The fermentation process is in principle very simple: a fermentation tank is charged under sterile conditions with a culture medium containing a suitable carbon source, such as sugar cane syrup, as well as the required nitrogen, sulfur, and phosphorus sources, and some trace elements. A culture of the production strain prepared in a pre-fermenter is added to the fermentation tank and stirred under specified conditions (temperature, pH, aeration). The L-glutamic acid released by the microorganism into the fermentation solution is then obtained by crystallization in the recovery section of the fermentation plant. MSG (1.5 million tons) is currently produced each year by this method, making L-glutamic acid the number one amino acid in terms of production capacity and demand.


6. ORGANIC ACIDS AS FOOD ADDITIVES The organic acids commonly used as food additives include, in order of decrease importance, citric, acetic, lactic, tartaric, malic, gluconic, propionic and fumaric acids. From the root word, acid, one can conclude that this class of compounds tends to lower pH of any food in which the compounds are incorporated. They also enhance desirable flavors, and in many cases, such as in pickled products, are the major taste component. Vinegar (acetic acid) is added to relishes, chili sauce, ketchup and condiments as a flavor component and to aid in the preservation of these products. Since the microbial spoilage of food is inhibited when the pH of a food is lowered, acidulants are used for that purpose in many cases. Many acidulants occur naturally in foods (e.g., citric acid in citrus fruits, malic acid in apples, acetic acid – the major component of vinegar). In addition to their preservative and flavor enhancing effects, organic acids are used to improve gelling properties and texture. They are also used as cleaners of dairy equipment. Acidulants may be used in the manufacturer of processed cheese and cheese spread for the purpose of emulsifications as well as to provide a desirable tartness. Acid salts may be added to soft drinks to provide a buffering action to prevent excess acidity. In some cases, acid salts are used to inhibit mold growth. 6.1. Citric acid Citric acid has several uses in the food industry. Some of them are presented in table 2. Citric acid was originally extracted from lemons and limes but it is now produced commercially by a fermentation process. Its production currently uses submerged or surface fermentation processes, with beet molasses or glucose syrup as the main raw material. The mould Aspergillus niger is Table 2: Examples of citric acid use in the food industry Use in the food industry

Associated products

generate the optimum conditions for the formation of gels

jams, jellies, confectionary and desserts

help give the conditions for the stabilization of emulsions

processed cheese and dairy products

prevent the browning

salads

enhance the action of antioxidants and prevent deterioration

frozen food

act as an antioxidant

fats and oils

preserve products and help modify their texture during their processing

meat products

provide sharp taste

soft drinks and sweets


used to ferment its carbohydrate source even if citric acid fermentation was first found as a fungal product in cultures of Penicillium glaucum on sugar medium by Wehmer in 1893. An interesting review on citric acid production patents (Anastassiadis et al., 2008) summarizes the developments in citric acid production technologies in world for the last 100 years. Citric acid is commercially produced by large scale fermentation mostly using selected fungal or yeast strains in aerobic bioreactors. It still remains one of the runners in industrial production of biotechnological bulk metabolites obtained by microbial fermentation since about 100 years, reflecting the historical development of modern biotechnology and fermentation process technology. Global citric acid production has reached 1.4 million tones, increasing annually at 3.5-4.0% in demand and consumption. Citric acid production by fungal submerged fermentation is still dominating, however new perspectives like solid-state processes or continuous yeast processes can be attractive for producers to stand in today’s strong competition in industry (Soccol et al., 2006). Many inexpensive by-products and residues of the agro-industry (e.g. molasses, glycerin etc.) can be economically utilized as substrates in the production of citric acid, especially in solid-state fermentation, enormously reducing production costs and minimizing environmental problems. Alternatively, continuous processes utilizing yeasts which reach 200-250 g.L–1 citric acid can stand in today’s strong competition in citric acid industry and replace the traditional discontinuous fungi processes (Anastassiadis et al., 2008). 6.2. Acetic acid Acetic acid is produced both synthetically and by bacterial fermentation. Today, the biological route accounts for only about 10% of world production, but it remains important for vinegar production, as many nations’ food purity laws stipulate that vinegar used in foods must be of biological origin. Acetic acid is widely used, particularly in the pickling industry. Naturally fermented vinegar has a variable pH and so acetic acid is added to this to form a pickling liquor with a specified acidity. It can also be used in confectionery goods and flavorings. The flavoring sodium diacetate is commonly known as ‘salt ‘n’ vinegar’ and is widely used in crisps. Acetic acid has excellent bacteriostatic properties and hence has considerable importance as a preservative. It is produced from sugar-rich materials by successive anaerobic and aerobic fermentation. The first step of the process involves production of alcohol solution from sugar through yeast enzymes. Then, the conversion of alcohol to acetic acid is classically made by acetic acid bacteria of the genus Acetobacter. Most vinegar today is made in submerged tank culture, first described in 1949 by Hromatka and Ebner. In this method, alcohol is fermented to vinegar in a continuously stirred tank, and oxygen is supplied by bubbling air through the solution. Using modern applications of this method, vinegar of 15% (w/w) acetic acid can be prepared in only 24 hours in batch process, and up to 20% in 60 hours fed-batch process. Species of anaerobic bacteria, including members of the genus Clostridium, can also convert sugars to acetic acid directly, without using ethanol as an intermediate. More interestingly from the point


of view of an industrial chemist, these acetogenic bacteria can produce acetic acid from one-carbon compounds, including methanol, carbon monoxide, or a mixture of carbon dioxide and hydrogen. This ability of Clostridium to utilize sugars directly, or to produce acetic acid from less costly inputs, means that these bacteria could potentially produce acetic acid more efficiently than ethanoloxidizers like Acetobacter. However, Clostridium bacteria are less acid-tolerant than Acetobacter. Even the most acid-tolerant Clostridium strains can produce vinegar of only a few per cent acetic acid, compared to Acetobacter strains that can produce vinegar of up to 20% acetic acid. At present, it remains more cost-effective to produce vinegar using Acetobacter than to produce it using Clostridium and then concentrating it. As a result, although acetogenic bacteria have been known since 1940, their industrial use remains confined to a few niche applications (Sim et al., 2007). 6.3. Lactic acid Lactic acid is widely used in almost every segment of the food industry, in particular in the production of boiled sweets, pickled foods and as a raw material in the manufacture of important emulsifiers for the baking industry. Its main functions are flavoring agent, pH regulator, improvement of microbial quality and mineral fortification. It is classified as GRAS for use as a food additive by the US FDA. As for acetic acid production, both synthetically and bacterial fermentation are used to produce lactic acid. The advantage of fermentation process is to obtain the desirable L(+)-lactic acid stereoisomer while the mixture of L(+)– and D(–)-isomers is generated by the synthetic route. The existing commercial production processes use the homolactic organisms, such as Lactobacillus amylophilus, L. delbrueckii, L. bulgaricus, L. helveticus, Enterococcus faecalis or Poediococcus damnosus, which are capable of fully converting glucose to lactic acid. Among the different fermentation techniques, such as fed-batch, repeated-batch and continuous cultures the batch fermentation stays the common method for the industrial production of lactic acid. The limitation of the development of lactic acid production by fermentation has been the availability of cheap resources. Lots of recent studies have shown the success of using renewable resources in fermentation processes and become more and more advantageous than the chemical route from an economical point of view (Wee & Ryu, 2009). 6.4. Gluconic acid Gluconic acid is a mild organic acid, which finds lots of applications in the food industry. As stated above, it is a natural constituent in fruit juices and honey and is used in the pickling of foods. Its inner ester, glucono-d-lactone imparts an initially sweet taste which later becomes slightly acidic. It is used in meat, dairy products, particularly in baked food as a component of leavening agent for pre-leavened products. It is used as a flavoring agent (for example, in sherbets) and it also finds application in reducing fat absorption in doughnuts and cones. Foodstuffs containing


D-glucono-d-lactone include bean curd, yoghurt, cottage cheese, bread, confectionery and meat. In the European Parliament and Council Directive No. 95/2/EC, gluconic acid is listed as a generally permitted food additive (E 574). The US FDA has assigned sodium gluconate a GRAS status and its use in foodstuff is permitted without limitation. There are different approaches available for the production of gluconic acid, namely, chemical, electrochemical, biochemical and bioelectrochemical (Ramachandran et al., 2006). There are several different oxidizing agents available, but still the process appears to be costlier and less efficient compared to the fermentation processes. Although the conversion is a simple one-step process, the chemical method is not favored. Thus fermentation has been one of the efficient and dominant techniques for manufacturing gluconic acid. Among various microbial fermentation processes, the method utilizing the fungus A. niger is one of the most widely used ones. A recent review exposes in more details the gluconic acid properties, applications and microbial production processes (Ramachandran et al., 2006). Another recent review (Anastassiadis & Morgunov, 2007) presents the comprehensive information of patent bibliography for the production of gluconic acid and compares the advantages and disadvantages of known processes. Numerous manufacturing processes are described in the international bibliography and patent literature of the last 100 years for the production of gluconic acid from glucose, including chemical and electrochemical catalysis, enzymatic biocatalysis by free or immobilized enzymes in specialized enzyme bioreactors as well as discontinuous and continuous fermentation processes using free growing or immobilized cells of various microorganisms, including bacteria, yeast-like fungi and fungi. 6.5. Fumaric acid Fumaric acid is the strongest tasting food acidulant. It has limited applications due to its very low solubility. In the main, it is used in gelatin dessert powders, cheesecake mixes and some powdered drinks. A substantial amount of fumaric acid is used in animal feedstuffs mainly because of its strong flavor and favorable price. Fumaric acid is mainly manufactured synthetically. But the potential of this compound as raw material in industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Production of fumaric acid by Rhizopus species and the involved metabolic pathways has been recently reviewed by Roa Engel et al. (2009). They indicate that submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production also include metabolic engineering approaches to achieve low pH fermentations.


6.6. Malic acid Malic acid is naturally found in apples, pears, tomatoes, bananas and cherries. It has applications similar to those of citric acid and is the preferred acidulant in low calorie drinks, such as cider and apple juices. However, it has the disadvantage of being slightly more expensive than citric acid. Racemic malic acid is synthesized petrochemically from maleic anhydride. Enantiomerically pure L-malic acid is produced from fumarate (synthesized from maleic anhydride) by enantioselective hydration with fumarase, using either immobilized cells or isolated enzyme. Increasing oil prices, concerns about climate change, and advances in the field of metabolic engineering have fueled renewed interest in the production of organic acids by microbial fermentation (Goldberg et al., 2006). In 2004, the U.S. Department of Energy included a group of 1,4-dicarboxylic acids, namely succinic, fumaric, and malic acids, in the top 12 most interesting chemical building blocks that can be derived (Zelle et al.,2008). 7. ENZYMES AS FOOD ADDITIVES Enzymes are nontoxic protein substances that occur naturally in foods or may be produced by microorganisms or biotechnology to catalyze various reactions. Their employment in food industry is based on three basic aspects which are the control of quality of certain foods, the modification of the properties of some food additives and of the foods itself and, the production of enzymes used as food additives (Aguilar et al., 2008). In the food industry, the use of enzymes is more and more important in food processing. The food enzymes is the second largest segment of the industrial enzyme market (25%) and include the enzymes employed in the dairy, brewing, wine and juice, fats and oils, and baking industries, and this sector is expected to grow around 3% (Binod et al., 2008). Amylases are the most important enzymes used in food industry and have great significance in extensive biotechnological applications in particular in the bread and baking industry (Pandey et al., 2000). They represent a quarter of the total demand for industrial enzymes. Their thermostabilities represent a major advantage and they have completely replaced the chemical hydrolysis of the starch in starch processing industries (Emmanuel et al., 2000). They are also used along with acids in the production of corn syrup. Alpha amylases and glucoamylases are preferentially used in the alcohol industry and their use has resulted in complete malt replacement. Glucose oxidase is added to foods such as egg whites in order to prevent Maillard browning. Several enzymes, such as chymosin, are utilized in the dairy industry to coagulate milk proteins during cheese making. Betagalactosidase and lactase are used to split milk lactose into glucose and galactose, which allows the resulting product to be consumed by the lactose intolerant population. Papain is used in meat industry as tenderizer of muscle tissues. Several hydrolytic enzymes are also utilized in fruit juice manufacturing such as pectinase, xylanase and cellulase


which improve, by breaking down cell walls, the liberation of the juice from the pulp. The pectinases and amylases are also used in the clarification of juice (Binod et al., 2008). Microorganisms used in the production of enzyme preparations should not leave any residues harmful to human health in the processed food under normal conditions of use. Microbial strains used in the production of enzyme preparations may be native or mutant strains derived from native strains using techniques such as serial culture and selection or mutagenesis and selection or by using recombinant DNA technology. Although nonpathogenic and nontoxigenic microorganisms are normally used in the production of enzymes in food processing, several fungal species are known to include strains capable of producing low levels of certain mycotoxins under fermentation conditions. Enzyme preparations derived from such fungal species should not contain toxicologically significant levels of mycotoxins that could be produced by these species. Microbial production strains should be taxonomically and genetically characterized and identified by a strain number or other designation. Enzyme preparations should be produced in accordance with good food manufacturing practice and cause no increase in the total microbial count in the treated food over the level considered to be acceptable for the respective food. Ideally, extra-cellular enzymes are preferred as the recovery and purification processes are much simpler compared to intracellular enzymes. Other important factor is that the organism should be able to produce high amount of the desired enzyme in a reasonable time frame (Binod et al., 2008). Most of the industrial enzymes are produced by only a relatively few microorganisms such as Aspergillus, Trichoderma, Streptomyces and Bacillus. Most enzyme manufacturers produce enzymes using submerged fermentation (SmF) or liquid surface (SLF) fermentation techniques with enzyme titers in the range of grams per liter. Such levels are a prerequisite if specific compounds are to be considered as commodities because product recovery costs are inversely proportional to concentration in a fermentation broth. There is however a significant interest in using solid-state fermentation (SSF) techniques to produce a wide variety of enzymes, mainly from mold origin, as indicated by the growing number of research papers in the literature and the marketing and development by a small but visible number of fermentation industries (Viniegra-González et al., 2003 ; Pandey et al, 2001). 8. BACTERIOCINS AS FOOD ADDITIVES Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They can be introduced into food either as a pure compound, or by the use of lactic acid bacteria that secrete bacteriocins. They were first discovered by A. Gratia in 1925. He called his first discovery a colicine because it killed E. coli (Delves-Broughton et al, 1996). Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and killing mechanism (pore forming, Dnase, nuclease, inhibition of murein synthesis, etc), genetics (large plasmids, small plasmids, chromosomal), molecular weight and


chemistry (large protein, polypeptide, with/without sugar moiety, containing atypical amino acids like lanthionine) and method of production (ribosomal, post ribosomal modifications, nonribosomal). A method of classification fits bacteriocins into Class I, Class IIa/b/c, and Class III (Cotter et al., 2006). Class I bacteriocins are small peptides and include nisin. The class II are small heatstable proteins. The class IIa (pediocin-like bacteriocins) are the largest subgroup and contain an N-terminal consensus sequence -Tyr-Gly-Asn-Gly-Val-Xaa-Cys within this group. The C-terminal amino acid is responsible for species-specific activity, causing cell-leakage by permeabilizing the target cell wall. Class IIa bacteriocins have a large potential for use in food preservation as well as medical applications, due to their strong anti-listerial activity, and broad range of activity. Class IIb (two-peptide bacteriocins) requires two different peptides for activity. Other bacteriocins can be grouped together as Class IIc (circular bacteriocins). These have a wide range of effects on membrane permeability, cell wall formation and pheromone actions of target cells. Class III is large and groups heat-labile proteins. The first bacteriocin called nisin was discovered in 1927. It is currently used as food additive (E234) for food preservation but many bacteriocins are also naturally present in a large number of foods (sausage) 8.1. Nisin Nisin is an antimicrobial peptide, produced by several strains of Lactococcus lactis, belonging to the Class I bacteriocins called lantibiotics which are small (